Anal. Chem. 1991, 63,611-614
811
Immobilized Enzyme Electrode for Creatinine Determination in Serum Vu Khue Nguyen, Ch-Michel Wolff,*J Jean Louis Seris? and Jean-Paul Schwing Laboratoire de Chimie Physique et Electrochimie, Unit6 associ6e au CNRS No. 405, Ecole des Hautes Etudes des Industries Chimiques de Strasbourg, 1, rue Blaise Pascal, 67084 Strasbourg Ceden, France
An lmmoblllzed enzyme electrode for contlnuous Creatinine determination In blood serum Is descrlbed. The enzymes creatinine amidohydrolase, creatlne amldlnohydrolase, and sarcoslne oxldase are colmmoblllred to the surface of the polypropylene membrane of a Clark-type electrode responsive to oxygen. The immoblllzed enzymes catalyze the decompoSnlon of creatinine wlth the conwmotlon of oxygen and thus permlt the creatlnlne measurement. The whole assay takes less than 1 mln. Effects of pH and temperature on electrode response are also described. The proposed technlque offers a rapid, simple, and inexpensive means to determine creatinine In blood serum within the normal and abnormal ranges. The repeatablllty of the creatine determlnatlon In serum Is 2.5 % (relative standard devlatlon), and the detectlon llmlt Is 3 X lod mol L-'. The results obtained by this method were compared to those obtalned wlth the Technkon AutoAnalyzer SMAC system based on the JafH reaction; the correlation factor between the two methods was found to be r = 0.9997.
INTRODUCTION The assessment of creatinine levels in human blood serum is a valuable indicator in estimating renal function (1). At present, the spectrophotometric methods widely used for routine creatinine determination are based on the Jaff6 reaction between creatinine and picric acid in alkaline solution (2,3). However, the specificity of the Jaff6 reaction has been questioned in many publications, so numerous modifications of this method have been published (4-10). Several enzymatic methods have recently been investigated to increase specificity (11-19). However, they are laborious, costly, and time-consuming, and therefore, they are not yet widely used in clinical laboratories. Several authors have described the development of rapid, simple, and specific creatinine selective enzyme electrodes for serum creatinine determination (20-22). However, these enzyme electrodes are relatively imprecise. Here we propose a new enzyme electrode system for the determination of serum creatinine. The following three enzymes are used in the present method: creatinine amidohydrolase (CA; EC 3.5.2.10) (23), creatine amidinohydrolase (CI; EC 3.5.3.3) (24),and sarcosine oxidase (SO; EC 1.5.3.1) (25). T h e coupling of these three enzymes allows the transformation of creatinine without coenzymes. The reaction sequence is as follows: creatinine creatine sarcosine
CA + H 2 0 F=
creatine
+ H ~ OSsarcosine + urea
+ O2 + H 2 0 formaldehyde
(1) (2)
+ glycine + H2O2(3)
*To whom corres ondence should be addressed. Present address:Eaboratoire de Biochimie, Institut de Biologie Mol6culaire et Cellulaire du CNRS, 15, rue Ren6 Descartes, 67084 Strasbourg Cedex, France. *Present address: Laboratoire d'Enzymologie, Centre de Recherches de Lacq, B.P. 34 Lacq, 64170 Artix, France.
In this system, apart from the three enzymes CA, CI, and SO, only water and oxygen, which are normally present in biological fluids, are required as reagents to allow the development of the three reactions in the presence of creatinine. The reaction of creatinine in reaction 1 leads to oxygen consumption in reaction 3. In the present work, we have studied the immobilization of these three enzymes on the membrane of a Clark oxygen gas-sensitive electrode (26) and the conditions for creatinine determination in blood serum.
EXPERIMENTAL SECTION Apparatus. All experiments were carried out with magnetic stirring in temperature-controlled cells at 25 "C by using a Haake FJ bath. The pH measurements were made with a Minisis 6000 Tacussel pH meter equipped with a Tacussel TBA llHA glass electrode. To study the amperometric responses to oxygen, we used a Clark oxygen gas-sensitive electrode (Radiometer, Type E 5046/0) covered with a polypropylene membrane (Safidief, France; thickness 8 wm). The potential applied to the working platinum cathode in oxygen determination is -0.6 V versus the reference electrode (silver/silver chloride anode) by using a 1201 Transidyne potentiostat. The current passing through the electrode is converted into voltage (20 mV for 1 pA in all the present work) and recorded by means of a Tacussel EPLl recorder equipped with a TV-11-GD potentiometric plug-in. Under our experimental conditions, the response time to oxygen (defined as the time needed to reach 95% of the final response and noted tg5%)is 28 s. Enzyme Immobilization Procedure. To immobilize the enzymes, the polypropylene membrane was first stretched horizontally and then washed with a 5 g/L aqueous solution of sodium dodecyl sulfate (SDS). Then, the excess SDS was wiped off by means of a smooth and absorbent paper. For immobilization, equal volumes of a phosphate buffer solution containing the enzyme(s) and a 50 g/L gelatin solution (prepared by placing the gelatin in water and leaving it for 10 h at 37 "C) were mixed and laid 1mm thick on the polypropylenemembrane surface and then dried in air. The gelatin gel layer containing the immobilized enzyme(s) so obtained was immersed in a 1.25% (v/v) glutaraldehyde solution (in sodium phosphate buffer 50 mM, pH 7.5) for cross-linking with this agent during 30 s and then thoroughly rinsed with water. After subsequent drying in air, the immobilized enzyme membrane was cut for use. The membrane was placed at the bottom of the plastic outer jacket of the Clark electrode and fixed by means of a rubber O-ring. The electrode does not require any preconditioning, and is ready for use. When not in use, it is stored at 4 "C in air. When the electrode is placed in a solution containing creatinine, this substrate diffuses into the gelatine gel layer of coimmobilized CA, CI, and SO enzymes. These three coupled enzymes catalyze the transformation of creatinine according to reactions 1-3. The creatinine determination is performed by amperometric measurement of oxygen consumption. Reagents. Creatinine amidohydrolase (CA), creatine amidinohydrolase (CI), both from Pseudomonas sp., and sarcosine oxidase (SO),from Corynebacterium sp., were supplied by Sigma Chemical Co. (St Louis, MO). All these enzymes were obtained as a lyophilized powder. All other chemicals used were of reagent grade. All enzymes and reagents were used without further purification. All solutions were prepared in sodium phosphate buffer (50 mM; pH 7.5), unless otherwise noted. Deionized water was used for the preparation of all solutions. The samples of human
0003-2700/91/0363-0611$02.50/00 1991 American Chemical Society
612
ANALYTICAL CHEMISTRY, VOL. 63, NO. 6, MARCH 15, 1991
r Addition of rircoainr
~
0
20
40
80
00
~-
loo
Time (8) Figure 1. Typical current-time response of the sarcosine electrode. The intensity of the oxygen reduction current (S) is on the y axis. The electrode response time is on the x axis. The electrode was equipped with an immobilized SO membrane of 0.63 U/cm2. The initial conmol L-'. centration of sarcosine was 8 X
blood serum were obtained by centrifugation of blood and stored at -20 "C until assayed. The enzymatic units (U) are defined following the supplier's indications, i.e., 1 U of enzyme corresponds to the amount of enzyme needed to catalyze the transformation of 1 pmol of substrate in 1min in the optimum conditions of pH and temperature. The final activity of the enzyme after immobilization was not experimentally determined. It is expressed as the initial activity of the free enzyme divided by the surface of the membrane where this enzyme has been immobilized and is noted as an enzymatic density: U/cm2. This density is a mean value for the membrane; no control has been performed to test the uniformity of the activity in the cast membrane. RESULTS AND DISCUSSION Preparation of the Immobilized Enzyme Electrode. For this study, the membranes containing SO alone, SO and CI, and then SO, CI, and CA were successively prepared. T o determine the optimum density of immobilized SO enzyme for the development of the sarcosine electrode, membranes of different densities of immobilized SO enzyme (ranging from 1.6 to 10.4 U/cm2) were prepared. As an example, Figure 1 shows the typical response curve obtained with the sarcosine electrode equipped with an immobilized SO membrane of 6.3 U/cm2. Before the addition of sarcosine, the experimental signal S is constant and equal to S,. After the addition of sarcosine, the signal decreases and tends to a lower value S, (steady state). We define A S m as the difference between So and S, (3.3- = So - S,). Measurement of AS- corresponds to a measure of the steady-state rate of sarcosine consumption inside the membrane. In Figure 2, the AS- values obtained with the membranes of different densities in immobilized SO enzyme and for different concentrations of sarcosine are shown. For the densities of SO enzyme of 5.2 U/cm2 and less, the dose-response curves obtained look similar to those of the reaction rate versus the substrate concentration in kinetic analysis with free enzymes. This is due to the rate of sarcosine consumption, which is slow when compared to the diffusion rate of this substrate into the gel layer. The resulting rate is then imposed by the rate of the enzymatic reaction. For the densities in SO enzyme of 5.7 U/cm2 and more, the curves present, for the lower concentrations of sarcosine, a perfectly linear region (linear correlation coefficient r = 0.9997 for all the dose-response curves). An upper limit of linearity is obtained, being up to 1.0 x mol L-I for a SO enzyme of 5.7 and 6.3 U/cm2 and up to 1.2 x mol L-' for 10.4 U/cm2. Moreover, the slope of the linear region is not very dependent
0
1
2
3
4
5
6
[Sarcosine] (la3mol L') Figure 2. Sarcosine electrode response to sarcosine concentration. The steady-state rate measurement, AS-,as defined in the text, is represented against the sarcosine concentration for different enzymatic activities in the membrane: (a) 1.6, (b) 3.2, (c)5.2, (d) 5.7,(e) 6.3, and (f) 10.4 U/cm2.
on the density of the immobilized SO enzyme (linear regression equations where y is the current measured in pA and x the concentration of sarcosine in mmol L-l: y = 5.45x, y = 6.634 and y = 7 . 2 ~for 5.7, 6.3, and 10.4 U/cm2 respectively). By increasing the amount of enzyme, one accelerates the rate of enzymatic reaction up to a limit imposed by the diffusion rate of sarcosine into the gelatin layer. The diffusion rate of a species being proportional to its concentration, the rate of enzymatic reaction (limited by the diffusion rate of the substrate) is then exactly proportional to the concentration of the substrate. This explains the linear region of the curves obtained with densities of SO enzyme of 5.7 U/cm2 and more. When the sarcosine concentration is sufficiently high, the diffusion of this substrate into the gelatin layer becomes rapid and the resulting rate, in this case, is imposed by the rate of enzymatic reaction. Concerning oxygen, the diffusion of this substrate into the gelatin layer is sufficiently rapid when compared to that of sarcosine and thus there is no limitation on the rate of the enzymatic reaction caused by oxygen diffusion. For this study, we looked for the sarcosine electrode giving the uppermost limit of linearity of the dose-response curve. Because of the poor mechanical properties of the immobilized SO membrane of 10.4 U/cm2, the electrode equipped with an immobilized SO membrane of 6.3 U/cm2 was chosen. In addition to steady-state measuremenh (AS,), two kinds of kinetic measurements were performed: "fixed time" measurements (fixed time was 25 s after sarcosine addition) (AS,,) and the response rate of the electrode (v) measured as the slope of the response curve a t the inflexion point ( u = dS/dt when d2S/dt2 = 0). By using this electrode, three calibration curves (AS-, AS,,, and u versus the initial sarcosine concentration in the sample) for different concentrations of sarcosine were constructed. The curves obtained were all linear up to mol L-l sarcosine, passing through the origin (linear regression equations where y is the current in pA and x the concentration of sarcosine in mmol L-l: y = 6.6x, r = 0.9997 and y = 4.7x, r = 0.9998 for AS, and ASz5 measurements, respectively; for rate measurements, y is the response rate of the electrode in pA s-l and the linear regression equation is y = 0.281x, r = 0.9997). The Michaelis constant, KM,of the free SO enzyme relating to sarcosine is 2.1 X mol L-' (25). The persistence of the linearity of the electrode response, in steady state, up to mol L-' can be explained by the fact that the rate of the enzymatic reaction
ANALYTICAL CHEMISTRY, VOL. 63, NO. 6, MARCH 15, 1991
is limited by the diffusion rate of sarcosine into the gelatin layer. Linear calibration curves at substrate concentrations close to KMhave also been observed by other authors working on immobilized-enzyme electrodes (27). By defining the detection limit as the concentration of sarcosine that produces a significant response (twice that corresponding to the background value), we determined this limit to be 5 x 7X and 2 X mol L-' for AS,, AS25, and rate measurements, respectively. For the preparation of the creatine electrode, membranes containing SO and CI enzymes were prepared. For this coimmobilization of SO and CI, the density of SO enzyme was fixed as 6.3 U/cm2. Regarding the CI enzyme, we observed that for a density of CI enzyme greater than 0.63 U/cm2, there is a separation of the immobilized enzyme membrane from the polypropylene membrane during the construction of the enzyme electrode. This phenomenon was observed not only for the coimmobilization of CI and SO but also for the immobilization of CI alone at densities greater than 0.63 U/cm2. We therefore limited the CI enzyme to densities lower than or equal to 0.63 U/cm2. Two membranes containing the same density of SO enzyme (6.3 U/cm2) and different densities of CI enzyme (0.63 and 0.32 U/cm2) were prepared for the construction of two electrodes. In the presence of a creatine solution, the experimental response curve obtained with each of these two creatine electrodes is similar to that obtained with the sarcosine electrode (see Figure 1). Using these two creatine electrodes, we have constructed three calibration curves (AS,, ASz5, and v versus creatine concentration in the sample). A perfectly linear region of the curve was not obtained by steady-state measurements, AS,. This can be explained by the low activity of the CI enzyme of the enzymatic membrane on the one hand and by the high value of the Michaelis constant of CI relating to creatine, KM= 1.3 X loV3mol L-' (24), on the other hand. However, the calibration curves obtained by kinetic measurements (AS,, and u ) were linear mol L-' creatine and passed through the origin. This up to is valid for both creatine electrodes (linear regression equations where x is the concentration of creatine in mmol L-' and y the current in pA for AS25 measurements and the response rate of the electrode in pA s-l for rate measurements: y = 0.80x, r = 0.9995; y = 0.54x, r = 0.9994 for ASzs measurements and y = O.O45Ox, r = 0.9996; y = O.O312x, r = 0.9995 for rate measurements with CI densities of 0.63 and 0.32 U/cm2, respectively). The electrode response was greater for the creatine electrode equipped with the membrane of higher density of CI enzyme (0.63 U/cm2). This density was then chosen for creatine determination. The detection limit was found to be 1.5 X lo4, 3 X lo4, and 8 X mol L-' for AS,, AS25, and rate measurements, respectively. The construction of the creatinine electrode was performed with an enzymatic membrane containing SO (6.3 U/cm2), CI (0.63 U/cm2), and CA (1.3 U/cm2). In the presence of creatinine solution, the experimental curve obtained with this creatinine electrode is also similar to that obtained for sarcosine (see Figure 1). Using this creatinine electrode, we have constructed three calibration curves (ASzs, AS,, and u versus creatinine concentration in the sample). As in the case of the creatine electrode, there is no perfectly linear region of the curve obtained by steady-state measurements, AS,. The curve obtained is almost superimposable on that obtained for creatine measurement. This indicates that the transformation rate of creatinine to creatine, catalyzed by the CA enzyme, is sufficiently rapid that it does not limit the later reaction, catalyzed by CI enzyme. The calibration curves obtained by kinetic measurements (AS25 and u ) were linear up to mol L-' creatinine, passing through the origin (for AS25 measurements, the linear regression equation where y is the
613
current in pA and x the concentration of creatinine in mmol L-l is y = 0.75x, r = 0.9996; for rate measurements, the linear regression equation where y is the response rate of the electrode in pA s-l and x the concentration of creatinine in mmol L-l is y = O.O53x, r = 0.9997). The detection limit was found to be 1 X 3X and 1 X lo* mol L-' for AS,, AS25, and rate measurements, respectively. The response time to creatinine (t,,%)is 60 s and remains stable for more than 80 s. The relative standard deviations (or coefficient of variation, CV) for 15 repeated measurements with the same sample, containing 8 x lo4 mol L-* of substrate, without rejecting any value, were found to be 1.6%, 2 % , and 1.8% for sarcosine, creatine, and creatinine electrodes, respectively. Under our storage conditions (4 "C in air), the long-term stability of the sarcosine, creatine, and creatinine electrodes was studied. No decrease in electrode response to a solution of substrate (8 x mol L-l) was observed within 3 months and after more than 100 assays. Effect of pH a n d Temperature. All three enzyme electrodes were subjected to a study of the effects of pH (ranging from 6.5 to 8.5) and temperature (ranging from 18 to 31 "C) on their response. The results obtained showed that the maximum activity of immobilized enzymes was found in the same pH region as for free enzymes, pH 8 (23-25). The described immobilization procedure had, therefore, no effect on the optimal pH for enzymatic activity. Concerning the temperature effect, the electrode response was higher at higher temperatures. By defining Ql0 (temperature coefficient) as the factor by which the steady-state rate measurement, AS,, is multiplied for an increase of temperature of 10 "C, we determined this coefficient to be 1.6, 1.9, and 2.1 for sarcosine, creatine, and creatinine electrodes, respectively. Furthermore, a straight line (r = 0.9996) using an Arrhenius plot (log, AS, versus inverse of absolute temperature, T )was obtained for each of the three electrodes. This result indicates that there is no denaturation of the immobilized enzyme molecules a t temperature lower than 31 "C. Although the enzyme electrode response is higher at higher temperatures, we performed the creatinine measurement in blood serum at 25 "C. This avoids an eventual loss of enzymatic activity following long-term use at higher temperatures. Determination of Creatinine i n Human Blood Serum. In biological fluids such as human blood serum, sarcosine is normally absent but creatine, in the case of muscle disease, can be present and thus interferes with the creatinine determination. To eliminate the interference of creatine, as well as other possible interferences, we performed differential measurements with the creatine and creatinine electrodes. To test the feasibility of creatinine determination in such a case, mixtures of different known amounts of creatinine and creatine in sodium phosphate buffer were prepared. The two electrodes were immersed into the solution for simultaneous measurements of these two substrates. Both steady-state (AS,) and kinetic measurements (ASz and u ) were performed. The creatinine concentrations were determined by direct comparison to the corresponding calibration curves. No response to creatinine was observed with the creatine electrode. The creatinine concentration was obtained by subtracting the response to the creatine electrode from the response of the creatinine electrode. As shown in Table I, the best results for creatinine determination were obtained with rate measurements, U. This rate mode was then chosen for creatinine determination in blood serum. The human serum samples were used without further treatment. Because of the small volume of available serum samples and the large size of the electrode, the simultaneous measurement of creatinine and creatine concentrations by means of the creatine and creatinine electrodes was not possible. The successive measure-
614
ANALYTICAL CHEMISTRY, VOL. 63, NO. 6, MARCH 15, 1991
T a b l e I. C r e a t i n i n e D e t e r m i n a t i o n Creatine
in
t h e Presence
of
substrate concn added, m o l L-'
creatinine concn found, 10"' m o l L-l, for measurement of
creatinine
creatine
AS,
ASz5
Y
0.50 0.20 2.00 2.00 3.00 4.00
0.20 0.20 0.50 2.00 3.00 1.00
0.77 0.00 2.00 1.28 2.00 3.86
0.56 0.00 1.63 1.20 2.27 4.50
0.41 0.01 1.91 1.87 3.40 4.40
ACKNOWLEDGMENT We thank Pierre Jost for valuable discussions and Maurice Offner (Laboratoire de Biochimie, Pavillon Poincarr6, Hospices Civils de Strasbourg) for providing the human blood serum samples and the results from the Technicon AutoAnalyzer. Registry No. CA, 37340-59-3;CI, 37340-58-2; SO, 9029-22-5; creatinine, 60-27-5.
70 i' 60
P
v
d
50
v)
40 'c1
m
compared to those obtained for the same samples by the classical method based on the Jaff6 reaction and with the use of the Technicon AutoAnalyzer SMAC system. A good correlation (the Pearson correlation coefficient is 0.9997) between the results was obtained (Figure 3). Our results averaged 12% lower than those obtained by the Jaff6 reaction method. This is in agreement with the results of most authors who have compared results of enzymatic creatinine determination with those obtained by the non-enzymatic method (17,B-32).This difference can be attributed to the higher specificity of the enzymatic method. The proposed method is demonstrated to be a simple, low-cost, accurate, rapid method (results obtained in less than one minute) for the assay of creatinine in blood serum within the normal and abnormal ranges.
LITERATURE CITED
30 20 10
0
10
20
30
40
50
60
Technicon AutoAnalyzer results
70
80
(mg/L)
Figure 3. Comparison of results obtained for 15 human serum samples
from the enzyme electrode and from the Technicon AutoAnalyzer (JaffB reaction). The line is drawn by linear regression ( r = 0.9997, y = 0.88~ + 0.06). ments of these two substrates were performed. First, the creatinine electrode is immersed in 0.5 mL of sodium phosphate buffer. When the electrode is stabilized, 0.2 mL of serum sample is added (zero time) for total creatinine measurement. The same operation is performed with the creatine electrode for creatine determination. Creatinine concentration in human blood serum was determined with the proposed method and by direct comparison to a calibration curve. This calibration curve was constructed by a standard addition method using the same procedure as described previously. Different known amounts of substrate (creatinine or creatine), after stabilization of the electrode response to serum sample, were added. The electrode response due to the standard of substrate was also exploited by the proposed method. The calibration curve for creatinine determination in blood serum so obtained is linear (linear regression equation where y is the response rate of the electrode in pA s-l and x the concentration of creatinine in mmol L-l: y = 0.050x, r = 0.9995) from 0 to 3X mol L-' creatinine. In any event, the normal level of creatinine in human blood serum ranges from 0.5 x loe5 mol L-' to 1.0 X lo-* mol L-l. The detection limit was found to be 3 x mol L-l. The CV calculated for 20 repeated measurements of the same solution of creatinine (2 x lo4 mol L-l) was 2.5%. No change in the response of either electrode was observed during a period of 3 months. The creatinine measurement in 15 human blood serum samples by the present method was performed. The results obtained were
Tietz, N. Fundamentals of Cllnical Chemistry, 2nd ed.;W. B. Sanders Co.: Philadelphia, 1976; pp 994-998. Jaff6, M. 2.Physio/. Chem. 1888. 70, 391. Folin, 0. 2.Physiol. Chem. 1904, 4 7 , 223. Brown. N. D.; Sing, H. C.; Neeley, W. E.; Koetitz, E. S. Clin. Chem. 1877, 2 3 , 1281. Soldin, S . J.; Hill, G. J. Clin. Chem. 1978, 2 4 , 747. Spierto, F. W.; MacNeil, M. L.; Culbreth, P.; Ducan, I.; Burtis, C. A. Clin. Chem. 1980, 26, 286. Morishita, Y.; Nakane, K. EiseiKensa 1979, 2 8 , 172. Ryan, E. D.; Walker, W. H. C. Microchem. J. 1980, 25, 500. White, W. A.; Attwood, E. C. Ann. Clin. Biochem. 1980, 77, 153. Muller, H. Fresenius 2.Anal. Chem. 1988, 332, 464. Wahlfeld, A. W.; HoIz, G.; Bergmeyer, H. U. Methods of Enzymatic Analysis: Academic Press: New York, 1974; Vol. 4, pp 1786-1790. Moss, G. A.; Bondar, R. J. L.; Buzzelli, D. M. Clin. Chem. 1875, 27, 1422. Konoshita, T.; Hiraga, Y. Chem. Pharm. Bull. 1980. 28, 3501. Tanganelli, E.; Prencipe, L.; Bassi, D.; Cambiaghi, S.;Murador, E. Clln. Chim. 1982, 2 8 , 1461. Torelli, G. Eur. Pat. Appl. EP 66, 552 (CL. C12Q1/34), Dec. 8, 1982, I T Appl. 81/82, 009, May 28, 1981, 18 pp (Chem. Abstr. 1983, 98, 103926Jl. Fossati, 6.;Prencipe, L.: Berti, G. Clin. Chem. 1983, 2 9 , 1494. Perakis, N.; Wolff, C. M. Clin. Chem. 1984, 30, 1792. Goren, M. P.; Osborne, S.;Wright, R. K. Clin. Chem. 1986, 3 2 , 548. Jeppesen, M. T.; Hansen, E. H. Anal. Chim. Acta 1986, 214, 147. Meyerhoff, M.; Rechnitz, G. A. Anal. Chem. Acta 1876, 85, 277. Guilbault, G. G.; Chen, S.P.; Kuan, S. S. Anal. Left. 1980, 73 (B18),
.
1607
iiihio, T.;
Kentaro, Y. Clin. Chem. 1963, 29, 51. Rikitake, K.; Oka. I.; Ando, M.; Yoshimoto, T.; Tsuru, D. J. Biochem. 1979. 8 6 . 1109. Yoshimoto, T.; Oka, I.; Tsuru, D. Arch. Biochem. Siophys. 1976, 777, 508. Hayashi, S.;Suzuki, M.; Nakamura, S.Biochem. Biophys. Acta 1983, 742, 630. Clark, L. C. Trans. Am. SOC. Artif. Intern. Organs 1956, 2 , 41. Carr, P. W.; Bowers, L. D. Immobilized Enzymes in Analytical and Clinical Chemistry, Fundamentals and Applications ; John Wiley and Sons, Inc.: New York. 1980; pp 148-310. Spierto, F. W.; MacNeil, M. L.; Burtis, C. A. Clin. Chem. 1979, 72, 18. Szasz, G.;Bomer, U.; Stahler, F.; Bablok, W. G. Clin. Chem. 1877. 2 3 , 1172. Szasz, G.; Bomer, U.: Bablok, W. G.; Busch, E. W. Clin. Chem. 1978. 2 4 , 997. Wolff, C. M.; Monola, H. A. Anal. Chem. 1978, 50, 94. Roguijic, A.; Posavec, M. Farm. Glas. 1980, 3 6 , 149.
RECEIVED for review July 2,1990. Revised manuscript received November 14,1990. Accepted December 7,1990. This work was supported by a grant from Elf Aquitaine Society, B.P. 34 Lacq, 64170 Artix, France, and was a part of the Doctorat d'Etat thesis of one of us (v.K.N.).
