Ion Electrode Based Enzymatic Analysis of Creatinine Huvin Thompson and G. A. Rechnitz Department of Chemistry, State University of New York, Buffalo, N . Y. 74274
A method for the selective determination of creatinine, based on the use of a commercially available membrane electrode for ammonia and the newly purified enzyme creatininase, is proposed and evaluated. The purified enzyme is shown to selectively break down creatinine in the presence of possible interferents such as urea, creatine, and arginine. The ammonia produced in the enzymatic reaction is sensed with the membrane electrode and gives rise to a potentiometric signal proportional to the creatinine concentration. Although limited by the present short supply of the purified enzyme, the proposed method is shown to yield useful analytical data over the physiological concentration range.
T h e recent commercial introduction of ammonia selective membrane electrodes makes possible the development of further specific enzymatic assays for a number of clinically important substances such a s creatinine. Creatinine is second only to urea in being t h e most important nitrogenous end-product excreted by animals a n d yet the basic routine assay method for this substance is subject to numerous interferences (1). Based on t h e production of a red colored complex (Jaffb color) between creatinine a n d picrate in alkaline solution, this method gives erroneous results in t h e presence of proteins, ketone substances, a n d glucose. T h u s methods employing preliminary separation or reaction kinetic procedures have been developed to minimize t h e effects of other Jaffb positive materials which are found in serum a n d urine (2-6). I t would be a d vantageous, therefore, to have a more selective, or even specific, reaction on which to base new methods of analysis. T h i s paper proposes such a method. I t has already been shown (7-26) t h a t t h e combination of enzymes, to provide selective biochemical action, a n d ion electrodes to monitor the extent or progress of t h e reaction, can result in effective new analytical procedures. In the case of creatinine, the development of a novel a p proach required the availability of a n ammonia or ammonium ion sensor not subject to alkali metal ion interference a n d t h e isolation of a suitable enzyme with high selectivity for creatinine. Both conditions have now been met. T h e existence of a single enzyme capable of selectively (1) J. T. Clarke, Ciin. Chem., 7, 271 (1961). (2) A . C. Teger-Nilsson. Scand. J. Ciin. Lab. invest.. 13, 326 (1961). (3) R. A. Rockerbie and K. L. Rasmussen, Clin. Chim. Acta. 15, 475 (1967). (4) R . J. Mitchell. Clin. Chem.. 19, 408 (1973). (5) K . Larsen. Clin. Chim. Acta, 41, 209 (1972). (6) J. Bierensde Haan, Anal. Lett., 5, 815 (1972). (7) S. A . Katz and G . A. Rechnitz, Fresenius' Z. Anal. Chem., 196, 248 ( 1 963) (8) R. P. Buck, Anal. Chem.. 44, 270R (1972). (9) R. L . Llenado and G . A . Rechnitz. Anal. Chem., 44, 486 (1972). (10) R. L. Llenado and G . A. Rechnitz, Anal. Chem.. 44, 1366 (1972). (11) T . Neubecker and G . A Rechnitz, Anal. Lett. 5, 653 (1972). (12) R. L. Llenado and G. A . Rechnitz, Anal. Chem., 45, 826 (1973). (13) B. Erlanger and R . Sack, Anal. Biochem., 33, 318 (1970). (14) G . Baum and F. Ward, Anal. Biochem., 43,947 (1971). (15) G . G . Guilbault. Pure Appl. Chem.. 25, 727 (1971). (16) G . J. Papariello, A. K. Mukherji, and C. M . Shearer, Anal. Chem., 45, 790 (1973).
246
breaking down creatinine to ammonia a n d N-methylhydantoin was demonstrated by Szulmajster in 1958 (17).He isolated this enzyme in quite a pure form from a bacterial source. Other workers had previously failed to d o so in various studies of a number of bacterial strains (28-23). In most of these cases, it is probable t h a t a number of enzymes were present in t h e microorganisms since end-products varied from urea a n d sarcosine, to glycine, ammonia, a n d carbon dioxide ( 2 4 ) . Beckman Inc. has recently introduced a similar enzyme, creatininase, which also produces ammonia a n d N-methylhydantoin stoichiometrically. Its use in a double Jaffb procedure for t h e determination of creatinine has been investigated ( 2 5 ) . T h e present paper investigates t h e application of a n ammonia gas electrode in conjunction with this enzyme to the development of a specific method for routine creatinine analysis. T h e selectivity of t h e crude enzyme preparation is examined a n d its partial purification a n d subsequent application t o analysis is described. Proper enzyme purification a n d selection of experimental conditions are vital to t h e success of t h e proposed method.
EXPERIMENTAL Chemicals and Reagents. Creatinine (anhydrous), creatine H20, urea, and arginine were obtained from the Sigma Chemical Company, St. Louis, Mo. 63118. One gram per cent stock solutions of these were prepared in deionized water and stored in a refrigerator. Fresh working solutions were prepared as required by suitable dilutions of stock solutions with NaH2P04/Na2HP04, NaH2PO4/NaOH, or NaHzP04/Tris buffer solutions. Fkggent grade chemicals were used to prepare all buffer solutions. Lyophilized creatininase was obtained from Beckman Inc. Microbics Operations, Fullerton, Calif., and reconstituted as required according to the directions supplied. Reconstituted portions were stored at -20 "C and thawed just before use. In this frozen state the enzyme is stable for at least six months, while at room temperature, there is an 80% loss of activity in six hourz. Diethylaminoethylcellulose, DE 52 (Whatman, Kent, England) was used in the chromatographic purification. The salt gradient solutions used for elution contained reagent grade sodium chloride and buffer components. Apparatus. Potential readings of the Orion ammonia electrode (No. 95-10) were taken on a Corning Model 12 pH meter and recorded using a Beckman 10-in. recorder. Technicon AutoAnalyzer modules (Technicon Corporation, Tarrytown, N.Y.) were used in automated routine enzyme and creatinine assays based on the colorimetric procedure. A Beckman DB-G Spectrophotometer was used in all manual assays for enzyme and protein. Dialyses were carried out using hollow fiber beakers or minibeakers (Dow Chemical Company) and chromatographic fractions were collected using an LKB Automatic Fraction Collector, Type 7000. Methods. Enzyme Assay. One unit of enzyme is defined as the amount catalyzing the hydrolysis of 1 pmole of creatinine per J. Szulmajster, Biochim. Biophys. Acta, 3 0 , 154 (1958). D. Ackerman, Z. Biol., 62, 208 (1913). R . Dubos and R. F. Miller, J. Biol. Chem., 121, 429 (1937). H . A . Krebs and L. V . Eggleston, Enzymoiogia, 1. 320 (1939). S. Akamatsu and R. Miyashita. Enzymoiogra, 15, 122 (1951). J. Roche. G . Lacombe, and H . Girard, Biochem. Biophys. Acta. 6, 210 (1950). (23) J. Szulmajster and R. C. Gardiner, Biochem. Biophys. Acta, 39, 167 (1960) (24) A. Brunel, G . Brunet-Capelle, and A. Graziana-Sautier, C. R. Acad. Sci., Ser. 0 . 273, 1799 (1971) (25) J. R. Riese and R. S. Davis, Beckman Microbics Operations, La Habra. Calif.,private communication, Sept. 1972.
(17) (18) (19) (20) (21) (22)
ANALYTICAL CHEMISTRY, VOL. 46, NO. 2, FEBRUARY 1974
t o S o m p l e r Wosh
n
PUMP
40ft, l 5 r n i n
RECORDER
2 5'C
Figure 1. Schematic of continuous flow apparatus for routine enzyme assay
minute at 37 "C and pH 8.5. The following procedure was used for the manual measurement of enzyme activity: 100 p1 of enzyme solution was incubated with an equal volume of 8 p M creatinine solution, pH 8.5, for the desired time (usually 30 minutes) at 37 "C in a water bath. Enzymatic action was arrested and color development initiated by the addition of 2 ml of alkaline picrate solution (0.2% picric acid, 0.175M NaOH freshly prepared daily). After a further 15-minute incubation at room temperature, the absorbance of the solution was measured at 540 nm against alkaline picrate solution in 5-mm cells. This reading was compared to a blank in which deionized water replaced the enzyme solution. An automated procedure using the AutoAnalyzer set up, detailed in Figure l , was used to assay all fractions obtained from the chromatographic column for enzyme activity. This was primarily used to determine the point at which enzyme wab being eluted from the column and was sensitive to 0.005 unit ml-I. Since no detailed quantitative information was sought, it was possible to run the assay a t 40 samples her hour with a 1:2 sample to wash ratio. This allowed individual peaks to be distinguished while at the same time conserving enzyme fractions. Jaff6 color development was measured a t 505 nm using a 15-mm flow cell in the filter colorimeter. Protein Assa? An estimate of the protein content of each fraction was made by taking absorbance readings at 280 nm and 260 nm us. water in 1-mm quartz cells. The protein content was then calculated using Warburg and Christian's expression (%), 2.303 per cent protein 1 c, = x - x A,,, d P280 100 relating concentration of protein, C,, to molar absorptivity 3280, cell path length d, absorbance A m and per cent protein in the protein-nucleic acid mixtures. The value of the expression
2.303
X
per cent protein
P280 100 is related to the ratio of absorbances at 280 nm and 260 nm, Azso/A260 and is found from tables (26). Electrode Creatinine Assay. For studies with the crude enzyme, 5-ml samples of 1 to 100 mg 70creatinine solutions, p H 8.5, were incubated with 0.25 ml of enzyme solution (approximately 1 unit) for the desired time, a t the chosen temperature and in tightly capped vials. The mixtures were then quenched with 50 p1 of 3.8M NaOH which adjusts the pH to approximately 12. At this pH, the ammonia-ammonium ion equilibrium is held almost 100% in favor of the ammonia form and thus the ammonia concentration measured using the electrode is that formed during incubation. When purified enzyme fractions are used, equal volumes (100 pl) of enzyme and creatinine solutions are mixed and incubated in sealed vials for 1 hour at 37 "C. Before measuring. the mixtures are quenched with 10 pl of 1M NaOH. Because of the small volume involved, care should be taken during measurement to ensure that no air bubbles are trapped next to the membrane of the electrode as these may cause errors or sluggish response. Dialysis and Ion Exchange Chromatograph?. Hollow fiber dialysis devices proved the most rapid and efficient means of reducing the initial salt concentration of the enzyme preparation to tolerable levels (approximately lW4M). The minibeaker device used had a capacity of 7 ml. The enzyme solution was placed in the
(26) 0. Warburg and W .
Christian, Biochem. Z., 310, 384
(1941)
1
0
5
IO
I5
20 IRACIION
25
30
35
40
45
NUMBIR
Figure 2. Protein and enzyme activity profile obtained by chromatographic fractionation Sohd line refers to enzyme activity while broken line refers to protein concentration
outer chamber and stirred vigorously by means of a magnetic stirrer while deionized water was passed through the fibers at a rate of 10 ml min-':The process, carried out in a cold room at 4 "C, was continued for at least 90 minutes. During dialysis, the inlets to the outer chamber were kept sealed to prevent dilution of the enzyme preparation by osmosis. A typical chromatographic separation of proteins, graphically represented in Figure 2, was carried out using a DEAE cellulose column 1.3 cm in diameter and 4.5 cm long. This was conditioned to the appropriate pH, 6.8 or 8.5, with 25mM buffer. It was found to have a capacity of about 150 mg of protein when loaded with the dialyzed enzyme solution in a 1-hour period. The column was eluted with a salt gradient produced by 50 ml of 25mM buffer and 50 ml of 0.4M NaCl in buffer, 2.5-ml fractions being collected a t a rate of 0.5 ml min-I. The entire operation was performed in a cold room at 4 "C.
RESULTS AND DISCUSSION Studies with Crude Enzyme. In preliminary experiments, t h e presence of relatively large concentrations of ammonium ions ( 10-2M) in t h e crude enzyme preparation was noticed, requiring dialysis of t h e preparation before further study. T h e results of these studies using the dialyzed product a n d different combinations of incubation time a n d temperature for t h e range of creatinine concentrations 1-100 mg YC are shown in Table I. It can be seen t h a t down t o 1 m g ?& (8.85 X 10-5M) creatinine, all calibrations are useful but for t h e short incubation time a t 25 "C, the plot becomes curved between 40 a n d 100 mg %. T h i s indicates t h a t there was incomplete conversion of all creatinine present t o ammonia. T h e slopes of t h e calibrations carried out with incubation at 25 "C for 60 minutes a n d a t 35 "C for 20 minutes are 47.9 and 49.0 mV per dec-
A N A L Y T I C A L C H E M I S T R Y , VOL. 46, N O . 2, FEBRUARY 1974
247
Table I. Creatinine Assay at Different Incubation Times and Temperatures Creatinine conm, mg ',
1 10
20 40 60 100
rnV iendirig -
~
6n R4in ?I I >
21 -21 --37 -53 -62 74
'r
7 9
__
____
-
!(I Min at 27 ' C
20 Min at ?i "C
21 5 -23 2
3
-Xi 6 -49 5 -55 6
1
-
1 4
.
CKPatinine
1
5 10 20 100
23
C)
-9 E
-22 0 -36 5 -70 0
24 1 - 10 1 -22 0
20 100
-88.8
-89.5
-?6.1
-45.7
~~~~
~~
..
~
. . ..
. ..
21) m p ' , arpninc
,*,!I mg '.; urea
-22 3 -31 5 -41.2 -89.5
-47.2 -50.8 -58.2 -91.6
~-
1 5 10
40
36 5 -69 7 -
a d e change in creatinine concentration, respectively. While these d o not agree with t h e theoretically predicted slope of 59.2 mV per decade, t h e discrepancy might be explained by a slight departure from stoichiometry of t h e enzyme reaction as was reported by Szulmajster ( I 7 . In addition the slight deviation from linearity observed below 10 mg % also serves t o reduce t h e overall slope as calculated by t h e least squares method. T h e reproducibilith- of t h e method over the same wide range is shown by t h e results in T a b l e 11. T h r e e replicate samples of each concentration were incubated under identical conditions of time a n d temperature, 40 minutes a t 25 "C. T h e subsequent potential readings show excellent agreement over the completp range. X similar set of e x p e r i n e n t s cnrrieri out in t h e presence of various a m o u n t s o f three different possible interfering substances 1x.hic.h are !i~iially found in serum anti urine gave t h e resu!ts shown in T a h l e 111. T h i s shows t h a t various other enzyme? must be present in the crude preparation since excess ammonia was formed in the case of each interferant tested, Even in t h e absence of creatinine, a m monia was produced from urea, arginine. a n d creatine. This. of course. is no problem when colorimetric monitore reaction is performed (25) but when ammonia production is t'nl!owed. 2s in t h e present study, some method of masking these other effects or of purifying t h e crude enzyme must be found. T h e fact t h a t the final s t e p in the production of ammonia from arginine is known t o involve urea and Grease a n d the same final s t e p is thoug!it to ncciir i n ihe bacterial t)reakdown of creatine (24) prompted the thijl.:ght tha; masking the urease enzyme present may eliminate these interferences. Ynfortunately. effective urease inhibitors such as silver ions (27) also were t'ound t o inhibit creatininase. It was, therefore. necessary t o purify the enzyme. Enzyme Purifiration Studies. .L\lthwgh the enzyme obtained from Heckman comes from a different source t h a n t h a t studied b:; Szulmajster, we a t t e m p t e d to modify Szulmajster's purification method for our purposes. Of the two recommended o;jerations--i.e., ammonium sulfate fractionation and inn exchantre chromatography--the former proved ineffecti\e in the present situation. Only small amounts of the protein were precipitated outside
248
~~~
~ : r e iti i ne
-30.4
r i c itinint, concn. m g
No J
( 2 7 ) J. F . Ambrose. G . B. Kistiakowsky. a n d A . G Chern S o c . 73. 1232 ( 1 9 5 1 ) .
!ilnnk
-1.4 -20 5 -36.1
5
-
22 4 -10 2 -21 9 3 0
mT' rendings ~
; mg
rnV reading -
No 2
-36 -iO
1
.T!> '
Table IV. Study of C J r w lnterfrrence When Purified Enzyme Is Used
-~
r n C rc.iclinjis A0 1
rreatininc concn, rnx ,'
24 0 -23 5 -38 0 -57 1 -64 8 -74 4
61 9
-_
.
.
~~
Table 11. ReproducibilitS of the Method cnnrn mg l i
Table 111. Study of the Effects of Urea, Arginine, and Creatine lmpurities
Kridl.. J
Arner.
100
~
151 -35 +22 -16 -36
~~
With 71)O rng
Illnnk
2 i
2 0
3
~
I
U K C ~
149 3 133 5 123 0 -10 5 -34 1
t h e recommended 40 t o 60% saturation range a n d even these still contained enzymatic activity. Chromatographic fractionation. however, proved somewhat more successful, though minor modifications had t o be made in Szuimaister's procedure. To increase column capacity. albeit a t some siight reduction in flow rate, microgranular DEAE was used as opposed to the fibrous varietv. In addition. t h e column was preconditioned with 2 5 m M buffer instead of distilled water. T h e fractions collected from t h e application of a 0-0.3M NaCi gradient, however, showed only one major enzyme peak occurring over t h e concentration range 0.09-0.12-kf NaCl and corresponding roughly to t h e initial stages of the second major protein peak (Figure 2 ) . T h e specific activity of the most active fractions of this peak was only a slight improvement on t h e starting specific activity of approximately 30 e u g - l . In l a t r r experiments, when t h e salt gradient was increased t o 0-0.5M NaCl a n d the p H reduced to 6.8, a very minor peak of activity, about, one fifth t h e size of the major peak, occurred around t h e 0.4.V NaCl point. This corresponded to a n area of low protein a n d , thus, the specific activity was in the region of 80 eu g - Total activity of this peak was very low and these fractions were not of use in t h e studies with the purified enzyme detailed below. While all fractions with creatininase activity displayed no urease activity when tested. t h e total activity recovered from each column was never greater t h a n 25-000/0 of t h a t initially bound to t h e cellulose. At t h e same time, protein recovcry was at least 8(?70. Such losses indicate t h a t further studies in the purification are necessary. Studies with Purified Enzyme. Preliminary experiments in which portions nf t h e purified enzyme fractions were incubated for two t o sixteen hours with 3.0 mg 70solutions of creatinine, creatine, urea. a n d arginine showed t h a t only creatinine solritions released any species to which the anmionia electrode was sensitive. I t is thus likely t h a t a t least t h e contaminating urease h a s been removed from the enzyme preparation. Figure 3 shows t h e trace obtained when a set of ammonia standards and t h e enzyme substrate mixtures, incubated for two hours a t 35 "C were measured with t h e electrode in a flowing system. T h e peak displayed by t h e creatinine,'enzynie mixture corresponds well t o almost complete conversion of the 50 mg % creatinine to ammonia while ammonia levels in the
A N A L Y T I C A L C H E M I S T R Y , VOL. 46, N O . 2, F E B R U A R Y 1974
io-'
N tij
1
1.0
0.5 LOG
CII&IIHIHI
15
20
CONC.
Figure 4. Calibration line for creatinine in 1-100 m g YO range
Figure 3. Electrode response to ammonia standards compared to ammonia formed by purified enzyme incubation with various substances
+ enzyme: 6 ,50 mg % creatine + enzyme; C , + enzyme; D. 50 mg % urea 4- enzyme
A , 50 mg % creatinine
50 mg YOarginine
creatine, arginine, a n d urea incubation mixtures are less t h a n 10-5M. sufficiently low for even 1 mg 90 creatinine determinations (8.85 x 10-5M) to be possible. Table IV shows t h e results of two parallel runs on a series of creatinine solutions. One series contained only creatinine in buffer while t h e other had, in addition, 200 mg % of urea. All mixtures were incubated 14 hours a t 25 "C before quenching a n d good agreement is shown between t h e two series. If slightly more concentrated enzyme solutions are used, approximately 0.15 eu m l - l , incubation for 30 minutes a t 37 "C is sufficient t o hydrolyze all creatinine in t h e 1-100 m g % range. Such enzvme solutions were ohtained by reducing t h e total volume of eluent used in t h e chromatographic fractionation t o 40 ml and collecting 1.5-ml fractions. Using such fractions t h e calibration plot shown in Figure 4 was obtained.
CONCLUSION T h e application of a n ammonia gas electrode in conjunction with t h e enzyme creatininase provides a good
basis for t h e development of a new method for t h e determination of creatinine in solution. T h e purification of the enzyme t o a point where arginine, creatine, a n d urea interferences were eliminated has been demonstrated. and t h e purified product used t o construct a calibration curve in t h e 1-100 mg 70 range. While this already covers t,he complete normal range of creatinine levels found in both serum a n d urine, it could easily be extended to 500 or 1000 mg %. given a more active enzyme preparation. T h i s possibility of direct measurement over such a wide range is a n improvement over the colorimetric procedure which is linear only over a very restricted concentration range. In addition, if purer, more concentrated enzyme preparations were available, t h e time required for the determinations could be reduced t o a few minut,es since no lengthy color development periods are required. As a result, the proposed method offers attractive advantages over presently used methods and awaits only the wider availability of purified creatininase enzyme as a condition for routine use.
ACKNOWLEDGMENT We gratefully acknowledge t h e loan of AutoAnalyzer components by t h e Technicon Corporation. Thanks are also due to S a m Otieno, Oliver Dolly, a n d colleagues for their assistance with, and helpful discussion of, the enzyme purification procedure. Received for review cJune 15, 1973. Accepted September 6 , 1973. Work supported by t h e National Institutes of Health.
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