852
(9) (10) (11) (12)
Anal. Chem. 1984, 56,852-854 Wang, J.; Freiha, B. A. Anal. Chim. Acta 1983, 148, 79-85. Cheng, H. Y.; Faiat, L.; Li, R. L. Anal. Chem. 1982, 5 4 , 1384-1388. Jarbawi, T. B.; Heineman, W. R . Anal. Chim. Acta 1982, f35, 359-362. Lubert, K. H.: Schnurrbusch, M.; Thomas A. Anal. Chim. Acta 1982, 144. 123-136. Kalvoda. R . Anal. Chim. Acta 1982. 738. 11-18. Wang, J.; Freiha, B. A. Anal. Chem. 1983, 55, 1285-1288. Chaney, E. N.; Baidwin, R. P. Anal. chem. 1983, 54, 2556-2560. Wang, J.; Freiha, 8. A. J . Nectroanal. Chem. 1983, 757, 273-275.
(13) (14) (15) (16) (17)
Wang, J.; Freiha, 6 . A. Anal. Chim. Acta 1983, 754, 87-94. Wang, J.; Freiha, B. A. J . Bioelectrochem. Bioenerg., in press. Davis, D. G.; Everhart, M. E. Anal. Chem. 1984, 36, 38-40. Kuwana, T.; French, W. G. Anal. Chem. 1964, 3 8 , 241-242. Chambers. C. A. H.; Lee, J. K. J . Nectroanal. Chem. 1987, 1 4 ,
--- - . ..
RnQ-Rid
RECEIVED
fori%view October 25, 1983. Accepted November
28, 1983.
Enzyme-Amplified Determination of Methotrexate with a pC0, Membrane Electrode Purneshwar Seegopaul and G. A. Rechnitz* Department of Chemistry, University of Delaware, Newark, Delaware 19711 Methotrexate, or N - [ 4 - [[(2,4-diamino-6-pteridinyl)methyllmethylaminol benzoyl]glutamic acid, is an antimetabolite used in the treatment of certain neoplastic diseases. Its principal mechanism of action is the competitive inhibition of the enzyme folic acid reductase, important in the process of DNA synthesis, and thus interferes with tissue cell reproduction. With careful dosage control, methotrexate may impair malignant growth without irreversible damage to normal tissues ( I ) . Methotrexate (MTX) has been measured by enzyme inhibition (2, 31, HPLC (4), ion-exchange chromatography (5), homogeneous and heterogeneous enzyme immunoassay (4,6-81, radioimmunoassay (4), fluorometry (9), and mass spectrometry (10). We now report the use of an enzyme cycling amplification procedure in conjunction with a pCOz membrane electrode for the determination of methotrexate. The method is based on the inhibition of dihydrofolate reductase enzyme which couples with 6-phosphogluconic dehydrogenase to recycle the NADP+/NADPH redox system (Figure 1). Inhibition of the reductase by methotrexate reduces the extent of cycling which is then directly related to the drug concentration.
EXPERIMENTAL SECTION Apparatus. Enzymatic reactions were carried out in a 10-mL jacketed glass cell thermcatated at 37 f 0.1 "C with a Haake Model FM constant temperature circulator. Potentiometric rate measurements were made with a Corning Model 12 Research pH/mV meter and recorded on a Heath Schlumberger Model SR-255B strip chart recorder operated at 5 mm min-' and a range of 100 mV. Carbon dioxide liberation from the enzymatic reaction was monitored by an Orion Model 95-02 carbon dioxide membrane electrode. Reagents. Reagent grade chemicals were used without further purification, unless otherwise noted, and deionized water was used to prepare all solutions. Enzymatic reactions were carried out in 0.2 M citrate buffer of pH 6.2 containing 0.3 M potassium chloride. 6-Phosphogluconic acid (catalog no. P-7877), @-nicotinamide adenine dinucleotide phosphate, reduced form, @-NADPH(catalog no. N-7505), and dithioerythritol, DTE (catalog no. D-8255), were all obtained from Sigma Chemical Co. The 0.01 M @-NADPH solutions were made up fresh each day in 0.1 M citrate buffer of pH 6.2 and stored in the dark in crushed ice. The 0.25 M 6phosphogluconic acid solution was stored in the dark at 4 "C following adjustment to pH 6.2. Dihydrofolic acid (Sigma, catalog no. D-7006) was bought in 25-mg ampules and kept at -20 "C. A stock solution was prepared by dissolving 25 mg of the substrate in 5 mL of water containing 5 mM HCl and 30 mM DTE. The resultant suspension was thoroughly stirred in the dark at 4 OC and then stored frozen in 0.5-mL aliquots. Working solutions were prepared each day by thawing the 0.5-mL aliquot in the dark followed by the addition of 0.5 mL of 0.3 M citrate buffer, pH 6.2, containing 0.2 M DTE
and stored in crushed ice. Excess working solutions were discarded after 8-10 h. Dihydrofolate reductase (E.C. 1.5.1.3) obtained in 5 units vials of 10 units mL-' (Sigma, catalog no. D-6385, bovine liver source) was stored at 4 "C in the dark and used undiluted. 6-Phosphogluconic dehydrogenase (E.C. 1.1.1.44) obtained in 25 unit vials of 47.2 units mL-' (Sigma catalog no. P-0632 yeast source) was stored at 4 OC and used undiluted. Methotrexate ((+)-amethopterin, Sigma catalog no. A-6770) was stored at -20 "C. A stock solution was made by dissolving 3 mg of methotrexate in 100 mL of water in the dark at 4 "C. Following dissolution, 5-mL aliquots in tightly sealed vials were stored frozen in the dark. Working solutions of 3000 pg L-' were prepared fresh each day by 1 in 10 dilution of the thawed stock solution and stored in the dark in crushed ice. Procedures. Determination of Optimum Dihydrofolate Reductase Activity Necessary for Inhibition Reaction with Methotrexate. A 1.5-mL portion of 0.2 M citrate buffer, pH 6.2, containing 0.3 M KC1 was added t o the thermostated glass cell containing a small Teflon-coated spinbar for solution mixing at 37 A 0.1 "C. The 100 pL of 0.25 M 6-phosphogluconic acid, 50 pL of 0.01 M fi-NADPH, and 35 pL of 6-phosphogluconic dehydrogenase (1.65 units) were added to the buffer solution. Various aliquots of dihydrofolate reductase (0.0054.08 units) were then added and the total solution volume was adjusted to 1.9 mL with deionized water. After a stable base line potential was obtained, 100 pL of 5.64 X M dihydrofolic acid was added to initiate the enzymatic reaction. The carbon dioxide produced was detected by the pCOz electrode and the initial rate in millivolts per minute was recorded. Blank determinations in the absence of the reductase enzyme were carried out for correction of the rate measurements. The initial rates were plotted against corresponding enzyme activities to provide standard curves. Similarly, for optimization procedures, each reagent concentration or kinetic parameter was varied in turn while maintaining all other conditions constant. Determination of Methotrexate. One hundred microliters of 0.25 M 6-phosphogluconic acid, 50 pL of 0.01 M @-NADPH,35 pL of 6-phosphogluconic dehydrogenase (1.65 units), and 2 pL of dihydrofolate reductase (0.02 units) were added to 1.5 mL of 0.2 M citrate buffer, pH 6.2, containing 0.3 M KCl. Following solution mixing at 37 f 0.1 "C, various aliquots of 3000 wg L-' working methotrexate standard (1-30 Fg L-') were added and the total solution adjusted to 1.9 mL with deionized water. After 5 min of preincubation, 100 pL of 5.64 X lo-* dihydrofolic acid was added to initiate the enzymatic reaction. Initial rates, in millivolts per minute, were then recorded. Blank determinations in the absence of methotrexate were carried out for correction of rate measurements. The initial rates were plotted against the corresponding methotrexate concentrations t o provide a standard calibration curve. Unknown methotrexate samples were similarly processed and their levels determined from the standard curve.
RESULTS AND DISCUSSION The kinetic parameters influencing the coupled enzyme reaction in the absence of methotrexate were first optimized
0003-2700/84/0356-0852$01.50/00 1984 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
c- pH 6,2 -t
E.C.151.3
Tetrahydrofolate
NADP'
A
4.0
E,C.l.l.lA 4
853
-
.-
C
6-phosphogluconate
s E
-.-
3.0-
0
Figure 1. Schematic diagram of cycling system for the determination of methotrexate, using dihydrofolate reductase (E.C. 1.5.1.3) and 6phosphogluconic dehydrogenase (E.C. 1.1.1.44) with the pC0, electrode.
m
K
.i
2.0-
C
1.0
I
I
I
I
I
Table I. Within-Run Precision and Recovery Studies of Methotrexate Added to Laboratory Samples amt of methotrexate added, pg L-* 1.5 0
5
10
D i hyd r o f 0 1 a t e
15
20
25
R e d u c t a s e , u n its/L
Flgure 2. Effect of dihydrofolate reductase on the rate of enzymatic M @-NADPH. catalysis at pH 6.2 and 37 OC with 2.5 X
by studying the influence of reagent concentrations, enzyme activities, pH, and temperature. Under the optimum conditions established, the drug was then determined through its inhibitory effect on dihydrofolate reductase enzyme activity. Figure 1 illustrates the coupled enzyme mediation of the NADP+/NADPH recycling system. In the presence of dihydrofolic acid, dihydrofolate reductase converted @-NADPH to @-NADP+which was then regenerated to @-NADPHby 6-phosphogluconic dehydrogenase. In the process, 6phosphogluconic acid was decarboxylated to yield ribulose 5-phosphate and the carbon dioxide monitored by the pCOz electrode. The overall reaction, therefore, provided a sensitive determination of the reductase enzyme through amplification and, consequently, the assay of methotrexate. Optimization of Kinetic Parameters. Figure 2 shows the effect of dihydrofolate reductase activity on the initial rate of COz liberation at p H 6.2 and 37 " C . Activity levels of 17 units L-l or higher were sufficient for optimum COz production and were used to establish other optimum reagent concentrations. However, since the methotrexate assay was based on the indirect measurement of reductase activity, only enzyme activity levels within the linear section of the calibration curve would prove to be useful in the assay. On this basis, therefore, 10 units L-l enzyme was used in the drug determination procedure, with the activity representing the upper limit of linearity. The other enzyme and reagent concentrationswere similarly optimized by varying one parameter while keeping all other conditions constant. It was found that maximum decarboxylation was obtained with 2.5 X lo4 M P-NADPH, 2.82 X lo4 M dihydrofolic acid, 12.5 X M 6-phosphogluconic acid, and 0.83 units mL-16-phosphogluconicdehydrogenase. These
2.4 4.5 7.5 9.0 12.0
recovery
wi thin-run precision, % RSD
93 95 102 98 96 96
i 5.2 i3.9 t3.2 i.3.5 +1.9 *2.4
%
concentrations ensured rate dependence only on the folate reductase and, indirectly, on the methotrexate. The net KC1 concentration of 0.23 M in the reaction buffer provided optimum activating effect on the 6-phosphogluconate enzyme and replaced cations (11)which could interfere with the other enzyme. The two enzymes utilized in the method have slightly differing pH optima and require a compromise pH compatible with the functioning of the pC0, electrode. Figure 3 shows the influence of pH on the recycling system with initial rate of COz liberation plotted against the corresponding pH. A pH of 6.2 proved to be the optimum value for the maximum enzyme activity. Temperature also affected the sensitivity of the assay procedure. Although maximum COz liberation was noted at a temperature around 43 "C, all studies were conducted at 37 "C to avoid base line potential drift at the higher temperatures. A t 22 OC, only a fourth of the maximum rate was obtained. Determination of Methotrexate. It has been reported that the methotrexate enzyme assay suffers from poor stability of the folate enzyme and substrate (11). In this study, it was found that dithioerythritol (DTE) employed in reagent preparation instead of the foul-smelling fi-mercaptoethanol used in previous work yielded more stable and reproducible results. Enzymes were used undiluted to reduce the risk of activity loss. Figure 4 shows a typical methotrexate calibration curve with percent enzyme activity plotted against the corresponding drug concentration. The enzyme activity decreased linearly up to 15 pg L-' inhibitor followed by a sharp deviation from linearity leading to a graduate leveling off in available activity. The practical lower limit under the reported conditions is in the 1.5 pg L-' methotrexate concentration region, e.g., well below
854
Anal. Chem. 1984, 56,854-856
f1.9% to =k5.2%. The recovery studies for the same concentration range showed values of 93-102% with an average of 96%. These results are in accord with previous studies of coupled enzyme systems using the pCOz electrode (14). Registry No. MTX, 59-05-2; dihydrofolate reductase, 900203-3.
100
80 21
t .->
LITERATURE CITED
3
2 60 0
5 C
40 hp
20
0
6
12 18 Methotrexate, pg/L
24
30
Flgure 4. Calibration curve for methotrexate determination at pH 6.2 and 37 O C with 2.5 X M P-NADPH and 10 units L-' dihydrofolate reductase.
the range of clinical interest (12, 13). Precision and Recovery Studies. Table I shows both the within-run precision and recovery of methotrexate added to synthetic laboratory samples. In the concentration range of 1.5 to 12.0 pg L-I methotrexate, the precision ranged from
(1) Stryer, Lubert "Biochemistry", 2nd ed.; W. H. Freeman & Co.: San Francisco, CA, 1981; pp 526-528. (2) Falk, L. C.; Clark, D. R.; Kalman, S. M.; Long, T. F.. Clin. Chem. (Winston-Salem, N.C.) 1976, 22, 785-788. (3) Brown, L. F.; Johnson, G. F.; Witte, D. L.; Feld, R. D., Clln. Chem. (Winston-Salem, N . C . ) 1980, 26, 335-338. (4) Buice, R. G.; Evans, W. E.; Karas, J., Nicolas, C. A,; Sidhu, P.; Straughn, A. B.; Meyer, M. C.; Crom, W. R . Clln. Chem. (Winston-Sa/em, N . C . ) 1980, 26, 1902-1904. (5) Lankelma, J.; Poppe, H. J . Chromatogr. 1978, 149, 587-598. (6) AI-Bassam, M. N.; O'Sullivan, M. J.; Bridges, J. W.; Marks, V. Clin. Chem. (Winston-Salem, N . C . ) 1979, 25, 1448-1452. (7) Wannlund, J.; Azari, J.; Levine, L.; DeLuca, M. Biochem. Slophys. Res. Commun. 1980, 96, 440-446. (8) Ferrua, B.; Milano, G.; Ly, B.; Guennec, J. Y.; Masseyeff, R. J . Immuno/. Methods 1983, 60, 257-268. (9) Kinkade, J. M., Jr.; Vogler, W. R.; Dayton, P. G. 8iOChem. Med. 1974, 10, 337-350. (10) Przybylski, M.; Preiss; Dannebaum, R.; Fischer, J. Biomed. Mass Spectrom. 1982, 9 , 22-32. (11) Pontremoli, S.;Grazi, E. Methods Enzymol. 1986, 9 , 137-141. (12) Davis. J. E.; Solskv. R. L.: Gierina. L.: Malhotra, S. Anal. Chem. 1983, 55, 202R-214R. (13) Scheufler, Eckhard Clin. Chlm. Acta 1981, 1 1 1 , 113-116. (14) Hassan, S. S. M.; Rechnitz, G. A. Anal. Chem. 1982, 54, 303-307.
RECEIVED for review October 21, 1983. Accepted December 5 , 1983. We are grateful for the support of NIH Grant GM25308.
Electrochemical Probe for Simultaneous Extraction and Identification of Elements in Metal Alloys Daniel Mario Alperin, Victor Idoyaga Vargas, and Hector Carminatti* Instituto de Investigaciones Bioquimicas "Fundacion Campomar" and Facultad de Ciencias Eractas y Naturales, Universidad Nacional de Buenos Aires, Obligado 2490 (1428) Buenos Aires, Argentina A method based on well-known electrochemical procedures is currently used for the extraction and identification of metal alloys (1-3). In this method, the elements are passed to the ionic state with a dc source by using two electrodes, one applied to the metal and the other to a wet paper which is in contact with the sample. After the extraction, the paper is submitted to detection procedures. Here we describe a probe in which the electrodes are placed differently from those of the methods mentioned above. In this probe both electrodes are in contact with the paper. The paper of the probe could be used for the simultaneous extraction and identification of ions, requiring only a small area of the sample (e.g., 1 mm2). Principle of the Method. The scheme describing the functioning procedure of the probe is illustrated in Figure 1. Two platinum electrodes contact one side of the paper, whereas the other side contacts the metal. The electric resistance across the thickness of the paper is lower than across its length, thus permitting the generation of anodic and cathodic areas on the metal surface. Due to the low resistance across the thickness of the paper, the electrodes may be placed on either side of the paper without noticeable differences in the detection procedure. As shown in Figure 2, the electrodes are placed to self-retain the paper strip.
Ions liberated from the metal are attracted to the cathodic electrode passing through the paper and when the latter is chemically sensitized, they interact with the reagents as described below. Alternatively, an unsensitized paper could be used. After extraction, unsensitized paper may be submitted to conventional identification procedures (4,5).
EXPERIMENTAL SECTION The construction diagram of the probe is illustrated in Figure 2. A pencil-sized acrylic square bar holds two electrodes made of platinum sheet 0.25 mm thick. The paper must be inserted between the electrodes as shown in Figure 2. The electrodes must be positioned symmetrically with respect to the center of the bar and should be shorter than the acrylic support to avoid contact with the sample. Platinum electrodes are silver soldered to copper wires and fixed to the acrylic body with plastic screws and/or glued with a latex based adhesive. Dimensions are not critical and depend on the availability of materials or special needs. A dc current source was constructed with four 9-V batteries disposed so as to obtain 36 V. The source was connected to the probe terminals in series with a push button switch. Polarity of the current applied to the probe is indifferent. The paper used in all cases is Whatman No. 3 MM. Unless stated otherwise, our description of the method applies to the simultaneous identification of nickel, iron, and chromium in steel alloys (Table I) and gold, copper, nickel, chromium, and iron in
0003-2700/84/0356-0854$01.50/00 1984 American Chemical Society
