Anal. Chem. 1983, 55. 2439-2440 (9) Grosjean, D.; Lewis, R.; Fung, K.; Swanson, R.; Countess, R. J. 75th Air Pollution Control Assoclation Annual Meeting, New Orleans, LA, June 20-25, 1982;Paper No. 82-33.3. (IO) Cable, J.; Kagel, S. A.; McLeod, J. K. Org. Mass Spectrom. 1972, 6 ,
301-307. (11) Selbl, J.; Vollmin, J. Org. Mass Spectrom. 1968, 1 , 713-737. (12) Budzikiewicz, H.; Djerassi, C.; Williams, D. H. ”Mass Spectrometry of Organic Compounds”; Holden-Day: San Franclsco. CA, 1967; pp 399-405. (13) McCann, J.; Simmon, V.; Streitwieser, D.; Ames, B. N. R o c . NaN. A-d. SC/. U . S . A . 1975, 72, 3190-3193. (14) Kringstad, K. P.; Ljungquist, P. 0.;de Sousa, F.; Stromberg, L. M. Environ. Sci. Techno/. 1981, 15, 562-566. (15) Williams, R. G. Anal. Chem. 1982, 5 4 , 2121-2122. (16) Grosjean, D.; Friedlander, S. K. In “The Character and Origins of Smog Aerosols”; Hldy, G. M., et al., Eds.; Wiley: New York, 1979;pp 435-473. (17) Grosjean, D.; Van Cauwenberghe, K.; Schmid, J. P.; Kelley. P. E.; Pitts, J. N., Jr., Environ. Sci. Techno/ 1978, 12, 313-317. (18) Mansfield, C. T.; Hodge, B. T.; Hege, R. B., Jr.; Hamlln, W. C. J . Chromatogr. Scl. 1977, 75, 301-302.
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(19) Moree-Testa, P.; SaintJalm, Y. J . Chromatcgr. 1981, 217, 197-208. (20) Katsuki, H.; Yoshida, T.; Tanegashima, C.; Tanaka, S. Anal. Biochem. 1971, 43. 349-356. (21) Kobayashi, K.; Tanaka, M.; Kawai, S.; Ohno, T. J . Chromatogr. 1979, 178, 118-122. (22) Koshy, K. T.; Kaiser, D. G.; Vanderslik, A. L. J . Chromatogr. Sc/. 1976, 13, 97-104. (23) Shriner, R. L.; Fuson, R. C.; Curtln, D. Y. “The Systematic Identification of Organic Compounds”, 5th ed.; Wiiey: New York, 1964. (24) “Rodds Chemistry of Carbon Compounds”, 2nd ed.; Eisevier: Amsterdam; Coffey, S., Ed.; Voi. 1, Part D, 1965;and Ansell, M. F., Ed.; Vol. 1, Part C-D, 1973. (25) Zitrin, S.; Yinon, J. Org. Mass Spectrom. 1976, 1 1 , 388-393. (26) Harrison, G. A,; Kailury, R. K. M. R. Org. Mass Spectrom. 1980, 15, 284-288. (27) Grosjean, D. Atmos. Environ ., in press.
RECEIVED for review March 14, 1983. Accepted August 22, 1983.
Flow Injection Analysis with an Enzyme Reactor Bed for Determination of Ascorbic Acid in Brain Tissue Charles W. Bradberry and Ralph N. Adams* Department of Chemistry, University of Kansas, Lawrence, Kansas 66045 The utility of immobilized enzymes is now well established (1). Immobilized enzyme reactors (IMERs) have been used in conjunction with flowing stream analyses (2-5). In most of these applications the detector monitors an enzymatic reaction product(s) or some coreactant. However, one can employ IMERs to remove the analyte of interest from a flowing stream. This report summarizes an application of the latter type for ascorbic acid (AA) using an ascorbic acid oxidase (AAO, 1.10.3.3)Sepharose IMER. It was specifically designed for the electrochemical analysis of AA in brain tissue in the presence of catecholamines and their metabolites. It is also applicable to a variety of other biological samples. AAO in soluble form has been employed previously for plant tissue assays (6). In addition, AAO has been immobilized on a Clark oxygen electrode (7), and its successful immobilization on Sepharose was recently reported (8). The principle of the method is illustrated in terms of the recorder responses shown in Figure 1B. If a soluble sample containing AA plus other electrooxidizable substances (e.g., catecholamines and metabolites in brain samples) is passed over an inactive reactor bed, a peak amperometric signal proportional to the sum of these concentrations is obtained (peak A). If exactly the same injection is now passed through an active bed, AA is selectively removed and the lesser signal (peak B) is observed. (The peak amperometric signals are similar to those observed in the flow injection analysis methods.) From precalibrations the differences between the two signals are an accurate measure of AA content.
EXPERIMENTAL SECTION Reagents and Solutions. All reagents for buffer solutions were reagent grade. AAO was obtained from BoehringerMannheim (Indianapolis, IN) and immobilized as previously reported (8). AA standard solutions were prepared by diluting stock (1-10 mM AA in 0.10 M HC104) solutions with the buffer used as the carrier stream. Two buffers were used; 0.1 M NaH2P04/Na2HP04,1 mM EDTA, pH 5.6, and 0.1 M acetate, 1 mM EDTA, pH 5.6. Apparatus. Two matched enzyme reactor bed holders, which are shown in Figure lC, were machined from Lucite. A coarse glass frit (Corning) was ground by hand on a stationary abrasive
disk until it just fit into the bottom section of the bed. Teflon tape was wrapped around the male threads to ensure a tight seal. The beds were loaded with AAO-Sepharose (8) (totalof 0.13 mL) by repeated injections of dilute slurries through the same loop injector used to introduce samples. AAO-Seph was inactivated for use in the dummy bed by boiling for ca. 5 min. The arrangement of the flowing stream components is shown in Figure 1A. Solutions were pumped with a Milton-Roy minipump through low-pressure plastic tubing and fittings. A sealed glass column 35 cm long and 2 mm i.d. was used to dampen pump pulsations. A loop injector (Rheodyne type 50, loop volume 500 pL) was used to introduce samples into the flowing stream. A Chromatronix 6-into-1 valve was used to select which reactor bed the stream would flow through. The electrochemical detection system was a thin-layer glassy carbon flow-through cell held at +0.8 V vs. Ag/AgCl (BioanalyticalSystems,Lafayette, IN). The potentiostat and current-to-voltage converter used was either a PAR 174 polarographicanalyzer or a laboratory-built potentiostat. Current output was displayed as a voltage on a Houston Omniscribe strip-chart recorder. Reactor Bed Characterization. Figure 2 summarizes the data showing the performance of the system. The solid circles show the linear response of Aip (representing peak current for samples through the inactive bed minus that for the active bed). Ascorbate levels up to 400 pM produced peak currents from the active bed of less than 1nA, demonstrating the effectiveness of the reactor bed. At higher concentrations the capacity of the enzyme bed is exceeded and current “break-through” is observed. With pure standards, the detection limit for ascorbate alone is ca. loF8M. For the technique to give accurate results in the presence of other oxidizable substances, the dispersion characteristics of the two beds must be matched. This can be shown to be true in this case by making duplicate injections [of a substance which is unaffected by the enzyme, e.g., dopamine (DA)]through the bed. It is found that calibration curves of i, vs. concentrations of DA injected through the active and inactive reactor beds are indistinguishable within experimental error. It has been previously shown (8-10) that DA and other catecholamines and their metabolites are unaffected by soluble and immobilized forms of AAO. Finally, the determination of AA is independent of catecholamines such as dopamine. Even in the “worst case” of very low AA and equimolar dopamine concentrations (indicated in Figure 2 by the “+”), the calibration curves with or without dopamine
0003-2700/83/0355-2439$01.50/00 1983 American Chemlcai Societv
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
A
n
Table I. Ascorbate Content of Rate Caudate Nucleus animal 1 R'"
sample wt, mg 7.9 9.4 9.9 8.8 5.7 7.7 9.3 12.1
AA content, pmol/g
1.95 1.80 2R 1.97 L 2.15 3R 2.12 L 2.49 4R 2.16 L 1.91 mean 2.07 2 0.08 SEM a Signifies right and left caudate nuclei.
La
Figure 1. (A) Schematic diagram of flowlng stream apparatus. (B) Amperometric slgnals from a mixture of AA and other oxidizable substances Injected through the reactor beds. (C) Details of reactor bed construction.
8
1
Aip(nA) 6-
Y
0
.1
.2
.3
.4
.5
JJM
Figure 2. Calibrationfor AA alone (0)and equimolar AA and dopamine AID represents peak current for sample through Inactive bed minus that for an identical sample through the active bed.
(+).
present never deviate by more than 5%. In real brain samples AA concentrations are 10-100 times those of dopamine or other catecholamines. Assay Procedures. Sprague-Dawley rats (250-400 g) were killed by decapitation and the brains quickly removed and cooled in ice-cold buffer. For whole brain analyses, one hemisphere (minus the cerebellum) at a time was either weighed immediately and homogenized in 3.5 mL 0.1 M HC104or frozen on dry ice until processed. Following homogenization by hand, using a groundglass tissue homogenizer (Kontes, 15mL capacity), 250-pL aliquots of the homogenate were centrifuged in 1.5-mL plastic micro test tubes at 18000 rpm in a Beckman J-21B centrifuge (JA-21 rotor) for 5 min. For analyses of the caudate nucleus, the brain was removed as before and coronal slices ca. 500 pm thick were made at the level of the anterior caudate from one hemisphere at a time. Five to ten milligrams of the caudate nucleus was sampled by peeling back the cortex and corpus callosum and cutting away any ventral tissue that might contain nucleus accumbens. The tksue was then sonicated in 250 p L of PO4 buffer. The sonicate was centrifuged as described above. For either whole or brain part samples, after centrifugation 50-pL aliquots of the supernatant were diluted with 1mL of the carrier stream buffer for injection. For each sample a 500-pL injection was made through the inactive reactor bed, followed by an identical injection through the active bed. The difference in peak height for the two injections is due to AA removed by the AAO-Seph. Calibration for caudate nucleus samples was ac-
complished by running standards of M AA in PO, buffer through the same workup as the tissue sample.
RESULTS AND DISCUSSION T o assess the reproducibility of the entire assay, a whole rai brain was divided into the two hemispheres (minus the cerebellum) and each was homogenized and centrifuged. Triplicate samples were taken from both hemispheres, each of which was considered to be equivalent in AA content. The right hemisphere samples showed 2.69 f 0.04 (standard deviation) pmol AA/g wet tissue. The left hemisphere samples gave a value of 2.54 f 0.07 (standard deviation) pmol/g. Thus, a relative deviation of 1-3% is observed for replicate analyses. The left hemisphere was kept on dry ice while the right was being analyzed, which may account for the slightly lower value of the former. The difficulties in protecting against loss of AA during sample freezing/thawing, storage operations, etc. are well-known by those engaged in tissue AA analyses (11). Table I summarizes the analyses for rat caudate nuclei from four animals (total of eight left-right caudates, dissected as described earlier). This region was chosen since the results could be compared with previous literature data. The mean values of 2.07 f 0.08 (SEM) Kmol/g agree well with previous reports of 1.74 f 0.26 pmol/g (12) and 2.03 f 0.71 pmol/g (9). (All values are reported here in terms of SEM (standard error of the means) since that is the prevalent usage in brain assays.) In conclusion, this new assay method for ascorbate employing an AAO-Seph reactor for difference measures offers a rapid, reliable determination for ascorbic acid. The reactor bed will also be of use in removing AA as a possible source of current in the direct electrochemical detection (without liquid chromatography) from neuronal tissue perfusates. It is this use for which the enzyme was initially immobilized. Registry No. AA, 50-81-7; AAO, 9029-44-1. LITERATURE CITED Carr, P. W.; Bowers, L. D. "Immobilized Enzymes in Analytical and Clinical Chemistry"; Wiley: New York, 1980. Karube, I.: Hara, K.; Satoh, I.; Suzuki, S. Anal. Chim. Acta 1979, 106, 243. Dahodwala, S . K.; Weibel, M. K.; Humphrey, A. E. Biotech. Bioeflg. 1979, 18, 1679. Cremonesl, P.; Bovara, R. Biotech. Bloeng. 1978, 18, 1487. Ogren, L. Anal. Ghim. Acta 1981, 125, 45. Hewllt, E. J.; Dickes. 0. J. Blochem. J . 1981, 78, 384. Posadka, P.; Macholan, L. Collect. Czech. Chem. Commufl. 1979, 44, 3395. Bradberry, C. W.; Borchardt, R.; Decedue, C. FEBS Left. 1982, 146, 348. Matsumoto, K.; Yamada, K.; Osajima, Y. Anal. Chem. 1981, 5 3 , 1974. Schenk, J. 0.; Miller, E.; Adams, R. N. Anal. Chem. 1982, 54, 1452. Allison, J. H.; Stewart, M. A. Anal. Biochem. 1971, 43, 401. Miiby, K.; Oke, A,; Adams, R. N. Neurosci. Left. 1982, 2 8 , 15.
RECEIVEDfor review June 7,1983. Accepted August 22,1983. The support of this work by the National Institutes of Health via Grant NS08740 and Biomedical Research Support Grant RR 5606 is gratefully acknowledged.
