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cumstances, thereby diminishing the number of times that blots need to be stripped and the potential epitope loss (6) associated with each stripping procedure. Acknowledgments. This work was supported in part by a grant from the NIH (CA69008). The gifts of immunological reagents from Guy Poirier, Leroy Liu, and Yuri Lazebnik; technical assistance of Phyllis Svingen, Timothy Kottke, Laura Bruzek, Rebecca Spieker, Chris Hallgren, and Kit Lee; secretarial assistance of Deb Strauss; and helpful suggestions of the two anonymous reviewers are gratefully acknowledged.

REFERENCES 1. Gershoni, J. M., and Palade, G. E. (1983) Protein blotting: Principles and applications. Anal. Biochem. 131, 1–15. 2. Beisiegel, U. (1986) Protein blotting. Electrophoresis 7, 1–18. 3. Stott, D. I. (1989) Immunoblotting and dot blotting. J. Immunol. Methods 119, 153–187. 4. Kaufmann, S. H., and Kellner, U. (1998) Erasure of Western blots after autoradiographic or chemiluminescent detection. in Methods of Molecular Biology, Vol. 80, pp. 223–235, Humana Press, Clifton, NJ. 5. Erickson, P. F., Minier, L. N., and Lasher, R. S. (1982) Quantitative electrophoretic transfer of polypeptides from SDS polyacrylamide gels to nitrocellulose sheets: A method for their re-use in immunoautoradiographic detection of antigens. J. Immunol. Methods 51, 241–249. 6. Kaufmann, S. H., Ewing, C. M., and Shaper, J. H. (1987) The erasable Western blot. Anal. Biochem. 161, 89 –95. 7. Parekh, B. S., Mehta, H. B., West, M. D., and Montelaro, R. C. (1985) Preparative elution of proteins from nitrocellulose membranes after separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Anal. Biochem. 148, 87–92. 8. Salinovich, O., and Montelaro, R. C. (1986) Reversible staining and peptide mapping of proteins transferred to nitrocellulose after separation by sodium dodecylsulfate–polyacrylamide gel electrophoresis. Anal. Biochem. 156, 341–347. 9. Brill, A. S., and Weinryb, I. (1967) Reactions of horseradish peroxidase with azide. Evidence for a methionine residue at the active site. Biochemistry 6, 3528 –3535. 10. Richardson, T. C., Chapman, D. V., and Heyderman, E. (1983) Immunoperoxidase techniques: The deleterious effect of sodium azide on the activity of peroxidase conjugates. J. Clin. Pathol. 36, 411– 414. 11. Ortiz de Montellano, P. R., David, S. K., Ator, M. A., and Tew, D. (1988) Mechanism-based inactivation of horseradish peroxidase by sodium azide. Formation of meso-azidoprotoporphyrin IX. Biochemistry 27, 5470 –5476. 12. Krajewski, S., Zapata, J. M., and Reed, J. C. (1996) Detection of multiple antigens on Western blots. Anal. Biochem. 236, 221– 228. 13. Navarre, J., Bradford, A. J., Calhoun, B. C., and Goldenring, J. R. (1996) Quenching of endogenous peroxidase in Western blot. Biotechniques 21, 990 –992. 14. Liao, J., Xu, X., and Wargovich, M. J. (2000) Direct Reprobing with anti-beta-actin antibody as an internal control for Western blotting analysis. Biotechniques 28, 216 –218. 15. Lamarre, D., Talbot, B., deMurcia, G., Laplante, C., Leduc, Y., Mazen, A., and Poirier, G. G. (1988) Structural and functional analysis of poly(ADP-ribose) polymerase: An immunological study. Biochim. Biophys. Acta 950, 147–160. 16. Kaufmann, S. H., McLaughlin, S. J., Kastan, M., Liu, L. F., Karp, J. E., and Burke, P. J. (1991) Topoisomerase II levels

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Detection of Intrahepatic Ethanol Using a Microbiosensor Hiroshi Matsumoto, 1 Yoko Nishitani, Yasushi Minowa, and Yuko Fukui Department of Legal Medicine, Kyoto University Faculty of Medicine, Kyoto 606-8501, Japan Received May 25, 2001; published online August 16, 2001

The determination of alcohol concentration is a frequently performed test in forensic and clinical laboratories. Blood alcohol concentration is presently measured using three methods, breath analyzers, gas chromatography, and enzymatic analysis on automated analyzers or spectrophotometers. Gas chromatography is a sensitive method (1), but requires expensive instrumentation. Selective spectrophotometric methods require alcohol dehydrogenase and a coenzyme, nicotine adenine dinucleotide, and deproteinization of the blood serum prior to the analysis. Alcohol dehydrogenase-based electrochemical detectors require additional enzymes for formation of electrochemically detectable substrates or products (2). These methods are, therefore, not suitable for real-time measurement of blood or tissue alcohol concentrations. Albery et al. (3) presented a microbiosensor for determination of extracellular neurotransmitters, i.e., glutamate, lactate, and ascorbate (4 –9). This sensor is a microdialysis probe (OD 230 ␮m), which includes an oxidase solution and three electrodes (Fig. 1). An enzyme solution can be infused into the dialysis electrode by means of the inlet tubing at the top of the electrode. This sensor has not been introduced to measure ethanol concentrations of tissues. The alcohol microbiosensor contains alcohol oxidase 1

To whom correspondence should be addressed at Department of Legal Medicine, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan. Fax: ⫹81-11-611-3935. E-mail: hmatsumo@ sapmed.ac.jp. Analytical Biochemistry 296, 286 –288 (2001) doi:10.1006/abio.2001.5220 0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1. A schematic diagram of the microdialysis alcohol biosensor. It consists of a dialysis electrode for the measurement of ethanol.

within the dialysis fiber, next to the platinum electrode. The enzyme catalyzes the reaction Alcohol oxidase

Alcohol ⫹ O2 ™™™™™™™ 3 acetaldehyde ⫹ H2O2 Pt ⫹ 650 mV

H2O2 ™™™™™™3 2H ⫹ ⫹ O2 ⫹ 2e ⫺. The nonspecific oxidation of interferents is a problem common to all hydrogen peroxide-detecting enzyme electrodes. Murphy and Galley reported electropolymerized coats of o-phenylenediamine have been shown to significantly decrease interference currents (10). In the present study, our purposes were to detect ethanol via a microbiosensor and, if possible, to determine the intrahepatic ethanol concentration in perfused rat livers. Materials and Methods Chemicals. Alcohol oxidase (EC 1.1.3.13) was purchased from Sigma Chemicals, U.S.A. and o-phenylenediamine was from Nacalei Tesque Inc., Kyoto, Japan. All other chemicals were from Nacalei Tesque Inc., Kyoto, Japan. Ethanol biosensor preparation. The dialysis electrode (0.25 mm diameter; 2 mm exposed membrane), which was purchased from Sycopel International Ltd. (London, UK), was used. Figure 1 shows the detailed structure of the microdialysis ethanol biosensor. The biosensor was prepared with some modification as described previously (11). The dialysis electrode was filled with Ringer’s solution. The dialysis membrane was immersed in a beaker of 5 mM o-phenylenediamine in Ring-

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er’s solution bubbled with 100% N2 for 15 min, while the Ringer’s solution was constantly stirred. The dialysis electrode was connected to an EPS-800 detector (EICOM Co., Kyoto, Japan), and a voltage clamp was switched on at ⫹650 mV for 15 min to induce electropolymerization, with continuous bubbling and stirring. Upon completion of electropolymerization, the potential was switched off. The dialysis electrode membrane was removed from the o-phenylenediamine solution, and the membrane portion was stored in Ringer’s solution. After 20 min, the solution in the dialysis electrode was replaced with fresh Ringer’s solution using a perfusion pump, and the current was set at ⫹650 mV and the solution was allowed to stabilize. Alcohol oxidase was dissolved in Ringer’s solution. The dialysis electrode was set at a potential of ⫹650 mV and filled with Ringer’s solution containing the alcohol oxidase (100 U/ml). An analysis of the calibration was then performed based on a concentrated ethanol solution. Liver perfusion. All animal experiments were conducted in accordance with local institutional guidelines for the care and use of laboratory animals. Male Wistar rats, weighing approximately 250 g, were given standard laboratory chow and water ad libitum. Rats were anesthetized with sodium pentobarbital (50 mg/kg ip), and their livers were isolated and perfused with a Krebs–Henseleit bicarbonate buffer (pH 7.4; 37°C; 36 ml/min) saturated with 95% O 2–5% CO 2 via a cannula inserted in the portal vein as described previously (12) with some modification. pH, PO 2, and PCO 2 in the outflow were determined with an oxygen analyzer. Aliquots of perfusate were collected to determine lactate hydrogenase release enzymatically. At the end of the experiment, tissue samples were collected and immediately fixed in a formaldehyde solution for subsequent histochemical analysis. In vivo ethanol monitoring. Fifteen minutes after perfusion was started, a dialysis electrode was inserted in the perfused rat liver and a reference electrode was placed under the liver. After stabilization of the electrode, ethanol was added in the perfusate to a concentration of 10 or 20 mM and the changes in current were monitored using the alcohol microbiosensor system. Results and Discussion For determination of how much ethanol can be detected, in vitro experiments were performed using the alcohol microbiosensor system. Figure 2 shows a standard curve in a typical experiment. A significant correlation is observed in Fig. 2. The limits of detection were in the range of 0.1 ␮M. However, an upper detection limit of 1 mM existed. The standard curve for the method is linear for ethanol concentrations up to 200 ␮M. Watanabe-Suzuki et al. (1) reported that by using head-space capillary gas chromatography with cryogenic oven trapping the detection limit of ethanol was

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Intrahepatic Ethanol Concentrations Ethanol concentration Perfusate (mM) 10 20

FIG. 2. A standard curve of detection of ethanol in vitro. The correlation was significant (P ⬍ 0.05).

estimated to be 0.01 ␮g/ml (0.2 ␮M). However, ethanol cannot be directly measured in blood and tissue, or from the cannule into the vessel using these methods. No tissue damage caused by the sensors was recognized microscopically in rat liver sections. Using the alcohol microbiosensor, tissue ethanol concentrations can be detected rapidly. Figure 3 shows detection of ethanol in perfused rat livers. After a 10 mM ethanol challenge, the current was increased. Therefore, the change of current represents ethanol uptake by the liver. Table 1 shows ethanol concentrations calculated from current changes. Our findings indicate that concentrations of ethanol in liver tissue were relatively

Intrahepatic (␮M) 59 ⫾ 37 137 ⫾ 43

low compared with a previous report (13), where liver ethanol values were approximately 10 mM and close to peripheral blood values after administration of 0.75 g/kg ethanol. This finding shows that ethanol absorbed from blood to tissue is eliminated quickly, which is very important when considering the intracellular effects of ethanol. The present study has demonstrated intrahepatic concentrations of ethanol using monitoring with high time resolution (millisecond order), utilizing an in vivo microdialysis electrode. In conclusion, the present alcohol microbiosensor has been shown to achieve its potential increase in sensitivity for the accurate measurement of ethanol, when compared with the head-space GC method with multiple sample preparations. We illustrate here the biosensor technique as a contributor to the monitoring of tissue ethanol. Acknowledgment. This work was supported by grants from the Ministry of Education, Science, and Culture of Japan.

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FIG. 3. Detection of ethanol in perfused rat livers. The tracings were obtained from typical experiments under perfusion of 10 mM ethanol (A) and KHS (B). The vertical and horizontal arrows shows 10 ␮A of the current and 2 min of time, respectively.

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