The alcohol oxidase membrane technique is available for measurement of ethanol in commercial fluids. In this paper ... sensitivity for analysis of cat blood to which known amounts of ethanol have ..... review of chemical and infrared methods.
Rapid measurement of blood ethanol concentrations using the alcohol oxidase membrane technique C. V . GREENWAY' AND K. PUSWKA Department of Pharmacology, University of Manitoba, 770 Bannatyne Ave., Winnipeg, Ma~t., Cianuda R3E OW3
D. S. SITAR Departments of P&aam~co&ogy and Internal Medicine, University of Manitoba, Winnipeg, Man., Canada W3E OW3
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L. K . ROGERS Department of Internal Medics'np, University of Manitoba, Winnipeg, Man., Canada R4E OW3 Received October 8, 1987 GREENWAY, C. V . , PUSHKA,K . , SITAR,B. S., and ROGERS,L. K. 1988. Rapid measurement of blood ethanol concentrations using the alcohol oxidase membrane technique. Can. 9. Physiol. P h m a c o l . 66: 307-31 1. The alcohol oxidase membrane technique is available for measurement of ethanol in commercial fluids. In this paper we exmined its usefulness for cat and human blood in comparison with gas-liquid chromatography (GLC). The membrane method proved to be simple, reproducible, accurate, and inexpensive. Analysis took 1-2 min per sample and required only 25 pL of whole blood for measurement of concentrations between 0.05 and 1.0 mM (0.25-5 mg/dL) and 10 pL of whole blood for measurement of concentrations between 1.O and 40 d ( 5 - 190 mg/dL). Background concentrations were undetectable in cats after extraneous sources of alcohols were removed. The alcohol oxidase membrane technique is less specific than GLC, but it may be useful when ethanol is administered after background samples have shown an absence of other nonspecific reactants. Its high sensitivity is useful for kinetic studies where blood ethanol concentrations are below or close to those required for maximal hepatic ethanol metabolism. GREENWAY, C. V . , PWSHKA, K., SITAR,D. S., et ROGERS,Lo K. 1988. Rapid measurement of blood ethanol concentrations using the alcohol oxidase membrane technique. Can. J. Physiol. P h m a c o l . 66 : 307-3 11. La technique avec membrane il alcool oxydase est utilisCe pour mesurer 1'Cthanol dans les liquides comerciaaax. Dans cet article, nous avons exmink son utilitC pour le sang du chat et de l'humain c o m p d e A celle de la chromatographie gaz-liquide (CGL). La mCthode avec membrane s9est avCrCe simple, reproductible, prkcise et Cconomique. L'analyse a d u d 2 min par Cchmtillon et n'a nCcessitC que 25 p L de sang pour mesurer des concentrations comprises entre 0,05 et 1,8 mM (0,25-5 mg/dL) et 10 p L de sang pour des concentrations comprises entre 1 ,O et 40 mM (5-190 mg/dL). Aucune miere-concentration n'a pu Ctre dCtwtCe chez les chats apr&savok CcmC les sources d'alcools. La technique avec membrane A alcool oxydase est moins sflcifique que la CGL, mais elle pourrait Stre utile lorsque 1'Cthanol est administrk a p e s que des Cchantillons aient montrk une absence d9autresrkactifs non sflcifiques en mi&re-plan.Sa grande sensibilitk est utile pour les Ctudes cinktiques dans lesquelles les concentrations d'kthanol sont sous ou p d s de celles nCcessaires au mktabolisme hCpatique maximal de 19Ct%nanol. [Traduit par la revue]
Introduction Several methods exist for the quantitation of ethanol in biological fluids. The most clinically useful include gas chromatography (GLC), enzymatic oxidation with alcohol dehydrogenase, oxidation with acid dichromate, and osmometry. These approachesto ethanol quantitation have been reviewed (Jain and Cravey 1972a, 1972b), and GLC was perceived to be the most selective of the available procedures (Dubowski 1980). At clinically relevant concentrations, as little as 10 pL of blood can be analyzed by the alcohol dehydrogenase enzymatic method with acceptable precision (coefficient of variation, 3.4% at 50 mg/dL; Jones 1985). By comparison, using head-space GLC, ethanol concentrations as low as 0.3 mg/dL can be accurately quantified from a blood volume of 20-50 FL (coefficient of variation, 4.6% between 0.3 and 120 rng/dL; Wilkinson et al. 1975). However, the technical skill required for this method is considerably greater than for the enzymatic method. We have previously reported the measurement of blood galactose concentrations using the model 27 andyser supplied by Yellow Springs Instruments, Ohio (Burczynski and Greenway 1986; Greenway and Burczynski 1987). This instrument incorporates a galactose oxidase membrane in a temperature' ~ u t h o rto whom correspondence may be addressed.
controlled chamkr. We were impressed by its speed, convenience, and sensitivity over a wide range of concentrations. We therefore examined the use of the alcohol oxidase membrane for measurement of blood ethanol concentrations. These membranes are available for measurement of ethanol in industrial fluids, but they have not yet been approved for blood ethanol measurements. In this paper, we report the accuracy and sensitivity for analysis of cat blood to which known amounts of ethanol have been added, and we report preliminary results of a comparison between this method and GLC on samples of cat and human blood. Since the method is oxygen dependent, we also examined the effect of blood Po2.
Methods Cat blmd studies Donor cat blood was obtained from cats anesthetized ketamine (20 mg/kg by intramuscular injection) or pentobarbital sodium (30 mg/kg by intraperitoneal injection). Sodium pentobarbital was freshly dissolved in distilled water (30 mg/mL) to avoid the ethanol used in c o m e r c i d l y available solutions~Heparin injection U. S. P. (I0 units/PnE blood) was added to the blood. En later experiments p w d m d heparin sodium (Sigma) was used to avoid the b e n z ~alcohol l present in the injectable hepain. Hn some experiments this blood was used directly, while in others it was equilibrated with known gas
CAN. J . PHYSICBL. PHARMACOE. VOL. 66. 1988
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LINEARITY - LOW RANGE
LINEAWITY - HIGH RANGE
ADDED (DM) FIG. 1. Measurements of ethanol added to cat blood samples. In the low range (62-750 FM) four measurements of each of four separate samples at each concentration are shown. In the high range (5-25 mM),three measurements of each of three separate samples at each concentration are shown. Lines of identity are shown for each graph. mixtures to modify the Po2levels. Blmd gas measurements were made on all samples using an IL 1302 pH/blmd gas analyser ('lnstrurnentation Lab. Inc.). Known volumes of ethanol were added to aliquots of this blood and analysed within 1 h. Analyses of all samples were done on the YSI 27 instrument (Yellow Springs Instruments, Ohio 45387, U.S. A.) with the alcohol adaptor (YSI 239 I), the alcohol sxidase membrane (YST 2386), and the ethanol buffer solution (YST 2387). After the p ~ l i m i n qexperiments, the ethanol buffer was prepared from laboratory reagents: disodium hydrogen phosphate, 15.31 g; sodium dihydrogen phosphate, 1-94 g; sodium chloride, 0.176 g; dipotassium ethylenediamine tetraacetic acid (Sigma), 0.088 g; and chlorhexidine acetate, 0.088 g made up to 500 d.with distilled water. For low ethanol concentrations (. 10 nA4 even smaller sample volumes could be used if an accurate injection pipette were available.
Acknowledgemelats We are grateful to the Medical Research Council and the Manitoba Meart Foundation for grants in support of this work. BLOMSTRAND, R. 1971. Observations on the formation of ethanol in the intestinal tract in mam. Life Sci. 10: 575-5852.
BURCZYNSKI, F. I . , and GREENWAY, C . V. 1986. Hepatic blood flow:
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GREENWAY ET .4&.
accuracy of estimation from galactose clearances in cats. Can J. Physiol. P h m a c o l . 64: 1310-1315. BURCZYNSM. F. J., SITAR,D. S . , and WILSON,A. 1985. Ethanol disposition in man: comparison of breathalyzer and two methods sf gas-liquid chromatography. Int. J . Addict. 20: 583-589. D u s c p w s ~ , 18. M. 1980. Alcohol determination in the clinical laboratorye Am. J. Clin. Pathol. 74: 747-750. GREENWAY, C. V . , and BURCZYNSKH, F. J. 1987. Effects of liver blood flow on hepatic uptake kinetics of galactose in anesthetized cats: parallel tube model. Can. S. Physiol. P h m a c s l . 65: 1 193- 1 199. JAIN,N. C., and CRAVEY,R. H. 1972a. Analysis of alcohol. I. A review of chemical and infrared methods. J. Chromatogr. Sci. 10: 257-262. 4972b. Analysis of alcohol. II. A review of gas chromatographic methods. J. Chromatogr. ScE. 10: 263-267.
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JONES, A. W. 1985. Electrochemical measurement sf breath-alcohol concentration: precision and accuracy in relation to blood levels. Clin. Chim. Acta, 146: 175- 183. KEHDING, S . , JOHANSEN, S . , MIBTBOLL,I . , RABOL,A., and CHRISTIANSEN, L. 1979. Ethanol elimination kinetics in human liver and pig liver in vivs. Am. S. Physiol. 237: E3 16-E324. LI, T. 1977. Enzymology of human alcohol metabolism. Adv. EnzymoE. 45: 427-483. LUNBQUIST, F., and WOLTHERS, PI. 1958. The kinetics of alcohol elimination in man. Acta Pharmacsl . Tsxicol . 14: 265-289. WILMNSON,P. K.,WAGNER,J . G . , and SEDMAN,A. J. 1975. Sensitive head-space gas chromatographic method for the determination of ethanol utilizing capillary b l o d samples. Anal. Chern. 47: 1506-1510.
