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In vivo inhibition of liver alcohol dehydrogenase by ethanol administration
Life Sciences, Vol. 35, pp. 2353-2357 Printed in the U.S.A.
Pergamon Press
IN VIVO INHIBITION OF LIVER ALCOHOL DEHYDROGENASE BY ETHANOL ADMINISTRATION Mahmoud Sharkawi D~partement de Pharmacologie Facult~ de M~decine Universit~ de Montreal C.P. 6128, Succ. "A" Montreal, Quebec H3C 3J7 (Received in final form September 25, 1984) Summary The activity of liver alcohol dehydrogenase (LADH) from rats sacrificed two hours after the administration of ethanol 3, 4 or 5 g/kg intraperitoneally was significantly inhibited compared to the activity of LADH from control rats. LADH activity was inversely related to the dose of ethanol administered, to the concentration of ethanol in the liver, and to the concentration of ethanol in the blood. The clearance of blood ethanol in rats was dose-dependent and was inversely related to the dose administered. The half-life of ethanol elimination increased as the dose of ethanol increased. These results suggest that ethanol-induced inhibition of LADH can occur in vivo and that the level of activity of this enzyme determines the rate of oxidation of ethanol. The oxidation of ethanol by liver alcohol dehydrogenase (LADH) is generally considered as the major pathway for the elimination of ethanol in mammals. It is also believed that the level of activity of LADH is a major factor limiting rates of ethanol oxidation in vivo (1). Substrate inhibition of LADH by ethanol can be demonstrated in vitro (2). The possibility that ethanolinduced inhibition of LADH can occur in vivo was explored. Here we report on the effects of ethanol administration on the level of activity of LADH and on the relationship between the activity of LADH and the rate of ethanol elimination in rats. Methods and Materials Male Sprague-Dawley rats (200-260 g) were used in all experiments. They were housed in temperature-regulated quarters (23oc to 25°C) on a 12-hour light-dark cycle (lights on from 0700 to 1900) and given free access to food and water. The animals were divided into groups of 5-6 rats each. For measuring the elimination of ethanol in conscious animals, groups of rats were lightly anesthetized with ether and a carotid artery was cannulated with a Silastic cannula (0.025 in.I.D., 0.047 in.O.D., Dow-Corning). The cannula was filled with heparinized saline, sealed and passed beneath the skin to the scapular region of the animal where it was exteriorized and secured within a mounted screw top plastic cylinder. Animals were allowed to recover over a 24-hour period. Groups of animals, 5 rats each, were injected with ethanol 2, 3, 4 or 5 g/kg intraperitoneally. Arterial blood samples (0.2 ml) were withdrawn at hourly intervals after ethanol administration. The ethanol 0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
2354
Ethanol-induced
Inhibition
of L A D H
Vol.
35, No.
23, 1984
concentration in blood was determined by gas chromatography as previously described (3). The clearance rates were determined by d i v i d i n g the dose of ethanol a d m i n i s t e r e d by the area under the ethanol c o n c e n t r a t i o n - t i m e curve for such a dose. For m e a s u r i n g the activity of LADH, groups of rats were injected intraperitoneally with ethanol 2, 3, 4 or 5 g/kg (20% v/v in water). Different groups of rats were injected w i t h vehicle and used as controls. The rats in all groups were d e c a p i t a t e d two hours after the injection of ethanol or vehicle. The blood was collected in heparinized tubes; the livers were immediately removed to measure the activity of LADH. The livers were rinsed in icechilled 1.15% KCI, and homogenized with 4 volumes of 0.25M sucrose in 0.i M Tris-HCl buffer pH 7.4. The homogenates were centrifuged at 105,000 x g for 1 h, and alcohol dehdyrogenase activity in the supernatant (cytosol) was determined by the m e t h o d of Bonnichsen and Brink (4). The reaction mixture contained 2.7 ml of 0 . 1 M glycine-NaOH pH 9.9, 0.1 ml ethanol solution to obtain a final c o n c e n t r a t i o n of 10 mM, 0.i ml of the supernatant, and to start the reaction 2 m g of NAD were added. The rate of NADH formation at 37°C was m e a s u r e d u s i n g a U n i c a m SP 8000 r e c o r d i n g spectrophotometer. The total volume of the reaction mixture was 3 ml. P r o t e i n c o n c e n t r a t i o n in the supernatant was measured by the method of Lowry et al. (5) u s i n g bovine serum albumin as standard. The ethanol concentrations in the blood and the liver were m e a s u red by gas-liquid chromatography (3). Results
and D i s c u s s i o n
Results presented in table I show that the activity of LADH from rats sacrificed two hours after the a d m i n i s t r a t i o n of ethanol 3, 4 or 5 g/kg was significantly inhibited as compared to the activity of LADH from control rats or rats pretreated with ethanol 2 g/kg i.p. The results also show that the inhibition of LADH seems to be dose-dependent and directly related to the c o n c e n t r a t i o n of ethanol in the liver and blood. The addition of excess NAD to the reaction mixture did not affect the activity of the inhibited enzyme. However the i n h i b i t i o n was completely reversed by dialysis of the inhibited enzyme against 0.25 M sucrose at 4°C for 4 hours. The recovery of the activity of the enzyme was associated with the d i s a p p e a r a n c e of ethanol from the dialyzed enzyme. The u n d i a l y z e d enzyme (cytosol) from rats sacrificed 2 hours after the a d m i n i s t r a t i o n of ethanol 5 g/kg i.p. contained about 580 ~g of ethanol/ml (12.5 Bmol/ml). No ethanol was detected in the cytosol after dialysis. TABLE
I
LADH Activity and Ethanol C o n c e n t r a t i o n in Liver and Blood from Rats Sacrificed 2 Hours After the A d m i n i s t r a t i o n of Different Doses of Ethanol. Ethanol C o n c e n t r a t i o n in the Reaction Mixture Was I0 mM. Ethanol dose g/kg 0 2 3 4 5
LADH activity ~mol NADH f o r m e d / m g 1.61 1.60 1.13 1.04 0.80
-+ ± + ± ±
0.14 0.06 0.04* 0.07* 0.08*
prot/hr
Ethanol Liver ( ~mol / $) 8.2 ± 2.9 33.8 ± 2.2 54.4 ± 1.6 68.4 ± 3.5
concentration Blood ( ~mol/ml) 16.4 _+ 5.3 54.4 i 3.1 58.2 ± 1.3 100.9 ± 5.5
*values are s i g n i f i c a n t l y different from control (p < 0.05) by two-tailed student's t-test. Values are the m e a n ± standard error of the mean from 4-6 rats.
Vol. 35, No. 23, 1984
Ethanol-induced Inhibition of LADH
2355
Results in table II show that the inhibition of LADH was reversible in vivo as well. The activity of LADH from rats sacrificed 12 hours after the administration of ethanol (5 g/kg i.p.) was similar to the activity of LADH from control rats. The recovery was associated with decreasing concentrations of ethanol in liver and blood as seen in table II. TABLE II LADH Activity and Ethanol Concentrations in Liver and Blood at Different Time Intervals After the Administration of Ethanol 5 g/kg i.p. Ethanol Concentration in the Reaction Mixture Was I0 mM. Time after ethanol administration 0 (no ethanol) 2 4 8 12
LADH activity ~mol NADH formed /mg prot/hr 1.61 0.80 i.Ii 1.24 1.60
± ± ± ± ±
0.14 0.08* 0.13" 0.11, 0.20
Ethanol concentration Liver Blood (~mol/~) (~mol/ml) 66.8 ± 3.6 100.9 ± 5.5 62.0 ± 2.0 89.1 ± 4.0 32.0 ± 8.0 47.3 ± 7.7 I0.0 ± 4.2 16.0 ± 5.7
*values are significantly different from control (p < 0.05) by two-tailed student's t-test. Values are the mean ± standard error of the mean from 4-6 rats. Figure 1 represents the elimination curves of ethanol from the blood after the administration of different doses of ethanol. The clearance of ethanol from blood decreased as the dose of ethanol increased. The clearance rates were 6.8 ± 0.3, 4.3 ± 0.I, 3.0 ± 0.I and 2.7 ± 0.2 ml/min for 2, 3, 4 and 5 g/kg ethanol i.p. respectively. The half-lives of elimination of ethanol increased with increasing the dose of ethanol. The half-lives were 1.35 ± 0.12, 1.74 ± 0.14, 3.65 ± 0.59 and 11.85 ± 2.12 hr for 2, 3,4 and 5 g/kg ethanol i.p. respectively. This phenomenon of dose-dependent elimination does not seem to be peculiar to ethanol. The same phenomenon has been previously observed with salicylate elimination in man (6). Dose-dependent elimination seems to be associated with drugs which are eliminated essentially by biotransformation (7). This phenomenon has been explained in terms of saturation of the biotransformation process as the concentration of the drug reaches certain levels (6). Table II shows that the inhibition of LADH increased as the dose of ethanol increased. Thus, the slower clearance seen with higher doses of ethanol could be due to the inhibition of the catalytic activity of LADH. This could also explain the dramatic increase in the duration of ethanol-induced loss of righting reflex as the dose is moderately increased (Figure 2). LADH is believed to be saturable at extremely low concentrations of substrate (8). Substrate inhibition of mammalian LADH by ethanol has been demonstrated in vitro (2). The present experiments demonstrate that mammalian LADH inhibition can occur in vivo after the administration of certain doses of ethanol and that this can greatly affect the rate of ethanol elimination. Such inhibition and its effects on the rate of ethanol oxidation were recently predicted in a study dealing with the steady state kinetics of purified rat LADH (9). The mechanism or mechanisms of in vivo ethanol-induced inhibition of LADH is beyond the scope of this report. However, the reversibility of the inhibition by dialysis indicates that the inhibition is induced by a dialysable substance which could be ethanol itself.
r-
0
20
40
80
2
Time
4 (hours)
6
8
10
FIG. 1 B l o o d e t h a n o l c o n c e n t r a t i o n s at d i f f e r e n t time i n t e r v a l s a f t e r the a d m i n i s t r a t i o n of d i f f e r e n t d o s e s of e t h a n o l intraperitoneal]y. E a c h p o i n t r e p r e s e n t s the m e a n v a l u e f r o m 4 to 5 rats. B l o o d s a m p l e s (0.2 ml) w e r e w i t h d r a w n at h o u r l y i n t e r v a l s a f t e r the a d m i n i s t r a t i o n of d i f f e r e n t doses of e t h a n o l . The e t h a n o l c o n c e n t r a t i o n in b l o o d w a s m e a s u r e d by gas c h r o m a t o g r a p h y .
0¢.-
0 0
~-
o
-~ ._
100
Dose of Ethanol gm/kg o2 o3
80
160
../z
i
Dose
3
l
I
(g/kg)
456
I
FIG. 2 The d o s e - r e s p o n s e r e l a t i o n s h i p of e t h a n o l in rats. G r o u p s of 6 rats e a c h w e r e i n j e c t e d w i t h d i f f e r e n t d o s e s of e t h a n o l i n t r a p e r i t o n e a l l y . The d u r a t i o n of s l e e p i n g time ( l o s s of r i g h t i n g r e f l e x ) was measured. A n i m a l s w e r e c o n s i d e r e d to h a v e r e g a i n e d t h e i r r i g h t i n g r e f l e x if they r e t u r n e d to the n o r m a l s u p i n e p o s i t i o n 3 s u c c e s s i v e t i m e s w i t h i n i min. V a l u e s are the m e a n s l e e p i n g time t s t a n d a r d e r r o r of the m e a n for six rats.
O0
•
n
.~_
""
240
E 320
._E
v
A
400
o
Z
~n
O
0
0
~t
H m~
I
O
m"
o~
Vol. 35, No. 23, 1984
Ethanol-induced Inhibition of LADH
2357
The implications of these results for man are difficult to assess. Whether human LADH would be affected in a similar fashion is difficult to predict. However, there is evidence showing that the rate of ethanol accumulation in man is disproportionately increased as the dose of ethanol (amount of ethanol consumed per unit time) is increased (10). Thus it has been found that subjects who consumed one ounce of 100 proof bourbon per 150 pounds of body weight per hour for five hours had blood ethanol concentrations not exceeding 6 mg/ml. However, subjects who consumed two ounces of 100 proof bourbon per 150 pounds of body weight per hour had blood ethanol concentrations exceeding 17 mg/ml. This observation could be a reflexion of decreased oxidation of ethanol due to a probable decrease in the activity of LADH induced by increased doses of ethanol. Acknowledgements We thank Suzanne Leroux for excellent technical assistance. Research funds of 1'Universite de Montreal.
Supported by
References 633-640 (1981). 1. T.J. BRAGGINS and K.E. CROW. Eur. J. Biochem. 2, 2. R.N. ZAHLTEN, C.J. JACOBSON and M.E. NEJTEK. Biochem. Pharmacol. 2, 1973-1976 (1980). 3. K.A. DAVIS. J. Forensic Sci. 11, 205-213 (1966). 4. R.K. BONNICHSEN and N.G. BRINK. In: Methods of Enzymology. Eds. S.P. Colowick and N.O. Kaplan, Vol. 1, pp. 495, Academic Press, New York (1955). 5. O.H. LOWRY, H.J. ROSENBROUGH, A.L. FARR and R.J. RANDALL. J. Biol. Chem. 193, 265-275 (1951). 6. G. LEVY. J. Pharmaceut. Sci. 54, 959-967 (1965). 7. M. GIBALDI. In: Biopharmaceutics and Clinical Pharmacokinetics, 2nd edition pp. 102, Lea 6 Febiger, Philadelphia 1977. 8. R.R. LEVINE. In: Pharmacology, Drug Actions and Reactions, 1st ed., pp. 208, Little, Brown & Company, Boston (1973). 9. D.W. CRABB, W.F. BOSRON & T.-K. LI. Arch. Biochem. & Biophys. 224, 299-309 (1983). 10. R.B. FOFCNEYand F.W. HUGHES. Clin. Pharmacol. Ther. 4, 619-621 (1963).
Pergamon Press
IN VIVO INHIBITION OF LIVER ALCOHOL DEHYDROGENASE BY ETHANOL ADMINISTRATION Mahmoud Sharkawi D~partement de Pharmacologie Facult~ de M~decine Universit~ de Montreal C.P. 6128, Succ. "A" Montreal, Quebec H3C 3J7 (Received in final form September 25, 1984) Summary The activity of liver alcohol dehydrogenase (LADH) from rats sacrificed two hours after the administration of ethanol 3, 4 or 5 g/kg intraperitoneally was significantly inhibited compared to the activity of LADH from control rats. LADH activity was inversely related to the dose of ethanol administered, to the concentration of ethanol in the liver, and to the concentration of ethanol in the blood. The clearance of blood ethanol in rats was dose-dependent and was inversely related to the dose administered. The half-life of ethanol elimination increased as the dose of ethanol increased. These results suggest that ethanol-induced inhibition of LADH can occur in vivo and that the level of activity of this enzyme determines the rate of oxidation of ethanol. The oxidation of ethanol by liver alcohol dehydrogenase (LADH) is generally considered as the major pathway for the elimination of ethanol in mammals. It is also believed that the level of activity of LADH is a major factor limiting rates of ethanol oxidation in vivo (1). Substrate inhibition of LADH by ethanol can be demonstrated in vitro (2). The possibility that ethanolinduced inhibition of LADH can occur in vivo was explored. Here we report on the effects of ethanol administration on the level of activity of LADH and on the relationship between the activity of LADH and the rate of ethanol elimination in rats. Methods and Materials Male Sprague-Dawley rats (200-260 g) were used in all experiments. They were housed in temperature-regulated quarters (23oc to 25°C) on a 12-hour light-dark cycle (lights on from 0700 to 1900) and given free access to food and water. The animals were divided into groups of 5-6 rats each. For measuring the elimination of ethanol in conscious animals, groups of rats were lightly anesthetized with ether and a carotid artery was cannulated with a Silastic cannula (0.025 in.I.D., 0.047 in.O.D., Dow-Corning). The cannula was filled with heparinized saline, sealed and passed beneath the skin to the scapular region of the animal where it was exteriorized and secured within a mounted screw top plastic cylinder. Animals were allowed to recover over a 24-hour period. Groups of animals, 5 rats each, were injected with ethanol 2, 3, 4 or 5 g/kg intraperitoneally. Arterial blood samples (0.2 ml) were withdrawn at hourly intervals after ethanol administration. The ethanol 0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
2354
Ethanol-induced
Inhibition
of L A D H
Vol.
35, No.
23, 1984
concentration in blood was determined by gas chromatography as previously described (3). The clearance rates were determined by d i v i d i n g the dose of ethanol a d m i n i s t e r e d by the area under the ethanol c o n c e n t r a t i o n - t i m e curve for such a dose. For m e a s u r i n g the activity of LADH, groups of rats were injected intraperitoneally with ethanol 2, 3, 4 or 5 g/kg (20% v/v in water). Different groups of rats were injected w i t h vehicle and used as controls. The rats in all groups were d e c a p i t a t e d two hours after the injection of ethanol or vehicle. The blood was collected in heparinized tubes; the livers were immediately removed to measure the activity of LADH. The livers were rinsed in icechilled 1.15% KCI, and homogenized with 4 volumes of 0.25M sucrose in 0.i M Tris-HCl buffer pH 7.4. The homogenates were centrifuged at 105,000 x g for 1 h, and alcohol dehdyrogenase activity in the supernatant (cytosol) was determined by the m e t h o d of Bonnichsen and Brink (4). The reaction mixture contained 2.7 ml of 0 . 1 M glycine-NaOH pH 9.9, 0.1 ml ethanol solution to obtain a final c o n c e n t r a t i o n of 10 mM, 0.i ml of the supernatant, and to start the reaction 2 m g of NAD were added. The rate of NADH formation at 37°C was m e a s u r e d u s i n g a U n i c a m SP 8000 r e c o r d i n g spectrophotometer. The total volume of the reaction mixture was 3 ml. P r o t e i n c o n c e n t r a t i o n in the supernatant was measured by the method of Lowry et al. (5) u s i n g bovine serum albumin as standard. The ethanol concentrations in the blood and the liver were m e a s u red by gas-liquid chromatography (3). Results
and D i s c u s s i o n
Results presented in table I show that the activity of LADH from rats sacrificed two hours after the a d m i n i s t r a t i o n of ethanol 3, 4 or 5 g/kg was significantly inhibited as compared to the activity of LADH from control rats or rats pretreated with ethanol 2 g/kg i.p. The results also show that the inhibition of LADH seems to be dose-dependent and directly related to the c o n c e n t r a t i o n of ethanol in the liver and blood. The addition of excess NAD to the reaction mixture did not affect the activity of the inhibited enzyme. However the i n h i b i t i o n was completely reversed by dialysis of the inhibited enzyme against 0.25 M sucrose at 4°C for 4 hours. The recovery of the activity of the enzyme was associated with the d i s a p p e a r a n c e of ethanol from the dialyzed enzyme. The u n d i a l y z e d enzyme (cytosol) from rats sacrificed 2 hours after the a d m i n i s t r a t i o n of ethanol 5 g/kg i.p. contained about 580 ~g of ethanol/ml (12.5 Bmol/ml). No ethanol was detected in the cytosol after dialysis. TABLE
I
LADH Activity and Ethanol C o n c e n t r a t i o n in Liver and Blood from Rats Sacrificed 2 Hours After the A d m i n i s t r a t i o n of Different Doses of Ethanol. Ethanol C o n c e n t r a t i o n in the Reaction Mixture Was I0 mM. Ethanol dose g/kg 0 2 3 4 5
LADH activity ~mol NADH f o r m e d / m g 1.61 1.60 1.13 1.04 0.80
-+ ± + ± ±
0.14 0.06 0.04* 0.07* 0.08*
prot/hr
Ethanol Liver ( ~mol / $) 8.2 ± 2.9 33.8 ± 2.2 54.4 ± 1.6 68.4 ± 3.5
concentration Blood ( ~mol/ml) 16.4 _+ 5.3 54.4 i 3.1 58.2 ± 1.3 100.9 ± 5.5
*values are s i g n i f i c a n t l y different from control (p < 0.05) by two-tailed student's t-test. Values are the m e a n ± standard error of the mean from 4-6 rats.
Vol. 35, No. 23, 1984
Ethanol-induced Inhibition of LADH
2355
Results in table II show that the inhibition of LADH was reversible in vivo as well. The activity of LADH from rats sacrificed 12 hours after the administration of ethanol (5 g/kg i.p.) was similar to the activity of LADH from control rats. The recovery was associated with decreasing concentrations of ethanol in liver and blood as seen in table II. TABLE II LADH Activity and Ethanol Concentrations in Liver and Blood at Different Time Intervals After the Administration of Ethanol 5 g/kg i.p. Ethanol Concentration in the Reaction Mixture Was I0 mM. Time after ethanol administration 0 (no ethanol) 2 4 8 12
LADH activity ~mol NADH formed /mg prot/hr 1.61 0.80 i.Ii 1.24 1.60
± ± ± ± ±
0.14 0.08* 0.13" 0.11, 0.20
Ethanol concentration Liver Blood (~mol/~) (~mol/ml) 66.8 ± 3.6 100.9 ± 5.5 62.0 ± 2.0 89.1 ± 4.0 32.0 ± 8.0 47.3 ± 7.7 I0.0 ± 4.2 16.0 ± 5.7
*values are significantly different from control (p < 0.05) by two-tailed student's t-test. Values are the mean ± standard error of the mean from 4-6 rats. Figure 1 represents the elimination curves of ethanol from the blood after the administration of different doses of ethanol. The clearance of ethanol from blood decreased as the dose of ethanol increased. The clearance rates were 6.8 ± 0.3, 4.3 ± 0.I, 3.0 ± 0.I and 2.7 ± 0.2 ml/min for 2, 3, 4 and 5 g/kg ethanol i.p. respectively. The half-lives of elimination of ethanol increased with increasing the dose of ethanol. The half-lives were 1.35 ± 0.12, 1.74 ± 0.14, 3.65 ± 0.59 and 11.85 ± 2.12 hr for 2, 3,4 and 5 g/kg ethanol i.p. respectively. This phenomenon of dose-dependent elimination does not seem to be peculiar to ethanol. The same phenomenon has been previously observed with salicylate elimination in man (6). Dose-dependent elimination seems to be associated with drugs which are eliminated essentially by biotransformation (7). This phenomenon has been explained in terms of saturation of the biotransformation process as the concentration of the drug reaches certain levels (6). Table II shows that the inhibition of LADH increased as the dose of ethanol increased. Thus, the slower clearance seen with higher doses of ethanol could be due to the inhibition of the catalytic activity of LADH. This could also explain the dramatic increase in the duration of ethanol-induced loss of righting reflex as the dose is moderately increased (Figure 2). LADH is believed to be saturable at extremely low concentrations of substrate (8). Substrate inhibition of mammalian LADH by ethanol has been demonstrated in vitro (2). The present experiments demonstrate that mammalian LADH inhibition can occur in vivo after the administration of certain doses of ethanol and that this can greatly affect the rate of ethanol elimination. Such inhibition and its effects on the rate of ethanol oxidation were recently predicted in a study dealing with the steady state kinetics of purified rat LADH (9). The mechanism or mechanisms of in vivo ethanol-induced inhibition of LADH is beyond the scope of this report. However, the reversibility of the inhibition by dialysis indicates that the inhibition is induced by a dialysable substance which could be ethanol itself.
r-
0
20
40
80
2
Time
4 (hours)
6
8
10
FIG. 1 B l o o d e t h a n o l c o n c e n t r a t i o n s at d i f f e r e n t time i n t e r v a l s a f t e r the a d m i n i s t r a t i o n of d i f f e r e n t d o s e s of e t h a n o l intraperitoneal]y. E a c h p o i n t r e p r e s e n t s the m e a n v a l u e f r o m 4 to 5 rats. B l o o d s a m p l e s (0.2 ml) w e r e w i t h d r a w n at h o u r l y i n t e r v a l s a f t e r the a d m i n i s t r a t i o n of d i f f e r e n t doses of e t h a n o l . The e t h a n o l c o n c e n t r a t i o n in b l o o d w a s m e a s u r e d by gas c h r o m a t o g r a p h y .
0¢.-
0 0
~-
o
-~ ._
100
Dose of Ethanol gm/kg o2 o3
80
160
../z
i
Dose
3
l
I
(g/kg)
456
I
FIG. 2 The d o s e - r e s p o n s e r e l a t i o n s h i p of e t h a n o l in rats. G r o u p s of 6 rats e a c h w e r e i n j e c t e d w i t h d i f f e r e n t d o s e s of e t h a n o l i n t r a p e r i t o n e a l l y . The d u r a t i o n of s l e e p i n g time ( l o s s of r i g h t i n g r e f l e x ) was measured. A n i m a l s w e r e c o n s i d e r e d to h a v e r e g a i n e d t h e i r r i g h t i n g r e f l e x if they r e t u r n e d to the n o r m a l s u p i n e p o s i t i o n 3 s u c c e s s i v e t i m e s w i t h i n i min. V a l u e s are the m e a n s l e e p i n g time t s t a n d a r d e r r o r of the m e a n for six rats.
O0
•
n
.~_
""
240
E 320
._E
v
A
400
o
Z
~n
O
0
0
~t
H m~
I
O
m"
o~
Vol. 35, No. 23, 1984
Ethanol-induced Inhibition of LADH
2357
The implications of these results for man are difficult to assess. Whether human LADH would be affected in a similar fashion is difficult to predict. However, there is evidence showing that the rate of ethanol accumulation in man is disproportionately increased as the dose of ethanol (amount of ethanol consumed per unit time) is increased (10). Thus it has been found that subjects who consumed one ounce of 100 proof bourbon per 150 pounds of body weight per hour for five hours had blood ethanol concentrations not exceeding 6 mg/ml. However, subjects who consumed two ounces of 100 proof bourbon per 150 pounds of body weight per hour had blood ethanol concentrations exceeding 17 mg/ml. This observation could be a reflexion of decreased oxidation of ethanol due to a probable decrease in the activity of LADH induced by increased doses of ethanol. Acknowledgements We thank Suzanne Leroux for excellent technical assistance. Research funds of 1'Universite de Montreal.
Supported by
References 633-640 (1981). 1. T.J. BRAGGINS and K.E. CROW. Eur. J. Biochem. 2, 2. R.N. ZAHLTEN, C.J. JACOBSON and M.E. NEJTEK. Biochem. Pharmacol. 2, 1973-1976 (1980). 3. K.A. DAVIS. J. Forensic Sci. 11, 205-213 (1966). 4. R.K. BONNICHSEN and N.G. BRINK. In: Methods of Enzymology. Eds. S.P. Colowick and N.O. Kaplan, Vol. 1, pp. 495, Academic Press, New York (1955). 5. O.H. LOWRY, H.J. ROSENBROUGH, A.L. FARR and R.J. RANDALL. J. Biol. Chem. 193, 265-275 (1951). 6. G. LEVY. J. Pharmaceut. Sci. 54, 959-967 (1965). 7. M. GIBALDI. In: Biopharmaceutics and Clinical Pharmacokinetics, 2nd edition pp. 102, Lea 6 Febiger, Philadelphia 1977. 8. R.R. LEVINE. In: Pharmacology, Drug Actions and Reactions, 1st ed., pp. 208, Little, Brown & Company, Boston (1973). 9. D.W. CRABB, W.F. BOSRON & T.-K. LI. Arch. Biochem. & Biophys. 224, 299-309 (1983). 10. R.B. FOFCNEYand F.W. HUGHES. Clin. Pharmacol. Ther. 4, 619-621 (1963).