Pharmacology Biochemistry & Behavior, Vol. 3, pp. 973--978. Copyright © 1975 by ANKHO International Inc. All rights of reproduction in any form reserved. Printed in the U.S.A.
Variations in Alcohol Metabolism: Influence of Sex and Age ' A L L A N C. C O L L I N S , T O N I N. Y E A G E R , M. E. L E B S A C K A N D S. S C O T T P A N T E R
School o f Pharmacy and Institute f o r Behavioral Genetics, University o f Colorado Boulder CO 80302 (Received 20 January 1975) COLLINS, A. C., T. N. YEAGER, M. E. LEBSACK AND S. S. PANTER. Variations in alcohol metabolism: influence o f age and sex. PHARMAC. BIOCHEM. BEHAV. 3(6) 9 7 3 - 9 7 8 , 1975. - Male and female C57BL/Ibg mice were divided into two age groups: 5 0 - 6 0 and 9 5 - 1 1 0 days of age. Alcohol-induced sleep time was measured subsequent to the intraperitoneal injection of a 3.5 g × k g ~ dose. The old male group had a sleep time approximately 4 times that of the young male group and approximately twice that of the old female group. Blood alcohol concentrations at time of awakening were nearly identical in all groups, indicating the difference in sleep time is not due to an altered CNS sensitivity. Measurement of in vivo alcohol disappearance rate indicates the old male group is different from the other groups because of a slower rate of alcohol metabolism. Although changes in hepatic alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities were seen, the changes do not explain the observed decrease in alcohol metabolism observed in the old male group. These data provide further evidence that hepatic ADH and ALDH activities are not rate limiting in alcohol metabolism. Alcohol
Alcohol dehydrogenase
Aldehyde dehydrogenase
A n u m b e r o f variables seem to be d i r e c t l y i n v o l v e d in c o n t r o l l i n g d u r a t i o n a n d i n t e n s i t y o f the effects o f alcohol. Central n e r v o u s s y s t e m (CNS) sensitivity t o t h e d e p r e s s a n t effects of this agent, rate o f e l i m i n a t i o n , a n d rate o f a b s o r p t i o n s h o u l d be o f p r i m a r y i m p o r t a n c e in c o n t r o l l i n g the d u r a t i o n o f a r e s p o n s e such as loss of the righting reflex, i.e., a l c o h o l - i n d u c e d sleep t i m e [ 1 3 ] . It is very likely t h a t t h e first t w o o f these p a r a m e t e r s are i n f l u e n c e d by t h e g e n o t y p e o f t h e test organism. F o r e x a m p l e , McClearn a n d K a k i h a n a [ 14] have o b t a i n e d b y selective b r e e d i n g f r o m a h e t e r o g e n e o u s s t o c k o f mice t w o lines o f mice w h i c h differ s u b s t a n t i a l l y in d u r a t i o n o f a l c o h o l - i n d u c e d sleep time. R e c e n t l y , it has b e e n r e p o r t e d t h a t these t w o selected lines differ in alcohol sleep t i m e because o f a difference in CNS sensitivity to alcohol [ 9 ] . While n o a t t e m p t s have b e e n m a d e to selectively b r e e d t w o lines o f mice w h i c h differ in rate o f a l c o h o l m e t a b o l i s m , it seems likely t h a t this w o u l d be possible. Various i n b r e d strains of mice d e m o n s t r a t e vast differences in a l c o h o l - i n d u c e d sleep t i m e following the i n ; e c t i o n o f an h y p n o t i c dose o f e t h a n o l [ 11 ]. Rogers et aL [ 16] a n d S h e p p a r d et al. [17] have d e t e r m i n e d t h a t these same i n b r e d strains differ in h e p a t i c alcohol d e h y d r o g e n a s e ( A D H ) in such a way t h a t an e x c e l l e n t inverse c o r r e l a t i o n exists b e t w e e n a l c o h o l - i n d u c e d sleep t i m e a n d A D H activity as m e a s u r e d in vitro. More r e c e n t l y , o t h e r s have r e p o r t e d differences in alcohol m e t a b o l i s m in t h r e e i n b r e d strains o f mice. [ 3 ]. Strain differences in alcohol m e t a b o l i s m are n o t restricted to mice. Eriksson [4] has r e p o r t e d differences in t h e in
Sex differences
Developmental processes
vivo e l i m i n a t i o n rate o f e t h a n o l in t w o lines o f rats w h i c h h a d b e e n selectively bred for high a n d low a l c o h o l preference [ 5 ] . Within b o t h o f these selected lines, Eriksson n o t e d t h a t t h e females h a d a greater e t h a n o l elimination rate t h a n did the males. Liver A D H activity was n o t assessed in these e x p e r i m e n t s . However, strain a n d sex differences a p p a r e n t l y do exist in these p a r a m e t e r s , since a sex difference in h e p a t i c A D H activity in Sprague-Dawley b u t n o t Wistar rats has b e e n observed [ 2 ] . Such o b s e r v a t i o n s s e e m e d of p r o b a b l e significance w h e n , in e x a m i n i n g t h e effects of various p h a r m a c o l o g i c a l agents on e t h a n o l - i n d u c e d sleep t i m e in o u r l a b o r a t o r y , it was o b s e r v e d t h a t sex differences in sleep t i m e m i g h t exist. These initial e x p e r i m e n t s h a d utilized sexually m a t u r e mice o f b o t h sexes, ranging in age f r o m 55 to 95 days, from the C 5 7 B L / I b g strain, The sex difference s e e m e d to be greater as a f u n c t i o n o f age. Thus, e x p e r i m e n t s were designed t o e s t i m a t e the m a g n i t u d e of this o b s e r v a t i o n a n d t o determine possible u n d e r l y i n g m e c h a n i s m s . METHOD
Animals The animals were divided i n t o 2 age groups: 5 0 - 6 0 days o f age a n d 9 5 - 1 1 0 days o f age. A l c o h o l - i n d u c e d sleep t i m e was assessed following the i n t r a p e r i t o n e a l i n j e c t i o n o f 3.5 g x Kg -~ e t h a n o l . The alcohol s o l u t i o n was p r e p a r e d in i s o t o n i c saline a n d c o n t a i n e d 0.35 g x ml-' o f e t h a n o l . A t this c o n c e n t r a t i o n , t h e a p p r o p r i a t e dose is achieved if each
~This work was supported in part by NIAA grants AA-00293, AA-00092 and an award to Allan C. Collins by the National Council on Alcoholism, and the Foundations Fund For Research in Psychiatry. 973
974 m o u s e is i n j e c t e d w i t h 0.01 ml x g l b o d y weight. T h e animals were i n j e c t e d i n t r a p e r i t o n e a l l y a n d r e s t r a i n e d for a p p r o x i m a t e l y 30 sec a f t e r w h i c h t h e y were placed on t h e i r backs in a trough. Sleep t i m e was j u d g e d t e r m i n a t e d w h e n the animals could right t h e m s e l v e s successfully 2 t i m e s in 30 sec.
Procedure Since differences in sleep t i m e m i g h t arise as a conseq u e n c e of a l t e r a t i o n s in CNS sensitivity to t h e d e p r e s s a n t effects o f e t h a n o l or as a c o n s e q u e n c e o f differing rates o f m e t a b o l i s m o f e t h a n o l , e x p e r i m e n t s were p e r f o r m e d to test these possibilities. A c o n v e n i e n t way to e s t i m a t e CNS sensitivity to e t h a n o l in individual animals is to measure alcohol c o n c e n t r a t i o n in the b l o o d at t h e time o f regaining t h e righting reflex. The mice were i n j e c t e d with the s t a n d a r d 3.5 g x K g ' e t h a n o l dose; i m m e d i a t e l y a f t e r t h e righting reflex was regained, a central v e n o u s b l o o d sample was o b t a i n e d f r o m each a n i m a l by p u n c t u r i n g t h e retroo r b i t a l sinus w i t h a 40 m i c r o l i t e r capillary pipet. The b l o o d sample was a d d e d to 0.96 ml of a h e p a r i n i z e d i s o p r o p y l a l c o h o l s o l u t i o n . The final i s o p r o p y l alcohol c o n c e n t r a t i o n was 40 m g p e r c e n t and served as an i n t e r n a l standard. A 5 m i c r o l i t e r a l i q u o u t of this s o l u t i o n was i n j e c t e d o n t o a Porapak Q c o l u m n in a B e c k m a n GC-45 gas c h r o m a t o g r a p h , e q u i p p e d w i t h a flame i o n i z a t i o n d e t e c t o r . The c o l u m n a n d d e t e c t o r t e m p e r a t u r e s were 175°C a n d 225~C, respectively. The gas m i x t u r e consisted of air, 300 ml x min-~: h y d r o g e n , 45 ml x min -~ ; a n d h e l i u m , 80 ml x min -J . Each sample was r u n in duplicate. Blood alcohol c o n c e n t r a t i o n was calculated b y e s t i m a t i n g by t r i a n g u l a t i o n the areas of t h e e t h a n o l a n d i s o p r o p y l alcohol peaks a n d c o m p a r i n g t h e ratio o b t a i n e d for each sample w i t h a s t a n d a r d curve. The rate o f e t h a n o l e l i m i n a t i o n was d e t e r m i n e d for animals f r o m each o f t h e 4 groups using a similar t e c h n i q u e . Each a n i m a l was i n j e c t e d w i t h the s t a n d a r d e t h a n o l dose a n d 4 0 microliter b l o o d samples were o b t a i n e d at 1 , 2 a n d 4 h r after injection. Blood alcohol c o n c e n t r a t i o n s were d e t e r m i n e d as described previously and the e l i m i n a t i o n rate calculated for each animal. Hepatic alcohol d e h y d r o g e n a s e , ADH, and a l d e h y d e d e h y d r o g e n a s e , A L D H , have long been c o n s i d e r e d the most i m p o r t a n t e n z y m e s in e t h a n o l m e t a b o l i s m . Certainly, o n a kinetic basis, this appears to be the case. These considerations led us to measure h e p a t i c A D H a n d A L D H activities in vitro. Animals f r o m each of the 4 groups w h i c h had n o t b e e n e x p o s e d to e t h a n o l were sacrificed by a blow o n the head, t h e livers quickly excised a n d placed in 9 v o l u m e s o f cold 0.25 M sucrose. The tissue was h o m o g e n i z e d a n d t h e 4 8 , 0 0 0 × g s u p e r n a t a n t fraction o b t a i n e d by c e n t r i f u g i n g for 1 h r in a Sorvall RC2B refrigerated centrifuge. A D H activity was d e t e r m i n e d in this s u p e r n a t a n t f r a c t i o n by m o n i t o r i n g s p e c t r o p h o t o m e t r i c a l l y the rate of N A D H f o r m a t i o n with e t h a n o l a n d NAD as c o s u b s t r a t e s using a m o d i f i c a t i o n o f the m e t h o d of B o n n i c h s e n and Brink [ 1 ]. The m o d i f i c a t i o n consisted of the a d d i t i o n of 0.2 M s e m i c a r b a z i d e to t h e i n c u b a t i o n s . Semicarbazide serves t o trap t h e a c e t a l d e h y d e w h i c h is f o r m e d w h e n e t h a n o l is oxidized; if this step is o m i t t e d , b o t h A D H and A L D H , w h i c h c o n v e r t a c e t a l d e h y d e to acetic acid using NAD as a c o s u b s t r a t e , will be measured. T o t a l h e p a t i c A L D H activity was d e t e r m i n e d utilizing a r a d i o c h e m i c a l assay w h i c h has r e c e n t l y b e e n d e v e l o p e d in o u r l a b o r a t o r y . T h e assay s u b s t r a t e , 14 C b e n z a l d e h y d e , was
COLLINS, Y E A G E R , LEBSACK AND P A N T E R o b t a i n e d f r o m New England Nuclear as a b e n z e n e s o l u t i o n . The b e n z e n e was r e m o v e d by e v a p o r a t i o n u n d e r a s t r e a m of n i t r o g e n at 0°C. The radiolabelled b e n z a l d e h y d e was resolubilized in 0.5 ml of a 1:1 a c e t o n e - w a t e r s o l u t i o n and r u n across a 3.5 × 0.8 cm Dowex AG-I c o l u m n , pH 7.0. The c o l u m n was washed with 2.5 ml of w a t e r a n d e f f l u e n t s c o m b i n e d . The b e n z a l d e h y d e was diluted with u n t a b e l l e d b e n z a l d e h y d e to a specific activity of 0.10 m i c r o c u r i e s x m i c r o m o l e -l . E n z y m e activity was d e t e r m i n e d b y i n c u b a t i n g 50 microliters of a 10 p e r c e n t h o m o g e n a t e in a m i x t u r e of 0.65 ml 10 mM p y r o p h o s p h a t e buffer, pH 9.6, 0.1 ml 10 mM NAD, 0.1 ml 10 mM p y r a z o l e and 0.1 ml 2.5 mM b e n z a l d e h y d e (0.25 microcuries). This m i x t u r e was incub a t e d for 5 rain at 37.5°C in a shaking i n c u b a t o r bath. The r e a c t i o n was t e r m i n a t e d by the a d d i t i o n of 0.1 ml of a 20 p e r c e n t t r i c h l o r o a c e t i c acid (TCA) solution. Each h o m o g e nate was assayed in duplicate along with a b l a n k w h i c h consisted o f the c o m p l e t e r e a c t i o n m i x t u r e with the TCA a d d e d prior to the e n z y m e . The s o l u t i o n was t h e n made alkaline (pH 12) b y the a d d i t i o n of a 20 p e r c e n t NaOH s o l u t i o n and e x t r a c t e d twice with 6 ml o f w a t e r s a t u r a t e d 1,2 d i c h l o r o e t h a n e . This e x t r a c t i o n removes u n r e a c t e d b e n z a l d e h y d e and any o t h e r n e u t r a l m e t a b o l i t e s leaving the r e a c t i o n p r o d u c t , b e n z o i c acid, in the a q u e o u s layer. The radioactivity o f a 0.1 ml aliquot of the a q u e o u s layer was d e t e r m i n e d by placing it in 10 ml of a T r i t o n X-100 t o l u e n e based scintillation cocktail a n d c o u n t i n g in a B e c k m a n LS-133 liquid scintillation c o u n t e r . C o r r e c t i o n s for c o u n t ing efficiency were made. R e c o v e r y of b e n z o i c acid was nearly 100 p e r c e n t u n d e r these c o n d i t i o n s . Protein concent r a t i o n s were d e t e r m i n e d using the b i u r e t m e t h o d . The activities of b o t h ADH a n d A L D I t were calculated in t e r m s o f activity per mg of p r o t e i n , per g liver, per liver a n d per g o f b o d y weight. Data were a n a l y z e d by analyses of variance and the m e a n s c o m p a r e d using D u n c a n ' s multiple range test. RESULTS T h e results o f those e x p e r i m e n t s in w h i c h the p o t e n t i a l i n f l u e n c e of age a n d sex on a l c o h o l - i n d u c e d sleep t i m e in C 5 7 B L / I b g mice was assessed are p r e s e n t e d in Table 1. No significant sex d i f f e r e n t i a l is evident in the groups tested at 50 60 days of age; however, t h e r e is a m a r k e d sex difference in the older group. T h e older males slept nearly twice as long as the females of the same age a n d a p p r o x i m a t e l y 4 times as long as the 5 0 - 6 0 - d a y - o l d males. A l t h o u g h m e a n sleep time in females also increased w i t h age, there was so m u c h variance in this measure t h a t a 45 p e r c e n t increase was n o n s i g n i f i c a n t . The increase in e t h a n o l - i n d u c e d sleep time observed in C 5 7 B L mice is t h e r e f o r e d e p e n d e n t u p o n the sex of the animal. It is observed in greatest m a g n i t u d e in male mice. An increase in sleep t i m e m i g h t arise from differing CNS sensitivities to t h e depressant actions of alcohol. The results o f the e x p e r i m e n t in w h i c h 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 was m e a s u r e d at t i m e of regaining the righting reflex are p r e s e n t e d in Table 2. N o n e of t h e groups differ significantly from the o t h e r s with respect to b l o o d a l c o h o l c o n c e n t r a t i o n at t i m e of awakening. T h e r e f o r e , t h e r e p r o b a b l y are n o t a n y remarkable sex- or a g e - d e p e n d e n t differences in CNS sensitivity to alcohol in C 5 7 B L / I b g mice: In a d d i t i o n , these data i m p l y t h a t the old males differed f r o m the o t h e r groups with
AGE, SEX AND A L C O H O L M E T A B O L I S M
975 TABLE 1
EFFECTS OF AGE AND SEX ON ALCOHOL-INDUCED SLEEP TIME IN C57BL/Ibg MICE
Age (days)
Sex
Sleep Time*
Comparison
p
50-60
(YF)
Female
48.4 ± 5.8 (16)
OM-YM
Variations in Alcohol Metabolism: Influence of Sex and Age ' A L L A N C. C O L L I N S , T O N I N. Y E A G E R , M. E. L E B S A C K A N D S. S C O T T P A N T E R
School o f Pharmacy and Institute f o r Behavioral Genetics, University o f Colorado Boulder CO 80302 (Received 20 January 1975) COLLINS, A. C., T. N. YEAGER, M. E. LEBSACK AND S. S. PANTER. Variations in alcohol metabolism: influence o f age and sex. PHARMAC. BIOCHEM. BEHAV. 3(6) 9 7 3 - 9 7 8 , 1975. - Male and female C57BL/Ibg mice were divided into two age groups: 5 0 - 6 0 and 9 5 - 1 1 0 days of age. Alcohol-induced sleep time was measured subsequent to the intraperitoneal injection of a 3.5 g × k g ~ dose. The old male group had a sleep time approximately 4 times that of the young male group and approximately twice that of the old female group. Blood alcohol concentrations at time of awakening were nearly identical in all groups, indicating the difference in sleep time is not due to an altered CNS sensitivity. Measurement of in vivo alcohol disappearance rate indicates the old male group is different from the other groups because of a slower rate of alcohol metabolism. Although changes in hepatic alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities were seen, the changes do not explain the observed decrease in alcohol metabolism observed in the old male group. These data provide further evidence that hepatic ADH and ALDH activities are not rate limiting in alcohol metabolism. Alcohol
Alcohol dehydrogenase
Aldehyde dehydrogenase
A n u m b e r o f variables seem to be d i r e c t l y i n v o l v e d in c o n t r o l l i n g d u r a t i o n a n d i n t e n s i t y o f the effects o f alcohol. Central n e r v o u s s y s t e m (CNS) sensitivity t o t h e d e p r e s s a n t effects of this agent, rate o f e l i m i n a t i o n , a n d rate o f a b s o r p t i o n s h o u l d be o f p r i m a r y i m p o r t a n c e in c o n t r o l l i n g the d u r a t i o n o f a r e s p o n s e such as loss of the righting reflex, i.e., a l c o h o l - i n d u c e d sleep t i m e [ 1 3 ] . It is very likely t h a t t h e first t w o o f these p a r a m e t e r s are i n f l u e n c e d by t h e g e n o t y p e o f t h e test organism. F o r e x a m p l e , McClearn a n d K a k i h a n a [ 14] have o b t a i n e d b y selective b r e e d i n g f r o m a h e t e r o g e n e o u s s t o c k o f mice t w o lines o f mice w h i c h differ s u b s t a n t i a l l y in d u r a t i o n o f a l c o h o l - i n d u c e d sleep time. R e c e n t l y , it has b e e n r e p o r t e d t h a t these t w o selected lines differ in alcohol sleep t i m e because o f a difference in CNS sensitivity to alcohol [ 9 ] . While n o a t t e m p t s have b e e n m a d e to selectively b r e e d t w o lines o f mice w h i c h differ in rate o f a l c o h o l m e t a b o l i s m , it seems likely t h a t this w o u l d be possible. Various i n b r e d strains of mice d e m o n s t r a t e vast differences in a l c o h o l - i n d u c e d sleep t i m e following the i n ; e c t i o n o f an h y p n o t i c dose o f e t h a n o l [ 11 ]. Rogers et aL [ 16] a n d S h e p p a r d et al. [17] have d e t e r m i n e d t h a t these same i n b r e d strains differ in h e p a t i c alcohol d e h y d r o g e n a s e ( A D H ) in such a way t h a t an e x c e l l e n t inverse c o r r e l a t i o n exists b e t w e e n a l c o h o l - i n d u c e d sleep t i m e a n d A D H activity as m e a s u r e d in vitro. More r e c e n t l y , o t h e r s have r e p o r t e d differences in alcohol m e t a b o l i s m in t h r e e i n b r e d strains o f mice. [ 3 ]. Strain differences in alcohol m e t a b o l i s m are n o t restricted to mice. Eriksson [4] has r e p o r t e d differences in t h e in
Sex differences
Developmental processes
vivo e l i m i n a t i o n rate o f e t h a n o l in t w o lines o f rats w h i c h h a d b e e n selectively bred for high a n d low a l c o h o l preference [ 5 ] . Within b o t h o f these selected lines, Eriksson n o t e d t h a t t h e females h a d a greater e t h a n o l elimination rate t h a n did the males. Liver A D H activity was n o t assessed in these e x p e r i m e n t s . However, strain a n d sex differences a p p a r e n t l y do exist in these p a r a m e t e r s , since a sex difference in h e p a t i c A D H activity in Sprague-Dawley b u t n o t Wistar rats has b e e n observed [ 2 ] . Such o b s e r v a t i o n s s e e m e d of p r o b a b l e significance w h e n , in e x a m i n i n g t h e effects of various p h a r m a c o l o g i c a l agents on e t h a n o l - i n d u c e d sleep t i m e in o u r l a b o r a t o r y , it was o b s e r v e d t h a t sex differences in sleep t i m e m i g h t exist. These initial e x p e r i m e n t s h a d utilized sexually m a t u r e mice o f b o t h sexes, ranging in age f r o m 55 to 95 days, from the C 5 7 B L / I b g strain, The sex difference s e e m e d to be greater as a f u n c t i o n o f age. Thus, e x p e r i m e n t s were designed t o e s t i m a t e the m a g n i t u d e of this o b s e r v a t i o n a n d t o determine possible u n d e r l y i n g m e c h a n i s m s . METHOD
Animals The animals were divided i n t o 2 age groups: 5 0 - 6 0 days o f age a n d 9 5 - 1 1 0 days o f age. A l c o h o l - i n d u c e d sleep t i m e was assessed following the i n t r a p e r i t o n e a l i n j e c t i o n o f 3.5 g x Kg -~ e t h a n o l . The alcohol s o l u t i o n was p r e p a r e d in i s o t o n i c saline a n d c o n t a i n e d 0.35 g x ml-' o f e t h a n o l . A t this c o n c e n t r a t i o n , t h e a p p r o p r i a t e dose is achieved if each
~This work was supported in part by NIAA grants AA-00293, AA-00092 and an award to Allan C. Collins by the National Council on Alcoholism, and the Foundations Fund For Research in Psychiatry. 973
974 m o u s e is i n j e c t e d w i t h 0.01 ml x g l b o d y weight. T h e animals were i n j e c t e d i n t r a p e r i t o n e a l l y a n d r e s t r a i n e d for a p p r o x i m a t e l y 30 sec a f t e r w h i c h t h e y were placed on t h e i r backs in a trough. Sleep t i m e was j u d g e d t e r m i n a t e d w h e n the animals could right t h e m s e l v e s successfully 2 t i m e s in 30 sec.
Procedure Since differences in sleep t i m e m i g h t arise as a conseq u e n c e of a l t e r a t i o n s in CNS sensitivity to t h e d e p r e s s a n t effects o f e t h a n o l or as a c o n s e q u e n c e o f differing rates o f m e t a b o l i s m o f e t h a n o l , e x p e r i m e n t s were p e r f o r m e d to test these possibilities. A c o n v e n i e n t way to e s t i m a t e CNS sensitivity to e t h a n o l in individual animals is to measure alcohol c o n c e n t r a t i o n in the b l o o d at t h e time o f regaining t h e righting reflex. The mice were i n j e c t e d with the s t a n d a r d 3.5 g x K g ' e t h a n o l dose; i m m e d i a t e l y a f t e r t h e righting reflex was regained, a central v e n o u s b l o o d sample was o b t a i n e d f r o m each a n i m a l by p u n c t u r i n g t h e retroo r b i t a l sinus w i t h a 40 m i c r o l i t e r capillary pipet. The b l o o d sample was a d d e d to 0.96 ml of a h e p a r i n i z e d i s o p r o p y l a l c o h o l s o l u t i o n . The final i s o p r o p y l alcohol c o n c e n t r a t i o n was 40 m g p e r c e n t and served as an i n t e r n a l standard. A 5 m i c r o l i t e r a l i q u o u t of this s o l u t i o n was i n j e c t e d o n t o a Porapak Q c o l u m n in a B e c k m a n GC-45 gas c h r o m a t o g r a p h , e q u i p p e d w i t h a flame i o n i z a t i o n d e t e c t o r . The c o l u m n a n d d e t e c t o r t e m p e r a t u r e s were 175°C a n d 225~C, respectively. The gas m i x t u r e consisted of air, 300 ml x min-~: h y d r o g e n , 45 ml x min -~ ; a n d h e l i u m , 80 ml x min -J . Each sample was r u n in duplicate. Blood alcohol c o n c e n t r a t i o n was calculated b y e s t i m a t i n g by t r i a n g u l a t i o n the areas of t h e e t h a n o l a n d i s o p r o p y l alcohol peaks a n d c o m p a r i n g t h e ratio o b t a i n e d for each sample w i t h a s t a n d a r d curve. The rate o f e t h a n o l e l i m i n a t i o n was d e t e r m i n e d for animals f r o m each o f t h e 4 groups using a similar t e c h n i q u e . Each a n i m a l was i n j e c t e d w i t h the s t a n d a r d e t h a n o l dose a n d 4 0 microliter b l o o d samples were o b t a i n e d at 1 , 2 a n d 4 h r after injection. Blood alcohol c o n c e n t r a t i o n s were d e t e r m i n e d as described previously and the e l i m i n a t i o n rate calculated for each animal. Hepatic alcohol d e h y d r o g e n a s e , ADH, and a l d e h y d e d e h y d r o g e n a s e , A L D H , have long been c o n s i d e r e d the most i m p o r t a n t e n z y m e s in e t h a n o l m e t a b o l i s m . Certainly, o n a kinetic basis, this appears to be the case. These considerations led us to measure h e p a t i c A D H a n d A L D H activities in vitro. Animals f r o m each of the 4 groups w h i c h had n o t b e e n e x p o s e d to e t h a n o l were sacrificed by a blow o n the head, t h e livers quickly excised a n d placed in 9 v o l u m e s o f cold 0.25 M sucrose. The tissue was h o m o g e n i z e d a n d t h e 4 8 , 0 0 0 × g s u p e r n a t a n t fraction o b t a i n e d by c e n t r i f u g i n g for 1 h r in a Sorvall RC2B refrigerated centrifuge. A D H activity was d e t e r m i n e d in this s u p e r n a t a n t f r a c t i o n by m o n i t o r i n g s p e c t r o p h o t o m e t r i c a l l y the rate of N A D H f o r m a t i o n with e t h a n o l a n d NAD as c o s u b s t r a t e s using a m o d i f i c a t i o n o f the m e t h o d of B o n n i c h s e n and Brink [ 1 ]. The m o d i f i c a t i o n consisted of the a d d i t i o n of 0.2 M s e m i c a r b a z i d e to t h e i n c u b a t i o n s . Semicarbazide serves t o trap t h e a c e t a l d e h y d e w h i c h is f o r m e d w h e n e t h a n o l is oxidized; if this step is o m i t t e d , b o t h A D H and A L D H , w h i c h c o n v e r t a c e t a l d e h y d e to acetic acid using NAD as a c o s u b s t r a t e , will be measured. T o t a l h e p a t i c A L D H activity was d e t e r m i n e d utilizing a r a d i o c h e m i c a l assay w h i c h has r e c e n t l y b e e n d e v e l o p e d in o u r l a b o r a t o r y . T h e assay s u b s t r a t e , 14 C b e n z a l d e h y d e , was
COLLINS, Y E A G E R , LEBSACK AND P A N T E R o b t a i n e d f r o m New England Nuclear as a b e n z e n e s o l u t i o n . The b e n z e n e was r e m o v e d by e v a p o r a t i o n u n d e r a s t r e a m of n i t r o g e n at 0°C. The radiolabelled b e n z a l d e h y d e was resolubilized in 0.5 ml of a 1:1 a c e t o n e - w a t e r s o l u t i o n and r u n across a 3.5 × 0.8 cm Dowex AG-I c o l u m n , pH 7.0. The c o l u m n was washed with 2.5 ml of w a t e r a n d e f f l u e n t s c o m b i n e d . The b e n z a l d e h y d e was diluted with u n t a b e l l e d b e n z a l d e h y d e to a specific activity of 0.10 m i c r o c u r i e s x m i c r o m o l e -l . E n z y m e activity was d e t e r m i n e d b y i n c u b a t i n g 50 microliters of a 10 p e r c e n t h o m o g e n a t e in a m i x t u r e of 0.65 ml 10 mM p y r o p h o s p h a t e buffer, pH 9.6, 0.1 ml 10 mM NAD, 0.1 ml 10 mM p y r a z o l e and 0.1 ml 2.5 mM b e n z a l d e h y d e (0.25 microcuries). This m i x t u r e was incub a t e d for 5 rain at 37.5°C in a shaking i n c u b a t o r bath. The r e a c t i o n was t e r m i n a t e d by the a d d i t i o n of 0.1 ml of a 20 p e r c e n t t r i c h l o r o a c e t i c acid (TCA) solution. Each h o m o g e nate was assayed in duplicate along with a b l a n k w h i c h consisted o f the c o m p l e t e r e a c t i o n m i x t u r e with the TCA a d d e d prior to the e n z y m e . The s o l u t i o n was t h e n made alkaline (pH 12) b y the a d d i t i o n of a 20 p e r c e n t NaOH s o l u t i o n and e x t r a c t e d twice with 6 ml o f w a t e r s a t u r a t e d 1,2 d i c h l o r o e t h a n e . This e x t r a c t i o n removes u n r e a c t e d b e n z a l d e h y d e and any o t h e r n e u t r a l m e t a b o l i t e s leaving the r e a c t i o n p r o d u c t , b e n z o i c acid, in the a q u e o u s layer. The radioactivity o f a 0.1 ml aliquot of the a q u e o u s layer was d e t e r m i n e d by placing it in 10 ml of a T r i t o n X-100 t o l u e n e based scintillation cocktail a n d c o u n t i n g in a B e c k m a n LS-133 liquid scintillation c o u n t e r . C o r r e c t i o n s for c o u n t ing efficiency were made. R e c o v e r y of b e n z o i c acid was nearly 100 p e r c e n t u n d e r these c o n d i t i o n s . Protein concent r a t i o n s were d e t e r m i n e d using the b i u r e t m e t h o d . The activities of b o t h ADH a n d A L D I t were calculated in t e r m s o f activity per mg of p r o t e i n , per g liver, per liver a n d per g o f b o d y weight. Data were a n a l y z e d by analyses of variance and the m e a n s c o m p a r e d using D u n c a n ' s multiple range test. RESULTS T h e results o f those e x p e r i m e n t s in w h i c h the p o t e n t i a l i n f l u e n c e of age a n d sex on a l c o h o l - i n d u c e d sleep t i m e in C 5 7 B L / I b g mice was assessed are p r e s e n t e d in Table 1. No significant sex d i f f e r e n t i a l is evident in the groups tested at 50 60 days of age; however, t h e r e is a m a r k e d sex difference in the older group. T h e older males slept nearly twice as long as the females of the same age a n d a p p r o x i m a t e l y 4 times as long as the 5 0 - 6 0 - d a y - o l d males. A l t h o u g h m e a n sleep time in females also increased w i t h age, there was so m u c h variance in this measure t h a t a 45 p e r c e n t increase was n o n s i g n i f i c a n t . The increase in e t h a n o l - i n d u c e d sleep time observed in C 5 7 B L mice is t h e r e f o r e d e p e n d e n t u p o n the sex of the animal. It is observed in greatest m a g n i t u d e in male mice. An increase in sleep t i m e m i g h t arise from differing CNS sensitivities to t h e depressant actions of alcohol. The results o f the e x p e r i m e n t in w h i c h 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 was m e a s u r e d at t i m e of regaining the righting reflex are p r e s e n t e d in Table 2. N o n e of t h e groups differ significantly from the o t h e r s with respect to b l o o d a l c o h o l c o n c e n t r a t i o n at t i m e of awakening. T h e r e f o r e , t h e r e p r o b a b l y are n o t a n y remarkable sex- or a g e - d e p e n d e n t differences in CNS sensitivity to alcohol in C 5 7 B L / I b g mice: In a d d i t i o n , these data i m p l y t h a t the old males differed f r o m the o t h e r groups with
AGE, SEX AND A L C O H O L M E T A B O L I S M
975 TABLE 1
EFFECTS OF AGE AND SEX ON ALCOHOL-INDUCED SLEEP TIME IN C57BL/Ibg MICE
Age (days)
Sex
Sleep Time*
Comparison
p
50-60
(YF)
Female
48.4 ± 5.8 (16)
OM-YM
