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0 45-6008/87/ 1 103-028 $2.00/0ALCOHOLISM: LINICALN D EXPERIMENTAL ESEARCH

Vol. 11 , No.May/June 1987

Developmental Changes in AlcoholPharmacokinetics in Rats

Sandra J. Kelly, PhD, Daniel J. Bonthius, BS, and James R. West, Ph D

Developmental changes in the pharmacokinetics of alcohol couldinfluence the outcome of alcohol exposure during diffe rent periodsof postnatal development. Hence, the development of the abi lity toabsorb and metabolize alcohol in the rat was examined by adminis-tering an acute dose (2.5 g/kg) of ethanol in milk formula by intra-gastric intubation o rats of 1, 2, 4, 6, 8, 10, 15, 21, 30, and 60 daysof age. Each animal in a particular litter was assigned a differen ttime point following intubation when its blood alcohol concentration(BAC) was determined from a tail blood sample. At all ages tested,maximum BACs occurred between 1.25 and 1.5 hr following intuba-tion. However, maximum BACs decreased with age from 155 mg/dlin 1-day-old rats to 111 mg/dl i n 60-day-old rats. Furthermore, therate of alcohol clearance was slower in the younger rats. By linear

regression analysis, the elimination rate of alcohol in 1-day-old ratswas estimated o be 7.5 mg/dl/hr which increased to 42.2 mg/dl/hrin 60-day-old-rats. By 8 hr following intubation, rats that were 21days of age and older had completely cleared the alcohol, whereasthe younger rats (1-15 days of age) had not. No consistent sexdifferences were seen in either the maximum BAC or clearance rate.Since developmental changes in the ability to clear alcohol occurthroughout the firs t 60 postnatal days i n the rat, controlling for thesechanges is essen tial when looking for cri tical periods of an organ'svulnerability to damage by alcohol.

DMINISTRATION of alcohol by a variety of methodsA to neonatal rats has been used by this laboratory'-3and a number of other lab~ratories~-'~ s a model systemfor the human third trimester to examine how alcoholaffects the development of the central nervous systemduring its period of most rapid g r ~ w t h . ' ~ - ' ~ uring thisperiod of brain development, alcohol has a number ofharmful effects, of which the most obvious is an overallreduction in brain 13 , 14, l 9 ndependent of re-ductions in body growth.59 9 The observed microence-phaly, in relationship to body size, is much greater follow-ing postnatal exposure to alcohol than that observed whenrats have been exposed to alcohol prenatally." There are

From the Alcohol and Brain Research Laboratory, Department of

Anatomy, Collegeo

Medicine, Universityof Iowa, Iowa C ity, Iowa.Received for publication June 27, 1986; revised m anuscript receivedSeptember 2, 1986;accepted September4 , 1986.

This work was supported in part by G rants AA0552 3 andAA061Wfrom the National Institute on Alcohol Abuse and Alcoholism (J.R.W. ) .D.J.B. was a recipient o training grants from the University of IowaNeuroscience program and fr om the National Institutes of Health Train-ing Grant GM 0733 7.

Reprint requests: Dr. James R. West, Alcohol and Brain ResearchLaboratory, Department o Anatomy, Collegeo Medicine, UniversityofIowa, lowa C ity,f A 52242.

Copyright0 1987 by The American Medical Societyon Alcoholismand The Research Society on Alcoholism.

at least two possible reasons for this finding. First, thebrain may be more vulnerable to alcohol during the neo-natal period. Second, the neonatal rat may be exposed toalcohol either at higher blood alcohol concentrations(BACs) or for longer periods of time than the fetus follow-ing comparable maternal ingestion of alcohol. This latterpossibility arises because the alcohol is metabolized by therat pup in the one case and by the dam in the other. Thecontribution of age-related differences in alcohol metab-olism to vulnerability to alcohol effects during develop-ment is unknown.

The majority of alcohol metabolism occurs in the liver,although a small percentage of alcohol elimination isaccounted for by the processes of renal filtration andexpiration.20x21 he most important enzyme involved inalcohol metabolism in the liver is alcohol dehydrogenase(ADH).20 his enzyme converts alcohol into acetaldehydeby using nicotinamide adenine dinucleotide (NAD) as acoenzyme. When the alcohol dose is low, this process islimited by the availability of NAD, which in turn isdetermined by the activity of a number of other liverdehydrogenases.22 n contrast, when the alcohol dose ishigh, the conversion of ethanol to acetaldehyde is limited

by the availability of ADH.22 Additional enzyme systemspossibly involved in alcohol metabolism are a catalaseand a microsomal ethanol-oxidizing system

(MEOS).25-27The development of the ability to clear alcohol must be

the result of the development of numerous physiologicalsystems that play a role in the elimination of alcohol inthe adult rat. Therefore, it would be difficult to predictdevelopmental changes in the rate of alcohol clearancesolely on the basis of the maturation of any one system.In spite of the increasing popularity over the last decadeof exposing neonatal rats to alcohol in order to studyaspects of the fetal alcohol syndrome, a perusal of theliterature reveals a paucity of studies on the developmentof the ability to clear alcohol in vivo. There are a fewstudies describing the development of particular en-zyme~.~*-~ ' owever, they may bear little or no resem-blance to the development of the in vivo metabolism ofa l c o h 0 1 ~ ' ~ ~ ~although see Makar and Mannerix~g~~), inceother enzymes and metabolic processes may be rate-lim-iting to the metabolism of alcohol during development.Therefore, as a first step in understanding the effects ofalcohol in the neonatal rat, we examined the pharmaco-

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282 KELLY ET AL .

kinetics of alcohol in rats of both sexes aged between 1and 60 days.

MATERIALS AND METHODS

Subjects

Groups of female Sprague-Dawley rats were housed with one male

per cage in a sound-attenuated temperature-controled room on a 12-hrlight-dark cycle. They were provided standard laboratory chow and tapwater ad libitum. The day that females were found to be sperm-positivewas assigned as gestational day 0. Since gestation is normally completedin 22 days, gestational day 22 was considered postnatal day 0, regardlessof whether the pups had actually yet been born. In order to maintainuniformity in terms of developmental age, all references to postnatal ageare relative to that day. The rats were kept with their dam until 2 1 daysafter birth.

Procedure

Rat pups, 1,2,4,6,8, 10, 15,2 1, 30, and 60 days of age were isolatedfrom food sources including their dams for I hr and were then adminis-tered an acute dose (2.5 g/kg) of ethanol by intragastric intubation. The

ethanol was administered in milk formula* in a volume of 27.8 ml/kg.This particular milk formula and volume were chosen because theycorrespond to that administered during each feeding in our artificialrearing experiments. Rats of up to 2 1 days of age were kept warm whileseparated from their mothers by placing them on a temperature-con-trolled heating pad. The rats were returned to their dams 2 hr followingintubation. After administration of alcohol, the rats exhibited signs ofinebriation. Young rats, aged 1 to 10 days, were flaccid after alcoholadministration. The alcohol increased the amount of time which olderrats spent sleeping and increased the amount of time required to rightafter placement in a supine position. Interestingly, observation of therighting reflex indicated that rats at postnatal day 30 were the mostintoxicated by the alcohol, even though their maximum BACs were notthe highest of the ages tested. The alcohol dose used did not cause anymortality.

Each animal in a litter o r group was assigned a different time followingintubation when its tail blood alcohol concentration (BAC) was deter-mined. The time points examined included 0.5,0.75, 1.0, 1.25, 1.5,2.0,4.0, and 8.0 hr after intubation. Five male and five female animals wereinvestigated for each age and for each time point. Each animal wassampled only once in order to preclude any alteration in alcohol metab-olism due to prior alcohol exposure or loss of blood. At the appropriatetime, the tip of the rat's tail was cut and 20 pl of blood was drawn intoa heparinized capillary tube. The blood was placed in trichloroacetic acidin order to denature the protein and individual samples were stored at4C until all samples from the litter had been collected. Alcohol wasdetermined from this extract enzymatically (Sigma Chemical Co, CatalogNo. 332-UV) in a glycine buffer containing NAD+ and alcohol dehydro-genase; NADH formation was measured with a spectrophotometer set at340 nm.

Statistical Analysis

Linear regression was performed on the linear portion of each curvein order to calculate the slope, which represents the alcohol eliminationrate. The l inear regression analysis was not conducted on points on thecurve that had reached zero such as the 8-hr time point of rats older than2 1 days. A two-way (sex by age) analysis of variance (ANOVA) was usedto analyze the maximum BACs. The maximum BAC was taken fromthe data at either 1.25 or 1.5 hr after intubation depending upon whichwas greater. Post hoc analysis was done by the Newman-Keuls test.ANOVAs were also performed to determine whether sex contributed tothe elimination rates at each age.

RESULTS

The blood alcohol concentration curves for rats of dif-ferent ages are shown in Figs. 1-5. The variance in thedata was quite small and there were no striking sex differ-ences. There were differences among rats at different ageswith regards to maximum BAC (Fig. 6), elimination rate(Fig. 7), and estimated time to eliminate completely the

alcohol from tail blood (Fig. 8).The maximum BACs occurred at either 1.25 or 1.5 hr

after administration regardless of age. The value of themaximum BACs differed across ages (F(9,82)= 32.4, p