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ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH

Vol. 11, No.3 May/June 1987

Developmental Changes in Alcohol Pharmacokinetics in Rats

Sandra J. Kelly, PhD, Daniel J. Bonthius, BS, and James R. West, PhD
Developmental changes in the pharmacokinetics of alcohol could influence the outcome of alcohol exposure during different periods of postnatal development. Hence, the development of the ability to absorb and metabolizealcohol in the rat was examined by administering an acute dose (2.5 g/kg) of ethanol in milk formula by intra, 2, 4, 6, 8, 10, 15, 21, 30, and 60 days gastric intubation to rats of 1 of age. Each animal in a particular litter was assigned a different time 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 intubation. However, maximum BACs decreased with age from 155 mg/dl in 1-day-old rats to 111 mg/dl in 60-day-old rats. Furthermore, the rate of alcohol clearance was slower in the younger rats. By linear regression analysis, the elimination rate of alcohol in 1-day-old rats was estimated to be 7.5 mg/dl/hr which increased to 42.2 mg/dl/hr in 60-day-old-rats. By 8 hr following intubation, rats that were 21 days of age and older had completely cleared the alcohol, whereas the younger rats (1-15 days of age) had not. No consistent sex differences were seen in either the maximum BAC or clearance rate. Since developmental changes in the ability to clear alcohol occur throughout the first 60 postnatal days in the rat, controlling for these changes is essential when looking for critical periods of an organ's vulnerability to damage by alcohol.

DMINISTRATION of alcohol by a variety of methods A to neonatal rats has been used by this laboratory'-3 and a number of other lab~ratories~-'~ as a model system for the human third trimester to examine how alcohol affects the development of the central nervous system during its period of most rapid g r ~ w t h . ' ~During -'~ this period of brain development, alcohol has a number of harmful effects, of which the most obvious is an overall 1 3 , 14, l9independent . of rereduction in brain ductions in body growth.59 l 9 The observed microencephaly, in relationship to body size, is much greater following postnatal exposure to alcohol than that observed when rats have been exposed to alcohol prenatally." There are
From the Alcohol and Brain Research Laboratory, Department of Anatomy, College o f Medicine, University of Iowa, Iowa City, Iowa. Received for publication June 27, 1986; revised manuscript received September 2, 1986; accepted September 4, 1986. This work was supported in part by Grants AA05523 and A A 0 6 1 W from the National Institute on Alcohol Abuse and Alcoholism (J.R.W.). D.J.B. was a recipient o f training grants from the University of Iowa f Health TrainNeuroscience program and from the National Institutes o ing Grant GM0733 7. Reprint requests: Dr. James R. West, Alcohol and Brain Research Laboratory, Department o f Anatomy, College o f Medicine, University of Iowa, lowa City, f A 52242. Copyright 0 1987 by The American Medical Society on Alcoholism and The Research Society on Alcoholism.

at least two possible reasons for this finding. First, the brain may be more vulnerable to alcohol during the neonatal period. Second, the neonatal rat may be exposed to alcohol either at higher blood alcohol concentrations (BACs) or for longer periods of time than the fetus following comparable maternal ingestion of alcohol. This latter possibility arises because the alcohol is metabolized by the rat pup in the one case and by the dam in the other. The contribution of age-related differences in alcohol metabolism to vulnerability to alcohol effects during development is unknown. The majority of alcohol metabolism occurs in the liver, although a small percentage of alcohol elimination is accounted for by the processes of renal filtration and expiration.20x21 The most important enzyme involved in alcohol metabolism in the liver is alcohol dehydrogenase (ADH).20 This enzyme converts alcohol into acetaldehyde by using nicotinamide adenine dinucleotide (NAD) as a coenzyme. When the alcohol dose is low, this process is limited by the availability of NAD, which in turn is determined by the activity of a number of other liver dehydrogenases.22 In contrast, when the alcohol dose is high, the conversion of ethanol to acetaldehyde is limited by the availability of ADH.22Additional enzyme systems possibly involved in alcohol metabolism are a catalase and a microsomal ethanol-oxidizing system (MEOS).25-27 The development of the ability to clear alcohol must be the result of the development of numerous physiological systems that play a role in the elimination of alcohol in the adult rat. Therefore, it would be difficult to predict developmental changes in the rate of alcohol clearance solely on the basis of the maturation of any one system. In spite of the increasing popularity over the last decade of exposing neonatal rats to alcohol in order to study aspects of the fetal alcohol syndrome, a perusal of the literature reveals a paucity of studies on the development of the ability to clear alcohol in vivo. There are a few studies describing the development of particular enzyme~.~*-~' However, they may bear little or no resemblance to the development of the in vivo metabolism of a l c o h 0 1 ~(although '~~~ see Makar and Mannerix~g~~), since other enzymes and metabolic processes may be rate-limiting to the metabolism of alcohol during development. Therefore, as a first step in understanding the effects of alcohol in the neonatal rat, we examined the pharmaco-

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The blood alcohol concentration curves for rats of different ages are shown in Figs. 1-5. The variance in the data was quite small and there were no striking sex differMATERIALS AND METHODS ences. There were differences among rats at different ages Subjects with regards to maximum BAC (Fig. 6), elimination rate Groups of female Sprague-Dawley rats were housed with one male (Fig. 7), and estimated time to eliminate completely the per cage in a sound-attenuated temperature-controled room on a 12-hr alcohol from tail blood (Fig. 8). light-dark cycle. They were provided standard laboratory chow and tap The maximum BACs occurred at either 1.25 or 1.5 hr water ad libitum. The day that females were found to be sperm-positive after administration regardless of age. The value of the was assigned as gestational day 0. Since gestation is normally completed BACs differed across ages (F(9,82)= 32.4, p < maximum in 22 days, gestational day 22 was considered postnatal day 0, regardless
of whether the pups had actually yet been born. In order to maintain uniformity in terms of developmental age, all references to postnatal age are relative to that day. The rats were kept with their dam until 2 1 days after birth.
175

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

RESULTS

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Procedure Rat pups, 1,2,4,6,8, 10, 15,21, 30, and 60 days of age were isolated from food sources including their dams for I hr and were then administered 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 they correspond to that administered during each feeding in our artificial rearing experiments. Rats of up to 2 1 days of age were kept warm while separated from their mothers by placing them on a temperature-controlled heating pad. The rats were returned to their dams 2 hr following intubation. After administration of alcohol, the rats exhibited signs of inebriation. Young rats, aged 1 to 10 days, were flaccid after alcohol administration. The alcohol increased the amount of time which older rats spent sleeping and increased the amount of time required to right after placement in a supine position. Interestingly, observation of the righting reflex indicated that rats at postnatal day 30 were the most intoxicated by the alcohol, even though their maximum BACs were not the highest of the ages tested. The alcohol dose used did not cause any mortality. Each animal in a litter or group was assigned a different time following intubation when its tail blood alcohol concentration (BAC) was determined. 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 were investigated for each age and for each time point. Each animal was sampled only once in order to preclude any alteration in alcohol metabolism due to prior alcohol exposure or loss of blood. At the appropriate time, the tip of the rat's tail was cut and 20 pl of blood was drawn into a heparinized capillary tube. The blood was placed in trichloroacetic acid in order to denature the protein and individual samples were stored at 4C until all samples from the litter had been collected. Alcohol was determined from this extract enzymatically (Sigma Chemical Co, Catalog No. 332-UV) in a glycine buffer containing NAD+ and alcohol dehydrogenase; NADH formation was measured with a spectrophotometer set at 340 nm. Statistical Analysis
Linear regression was performed on the linear portion of each curve in order to calculate the slope, which represents the alcohol elimination rate. The linear regression analysis was not conducted on points on the curve that had reached zero such as the 8-hr time point of rats older than 2 1 days. A two-way (sex by age) analysis of variance (ANOVA) was used to analyze the maximum BACs. The maximum BAC was taken from the data at either 1.25 or 1.5 hr after intubation depending upon which was greater. Post hoc analysis was done by the Newman-Keuls test. ANOVAs were also performed to determine whether sex contributed to the elimination rates at each age.

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Time (hours) Fig. 1. Alcohol concentration over time in tail M o o d after gastric intubation of 2.5 g/kg of ethanol in male and female rats at postnatal days 1 and 2. Error bars represent standard error of the mean (SEM).

4 Day Olds 6 Day Olds

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Time (hours) Fig. 2. Alcohol concentration over time in tail blood after gastric intubation of 2.5 g/kg of ethanol in male and female rats at postnatal days 4 and 6. Error bars represent SEM.

DEVELOPMENTAL CHANGES IN PHARMACOKINETICS

8 Day Olds 10 Day Olds

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Fig. 3. Alcohol concentration over time in tail blood after gastric intubation of 2.5 g/kg of ethanol in male and female rats at postnatal days 8 and 10. Erfor bars represent SEM.

Time (hours)
Fig. 5. Alcohol concentration over time in tail blood after gastric intubation of 2.5 g/kg of ethanol in male and female rats at postnatal days 30 and 60. Error bars represent SEM.

15 Day Olds 21 Day Olds

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Time (hours)
Fig. 4. Alcohol concentration over time in tail blood after gastric intubation of 2.5 g/kg of ethanol in male and female rats at postnatal days 15 and 21. Error bars represent SEM.

significantly less than those of the I-, 2-, and 4-day-old rats (ps < 0.05). Elimination rates of alcohol were calculated by linear regression analysis and are shown in Fig. 7. The elimination rates increased with age. There were no significant sex differences in the elimination rates except at day 30 (F(1,43) = 5.7, p < 0.02). The time of complete clearance of alcohol from the rats body varied with age. The longest time in which alcohol was still estimated to be present was 23 hr at 1 day of age, and this shortened to only 4 hr by 60 days of age. Again, in terms of the time of complete clearance of the 2.5 g/kg dose of alcohol, there were no striking sex differences. An attempt was made to analyze the alcohol concentration curves using the Widmark formulation as described by Kalant. However, all the Widmark rs were greater than 1 indicating a violation of the assumptions in the This will be discussed in further detail LIB f~rmulation.~ ,~~ the following section of this paper. The mean body weights (in g) with SEM of male and female rats increased from 7.1 f 0.1 and 6.8 f 0.1 on day 1 to 278.0 f 2.3 and 199.5 & 1.6 on day 60. Sex differences in body weight were apparent by 30 days of age.

0.001) but not sexes (see Fig. 6). Newman-Keuls analysis DISCUSSION showed that there were no significant differences in the maximum BAC from days 15-60. The maximum B A ~ s The ability to metabolize intragastrically administered exhibited by rats aged 1-10 days were all greater than the alcohol changes over development. Given the same dose maximum BACs exhibited on days 15-60 (ps < 0.05). of alcohol, rats on postnatal days 1-4 exhibit higher max,Rats aged 1 and 2 days exhibited similar maximum BACs. imum BACs than rats on postnatal days 6-10, which, in The maximum BACs of 4-day-old rats were higher than turn, exhibit higher BACs than rats on days 15-60. The those of 1- and 2-day-old rats ( p < 0.05). Rats of 6,8, and linear portions of the curves increase in steepness with age 10 days of age exhibited maximum BACs which were not with the biggest changes occurring between days 2 and 4 significantly different from each other, but they were and days 15 and 21,21 and 30, and 30 and 60. Further-

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Males

Females

Fig. 6. The maximum BACs measured over postnatal days 1 to 60 after intragastric administration of 2.5 g/kg of ethanol. Error term is SEM.

60

AGE (DAYS)
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Fig. 7. Elimination rates over postnatal days 1 to 60 after intragastric administrationof 2.5 g/kg of ethanol. Error term is the standard error of the regression coefficient.

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Fig. 8. Estimated time to reduce BACs to zero over days 1 to 60 after intragastric administration of 2.5 g/ kg of ethanol.

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30

60

AGE (DAYS)

DEVELOPMENTAL CHANGES IN PHARMACOKINETICS

285

more, complete elimination of alcohol from tail blood samples takes much longer in younger rats than in older rats. In sum, this implies that very young rats will have higher BACs and have alcohol in their bodies for a much longer time than older rats, given the same dose of alcohol. In general, with intragastric administration of alcohol, the time of peak concentration of alcohol in the brain will occur earlier than in the blood.20,2 However, the purpose of this study was to examine the changes over development of alcohol metabolism and, to this extent, the changes in alcohol content in the blood over development probably reflect relative changes in the exposure of the brain to alcohol. However, it should be noted that there is a possibility that the b1ood:brain ratio of alcohol concentration may also change over development. As mentioned earlier, there is a paucity of data on the in vivo development of the metabolism of alcohol. BauerMoffett and Altman did not find any changes in blood ethanol concentrations from postnatal days 5-20, but the alcohol was administered by vapor inhalation making it difficult to determine whether the rats were actually exposed to the same dose of alcohol at different ages. Hollstedt and Rydberg3 administered 1.5 g/kg of alcohol intraperitoneally (ip) to male rats weighing 25 g (approximately 12 days of age) to 250 g (approximately 60 days of age). They found changes in peak blood alcohol concentration and in the elimination rate between 12 and 24 days followed by very little change in the alcohol elimination curves between 24 and 40 days. These findings are in agreement with the data presented here. Hollstedt and Rydberg3 reported that the maximum BAC increased and rate of elimination slowed after 40 days of age in male rats, which they argued was due to hormonal changes with the onset of adolescence and was a sex-related effect. We did not find any indication of a slowing of alcohol metabolism between the ages of 30 and 60 days and the only sex effect found in this study, which is a slower elimination rate in females at day 30, is opposite to that reported in the literature. Others have found that female rats metabolize alcohol faster and have higher ADH activities than male^,^^,^^ but that is not true for all strain^^^'^^ nor necessarily consistent within strains.38 Furthermore, there is evidence in mice that the sex difference may not occur until quite late in d e ~ e l o p m e n tThere.~~ fore, the general lack of sex differences in our study may be because we did not test rats older than 60 days of age, because sex differences cannot be detected when the alcohol is administered intragastrically, or because sex differences in alcohol metabolism afe not large in Sprague: Dawley rats.38 A number of researchers have found that ADH activity in the liver reaches adult levels between 2 1 and 30 days of age.28.29,40 However, it is clear that there are still developmental changes in alcohol metabolism after 30 days of age. This is probably because ADH is not rate-limiting in the metabolism of alcohol. The dissociation of ADH

activity from the in vivo metabolism of alcohol has been shown under other circumstances as ell.^',^^ Changes in the NAD/NADH2+ ratio in the liver,35various liver enzyme activities, renal filtration, expiration,21 and body fat can all have marked effects on blood alcohol concentrations, and these factors all change over development. For these reasons, our results are not surprising. One way to examine curves showing blood alcohol concentrations over time is to apply the equation proposed by Widmark (as described by Kalant and Wallg~-en~~), which predicts whole body alcohol concentration and metabolism from blood alcohol concentrations. However, when this formulation is applied to the present data, the resulting numbers describing the fraction of body mass into which alcohol is distributed (referred to as Y) are greater than 1. The most probable reason for this finding is that the assumptions of the Widmark formulation, that complete absorption and distribution have occurred before calculations are made and that intake is quicker than eliminati~n cannot , ~ ~ be met in this study, thus leading to an overestimate of Y. Intragastric administration of alcohol in milk formula could easily lead to violations of these assumptions. Alcohol is largely absorbed through the small intestine and not the However, alcohol concentrations as high as those used in this experiment slow the passage of alcohol through the stomach because alcohol disrupts muscular contractions of the stomach.34 Furthermore, milk will also slow the absorption of alcohol from the gastrointestinal t r a ~ t . 4 The ~ probable retardation of absorption in this experiment violates the assumptions of the Widmark formulation, rendering it inappropriate. However, since liquid diets are utilized commonly for both prenatal and early postnatal alcohol administration, the present data are informative and justify the method of administration. In summary, given a specific dose of ethanol, both the maximum BAC and duration of total exposure to alcohol vary over development. Young rats are much less efficient than older rats at eliminating alcohol from their bodies. The results have important implications for studies attempting to compare the effects of alcohol during different periods of development. Since alcohol is eliminated from a fetal rat predominantly via the mothers liver, the fetus is probably exposed to the same BAC for the same amount of time as the mother (an adult rat) would be. Thus, for a specific dose of alcohol administered orally in a liquid diet, the fetus would be exposed to a less severe insult than a neonatal rat pup forced to metabolize the alcohol on its own. Further, during the early postnatal period, rat pups would be exposed to a more severe insult by a given dose than animalsjust a few days older. Therefore, when critical periods are suggested for the effect of alcohol on organs, such as the brain, that develop over a prolonged period, it is important to determine whether observed critical periods actually occur because of changes in the metabolism

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of alcohol rather than differences in temporal vulnerability of the organ.

ACKNOWLEDGMENT
The authors would like to thank Dr. Ronald L. Alkana for his helpful criticisms of an early version of this manuscript.

REFERENCES
I. Pierce DR, West JR: Blood alcohol concentration: a critical factor for producing fetal alcohol effects. Alcohol 3:269-272, 1986 2. West JR, Hamre KM, Pierce DR: Delay in brain growth induced by alcohol in artificially reared rat pups. Alcohol 1:2 13-222, 1984 3. West JR, Hamre KM: Effects of alcohol exposure during different periods of development: changes in hippocampal mossy fibers. Dev Brain Res 17:280-284, 1985 4. Bauer-Moffett C, Altman J Ethanol-induced reductions in cerebellar growth of infant rats. Exp Neurol48:378-382,1975 5. Bauer-Moffett C, Altman J: The effect of ethanol chronically administered to preweanling rats on cerebellar development: a morphological study. Brain Res 119:249-268, 1977 6. Borges S, Lewis PD: A study of alcohol effects on brain during gestation and lactation. Teratology 25:283-289, 1982 7. Diaz J, Samson HH: Impaired brain growth in neonatal rats exposed to ethanol. Science 208:751-753, 1980 8. Grant KA, Choi EY, Samson HH: Neonatal ethanol exposure: effects on adult behavior and brain growth parameters. Pharmacol Biochem Behav 18:331-336, 1983 9. Grant KA, Samson HH: Ethanol and tertiary butanol induced microencephaly in the neonatal rat: comparison of brain growth parameters. Neurobehav Toxicol Teratol4:315-321, 1982 10. Samson HH, Grant KA: Fetal alcohol effects: Alcohol and the developing nervous system of the rat, in Parves H, Burns E, Burov V, Parves S (eds): Progress in Alcohol Research, vol. 1. Utrecht, VNU International Press, 1985, pp 1-35 1 1. Phillips SC, C r a g B G A change in susceptibilityof rat cerebellar Purkinje cells to damage by alcohol during fetal, neonatal and adult life. Neuropathology and Applied Neurobiology 8:44 1-454, 1982 12. Jones DG, Colangelo W Ultrastructural investigation into the influence of ethanol on synaptic maturation in rat neocortex. 11. Quantitative Analysis. Dev Neurosci 7:107-119, 1985 13. Samson HH, Diaz J: Effects of neonatal ethanol exposure on brain development in rodents, in Abel EL (ed): Fetal Alcohol Syndrome, Vol. 111, Animal Studies. CRC Press, Boca Raton, FL, 1982, pp 131- 150 14. Samson HH, Grant KA: Ethanol-induced microcephaly in neonatal rats: relation to dose. Alcoholism: Clin Exp Res 8:201-203, 1984 15. Sonderegger TB, Calmes H, Corbitt S, Zimmermann EG: Lack of persistent effects of low dose ethanol adminkration postnatally in rats. Neurobehav Toxicol Teratol4:463-468, 1982 16. Stibler H, Tabakoff B, Bums E, Cerven E, Borg S, Kruckeberg T, Gaetano P: Effect of ethanol treatment during synaptogenesison synap tosomal sialic acid and sialytransferase,in Pems C, Struwe G, Jansson B (eds): Biological Psychiatry. New York, North/Holland Biomedical Press, 1981, pp 929-932 17. Dobbing J: The later development of the brain and its vulnerability, in Davis JA, Dobbing J (eds): Scientific Foundations of Paediatrics, (ed 2). London, William Heinemann Medical Books, 1981, pp 744-758 18. Dobbing J, Sands J: Comparative aspects of the brain growth spurt. Early Human Develop 3:79-83, 1979 19. Samson HH. Microcephaly and fetal alcohol syndrome: human and animal studies, in West JR (ed): Alcohol and Brain Development. New York, Oxford University Press, 1986, pp 167-183 20. Goldstein D B Pharmacology of Alcohol. New York, Oxford University Press, 1983 2 1. Kalant H: Absorption, diffusion, distribution, and elimination of

ethanol: effects on biological membranes, in Kissin B, Begleiter H (eds): The Biology of Alcoholism. New York, Plenum Press, 1971, pp 1-46 22. Wendell GD, Thurman RG. Effect of ethanol concentration on rates of ethanol elimination in normal and alcohol-treated rats in vivo. Biochem Pharmacol28:273-279, 1979 23. Keilin D, Hartree E F Properties of catalase. Catalysis of coupled oxidation of alcohols. Biochem J 39:293-301, 1945 24. Smith ME: Interrelations in ethanol and methanol metabolism. J Pharmacol Exp Ther 134:233-237,1961 25. Lieber CS, DeCarli LM: Ethanol oxidation by hepatic microsomes: adaptive increase after ethanol feeding. Science 162:9 17-9 18, 1968 26. Lieber CS, DeCarli LM: The role of the hepatic microsomal ethanol oxidizing system (MEOS) for ethanol metabolism in viva J Pharmacol Exp Ther 181:279-287, 1972 27. Shigeta Y, Nomura F, Iida S, Leo MA, Felder MR, Lieber C S Ethanol metabolism in vivo by the microsomal ethanol-oxidizingsystem in deer mice lacking alcohol dehydrogenase(ADH). Biochem Pharmacol 332307414, 1984 28. Lad PJ, Shoemaker WJ, Lefferet H L Developmental changes in rat liver alcohol dehydrogenase. Dev Biol 105526-529, 1984 29. Raiha NCR, Koskinen M, Pikkarainen P Developmental changes in alcohol-dehydrogenase activity in rat and guinea-pig liver. Biochem J 103:623-626, 1967 30. Sjoblom M, Pilstrom L, Msrland J: Activity of alcohol dehydrogenase and acetaldehyde dehydrogenases in the liver and placenta during the development of the rat. Enzyme 23:108-115, 1978 3 1. Wilson E C Ethanol metabolism in mice with different levels of hepatic alcohol dehydrogenase,in Maickel RG (4): Biochemical Factors in Alcoholism. New York, Pergamon Press, 1967, pp 115-124 32. Edwards JA, Price Evans DA: Ethanol metabolism in subjects possessing typical and atypical liver alcohol dehydrogenase. Clin Pharmacol Ther 8:824-829, 1967 33. Makar AB, Mannering GJ: Kinetics of ethanol metabolism in the intact rat and monkey. Biochem Pharmacol 19:2017-2022, 1970 34. Wallgren H: Absorption, diffusion, distribution and elimination of ethanol. Effect on biological membranes, in Tremoli&resJ (ed): Alcohols and their Derivatives. Oxford, Pergamon Press, 1970, pp 16I 188 35. Hollstedt C, Rydberg US: Ethanol metabolism in the growing rat. Arch Int Pharmacodyn Ther 188:341-348, 1970 36. Erickson CK: Ethanol clearance in nine inbred rat strains. Alcohol Clin Exp Res 8:491-494, 1984 37. Rachamin G, MacDonald JA, Wohid S, Clapp JJ, Khanna JM, Israel Y: Modulation of alcohol dehydrogenase and ethanol metabolism by sex hormones in the spontaneously hypertensive rat. Biochem J 186:483-490, 1980 38. Gillion RB, Crow KE, Batt RD, Hardman MJ: Effects of ethanol treatment and castration on liver alcohol dehydrogenaseactivity. Alcohol 2:39-41, 1985 39. Collins AC, Yeager TN, Lebscock ME, Panter S S Variations in alcohol metabolism: influence of sex and age. Pharmacol Biochem Behav 31973-978, 1975 40. Horton AA, Mills DJ: Developmental patterns of alcohol dehydrogenase and aldehyde dehydrogenasesin homogenates and subcellular fractions of rat liver. Mech Ageing Dev 11:363-370, 1979 41. Bloom F, Lad P, Pittman Q, Rogers J: Blood alcohol levels in rats: non-uniform yields from intraperitoneal doses based on body weight. Br J Pharmacol75:251-254, 1982 42. York J L Increased responsiveness to ethanol with advancing age in rats. Pharmacol Biochem Behav 19:687-691, 1983 43. Harger RN, Hulpieu HR: The pharmacology of alcohol, in Thompson GN (ed): Alcoholism. Springfield, IL, Charles C Thomas pp 103-232 44. Mellanby E Alcohol: its absorption into and disappearance from the blood under different conditions. Med Res Council Spec Rep Ser 31:l-48, 1919