Drug and Alcohol Dependence,
Elsevier Scientific Publishers
31(1992) 57-64 Ireland Ltd.
57
Effects of acute and repeated alcohol ingestion on hypothalamic-pituitary-gonadal and hypothalamic-pituitaryadrenal functioning in normal males Yoshishige
Ida, Shusaku Tsujimaru, Katsura Nakamaura, Ishou Shirao, Hirotaka Mukasa, Hideaki Egami and Yoichi Nakazawa Department
of Neuropsychiatry,
Kurum.e
University School of Medicine,
Kurum
880 (Japan)
(Accepted April 2Oth, 1992)
We investigated the effects of acute and repeated alcohol ingestion on plasma levels of hormones associated with the functioning of the hypothalamic-pituitary-gonadal (I-IPG) and hypothalamic-pituitary-adrenal (HPA) systems in normal males. In the first experiment, 7 normal male subjects were given ethanol (1.3 g/kg) in the form of a 43% alcohol solution of whiskey and water over a 30-min period (from 19:00 h to 19:30 h); blood samples were collected 30 min and immediately before the beginning of alcohol ingestion and then at intervals of 30 min for 180 min. Blood ethanol levels rose sharply and reached their maximum at 60 min, remaining above 1.0 mg/ml until 180 min. Prolactin levels increased, reaching a peak at 60 min, gradually returning to the initial value at 180 min. Decreased testosterone levels were observed only at 30 min. Luteinizing hormone (LH), adrenocorticotrophic hormone (ACTH) and cortisol levels did not show any increases. In the second experiment, 9 normal males were given the same dose of alcohol, but this was given on 7 consecutive evenings and the hormonal changes were examined on the 1st and 7th days, only at 30 and 60 min after alcohol ingestion began (during the period that blood ethanol levels were ascending to their peak). The results on the 1st day reconfirmed the findings in the first experiment and on the 7th day, the last alcohol ingestion produced increases in prolactin levels and decreases in testosterone levels at 30 and 60 min, but did not change other hormone levels. The present findings indicate that (1) acute alcohol ingestion produces increases in plasma prolactin levels and decreases in plasma testosterone levels; (2) these changes in hormone levels occur during the period when blood ethanol levels are ascending, but hormone levels return to their respective initial values even when the level of blood ethanol remains high (above 1.0 mg/ml); (3) increases in prolactin levels appear to be induced by the pharmacological action of alcohol, but not by non-specific factors such as stress; (4) alcohol may reduce testosterone levels by acting on peripheral sites, but not by suppressing LH secretion; (5) alcohol at doses sufficient to affect plasma prolactin and testosterone levels does not produce any increases in plasma ACTH and cortisol levels; (6) repeated alcohol ingestion over 7 consecutive evenings did not lead to the development of any tolerance with regard to these hormonal changes. The present study thus suggests that acute and repeated alcohol ingestion in normal males affects the plasma levels of hormones associated with the functioning of the HPG system, but not those associated with the functioning of the HPA system. Key words: alcohol; prolactin;
luteinizing hormone; testosterone;
Introduction It is well established that long-term alcohol consumption induces endocrine abnormalities, especially in the hypothalamic-pituitary-gonadal Correspondence to: Yoshishige Ida, Department of Neuropsychiatry, Kurume University School of Medicine, Kurume 830, Japan. 0376-8716/92/$05.00 0 1992 Elsevier Scientific Publishers Printed and Published in Ireland
adrenocorticotrophic
hormone; cortisol
(HPG) and hypothalamic-pituitary-adrenal(HPA) systems (Ylikahri et al., 1980; Noth and Walter, 1984). Hypogonadism in alcoholics is thought to be partly due to the dysfunction of the HPG system as characterized by decreased testosterone levels, which are accompanied by increased LH levels (Lester and Van Thiel, 1977; Van Thiel et al., 1978; Castilla-Garcia et al., 1987). Prolactin has a minor inhibitory effect on Ireland Ltd.
58
the HPG system (Findling and Tyrrell, 1991) and its elevation in alcoholics may be partly associated with clinical manifestations of hypogonadism (Van Thiel et al., 1975; Lester and Van Thiel, 1977). The hypothalamic neural mechanism that results in increased prolactin levels and its relationship with psychiatric and somatic alcohol-withdrawal symptoms have also been investigated (Borg and Weinholdt, 1982; Miller et al., 1986). Another line of investigation has found hyperactivity of the HPA system during the withdrawal period in some groups of alcoholics, as indicated by findings of hypercortisolism (Burov et al., 1986; Holsboer et al., 1987; Risher-Flowers et al., 1988; Adinoff et al., 1991), negative dexamethasone suppression tests (Burov et al., 1986), and blunted responses of ACTH to CRH challenge test (Holsboer et al., 1987). The hypercortisolism has been suggested to be related to anxiety and dysphoria (Burov et al., 1986) or to the degree of severity of the withdrawal syndrome (Adinoff et al., 1991). However, to our knowledge, there have been few reports of simultaneous investigations into dysfunctions of the HPG and HPA systems induced by long-term alcohol consumption, and therefore, it is not known which of these systems is more vulnerable to the toxic effects of alcohol. Many studies have shown that acute and short-term alcohol ingestion in normal subjects causes similar hormonal changes to those observed in alcoholics, i.e., decreased testosterone (Gordon et al., 1976; Mendelson et al., 1977; Gordon et al., 1978), increased prolactin (Ellingboe et al., 1980) and increased cortisol (Fazekas 1966; Jenkins and Connolly, 1968 Merry and Marks, 1969; Bellet et al., 1970; Farmer and Fabre, 1975; Ylikahri et al., 1978), although these changes are not so continuous or severe. However, some of these findings in normal subjects are conflicting and are currently the subject of discussion. Accordingly, the present study was undertaken to investigate the effects of acute and repeated alcohol ingestion on plasma levels of hormones associated with the functioning of the HPG and HPA systems in normal male subjects. In the first experiment,
we examined the effects of acute alcohol ingestion on plasma prolactin, LH, testosterone, ACTH and cortisol levels by concomitant measurement of these hormone levels and blood ethanol levels. In the second experiment, we examined the effects of repeated alcohol ingestion (over 7 consecutive evenings) on these hormone levels. Subjects and Methods Subjects Adult male volunteers gave their informed consent to participate in these studies. The subjects in the first experiment were seven healthy men, aged 26- 37 years and weighing 62 - 75 kg, and those in the second experiment were 9 healthy men, aged 22 -27 years and weighing 57 - 68 kg. None of the subjects had any past or current history of alcohol or drug abuse or mental or physical illness. They were ‘social drinkers’ whose weekly consumption of alcohol did not exceed 80 g; they had not consumed alcohol (ethanol) for at least 3 days prior to the beginning of the study. The weight of the volunteers remained unchanged during the study. Procedures Taking into account the circadian rhythm of the endocrine systems, the present study was performed in the evening, when plasma hormone levels are relatively low and stable (Findling and Tyrrell, 1991). In the first experiment, after they had fasted for 6 h, all subjects were given ethanol (1.3 g/kg body wt.), in the form of a 43% alcohol solution of whiskey and water, over a 30-min period from 19:00 h to 19:30 h. An indwelling intravenous catheter (inserted into an antecubital vein at 18:00 h) was kept open with heparinized saline solution. Blood samples were collected 30 min and immediately before the beginning of alcohol ingestion and then at 30-min intervals for 180 min. In the second experiment, alcohol at the same dose and using the same time schedule was given on 7 consecutive evenings. On the first and seventh days, blood samples were collected at 18:30 h,
59
19:00 h, 19:30 h and 20:00 h. During the experimental period, all subjects stayed in our university hospital and their mental and physical conditions were carefully monitored by two medical physicians. Assays Blood ethanol levels were determined using a gas chromatographic method (Yamano et al., 1968); the detection limit of ethanol was 0.1 mg/ml. Plasma hormone levels were determined commercial radioimmunoassay kits with (Mitsubishi-Yuka Biochemical Laboratory Inc., Tokyo). The detection limits were: prolactin, 0.5 ng/ml; LH, 0.2 mIU/ml; testosterone, 12.5 pgltube; ACTH, 3.2 pg/ml; cortisol, 0.23 pgldl. The intra-assay and inter-assay coefficients of variation were: prolactin, 4% and 2%, respectively; LH, 4% and 7%; testosterone, 8% and 6%; ACTH, 5% and 6%; cortisol, 4% and 6%. Statistical analysis All statistical comparisons were made between values at 0 min and values at each time point, using Student’s paired t-test. Results The results of the first experiment are shown in Figs. 1, 2 and 3. After alcohol ingestion began, blood ethanol levels rose sharply, reaching a peak (1.27 + 0.11 (S.E.M.) mglml) at 60 min, and remaining at above 1.0 mg/ml until 180 min. Plasma prolactin levels, which showed significant elevation from 30 to 150 min after the beginning of alcohol ingestion, reached a maximum at 60 min and gradually returned to the normal initial value at 180 min (Fig. 1). Plasma LH levels showed a slight tendency to increase from 30 to 60 min, but did not show significant changes at any time points when compared with the initial value (Fig. 2). Plasma testosterone levels decreased significantly at 30 min after alcohol ingestion began, but did not show a significant difference from the initial value at 60 min (Fig. 2). Plasma ACTH and cortisol levels showed a slight tendency to decrease, and ACTH levels at 120 and 150 min and cor-
tisol levels at 150 and 180 min showed significant differences from their respective initial values (Fig. 3). All subjects displayed a moderate degree of intoxication after alcohol ingestion during the experiment, but none had complaints of dysphoria or gastrointestinal symptoms such as nausea and vomiting. However, about 20 min after the last blood samples were collected, one subject indicated that he felt discomfort and nausea and he vomited. We collected an additional sample from him to examine the effect of non-specific physical stress. Some of his plasma hormone levels were remarkably elevated, compared with levels at 180 min (values at 180 min vs. values after vomiting: prolactin 4.4 vs. 13.5 (ng/ml); LH 3.3 vs. 6.4 (mIU/ml); testosterone 5.1 vs 4.4 (ng/ml); ACTH 24 vs. 58 (pg/ml) and cortisol4.1 vs. 20.2 (lLg/dl)). The results of the second experiment are shown in Figs. 4, 5, and 6. On the first and seventh experimental days, blood ethanol levels rose sharply, reaching levels above 1.0 mg/ml. Significant increases in plasma prolactin levels were observed at 30 and 60 min after alcohol ingestion began on the first and last days (Fig. 4). The first and last alcohol ingestion had no significant effect on plasma LH levels when compared with the initial values (Fig. 5). On the first day, plasma testosterone levels were significantly decreased at 60 min after alcohol ingestion began and, on the seventh day, these levels were significantly decreased at both 30 and 60 min (Fig. 5). Plasma ACTH and cortisol levels were not changed by acute or repeated alcohol ingestion (Fig. 6). During the ‘I-day experiment, none of subjects were dysphoric or had gastrointestinal symptoms of alcohol ingestion. Discussion Both experiments revealed that acute alcohol ingestion produced increases in prolactin levels and decreases in testosterone levels, while no increases were shown in LH, ACTH and cortisol levels. The later decreases in ACTH and cortisol levels observed 2 h and more after alcohol
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Fig. 5. Changes in plasma LH and testosterone levels after repeated alcohol ingestion. Alcohol (1.3 g/kg ethanol) was given over a 30-min period, from 19:00 h to 19:30 h, on 7 consecutive days. The results of the 1st and 7th experimental days are shown. Each value indicates the mean * S.E.M. of 9 subjects. Significantly different from the values at 0 min on the respective experimental days; **P < 0.01.
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ingestion in the first experiment were probably a reflection of the circadian rhythm of HPA activity, in which plasma ACTH and cortisol levels are highest early in the morning and lowest late in the evening (Findling and Tyrrell, 1991). In the second experiment, the hormonal changes induced by acute alcohol ingestion were also observed after the last ingestion on the 7th day. The present findings suggest that alcohol affects plasma levels of hormones associated with the functioning of the HPG system rather than the HPA system. The findings also suggest that repeated alcohol ingestion over a 7-day period does not lead to the development of tolerance with regard to either the increase of prolactin levels or the decrease of testosterone levels. In the first experiment, prolactin levels reached their maximum at maximum blood ethanol levels, but returned to the initial value even when blood ethanol remained at levels above 1.0 mg/ml. A decrease of testosterone levels was observed at only one time point during the ascending period of the blood ethanol curve. In the second experiment, however, we found that this decrease continued until 60 min, when blood ethanol levels probably reached maximum. These results are virtually in agreement with previous findings, which showed that after ingestion of alcohol at 1.0 g/kg over a 15 minperiod, plasma prolactin and testosterone levels began to change during the ascending period of the blood ethanol curve, being most elevated and depressed, respectively, at peak blood ethanol levels above 1.0 mglml (Mendelson et al., 1977; Ellingboe et al., 1980). In other studies, no changes in either prolactin or testosterone levels were observed after acute ingestion of alcohol at 1.5 glkg over a 3-h period (Ylikahri et al., 1978; Ylikahri et al., 1980). Taken together with these studies, the present findings suggest that changes in plasma prolactin and testosterone levels may occur following rapid and sufficient elevation of blood ethanol levels during a short period of alcohol consumption, probably within 30 min; however, such hormonal changes may recover even when blood ethanol is sustained at high levels. Prolactin secretion is predominantly under
the inhibitory control of the tuberoinfundibular dopamine (TIF-DA) system in the hypothalamus and although prolactin does not appear to play a physiologic role in the regulation of gonadal function, hyperprolactinemia leads to hypogonadism (Findling and Tyrrel, 1991). The exact mechanisms of prolactin inhibition of gonadal functions are unclear, but the principal one appears to be an inhibitory effect on LH-RH secretion from the hypothalamus, produced by activation of the TIF-DA system via a feedback mechanism (Findling and Tyrrel, 1991). Therefore, the increased prolactin levels observed in alcoholics could be partly associated with hypogonadism (Van Thiel et al., 1975; Lester and Van Thiel, 1977). The authors of a previous study (Borg and Weinholdt, 1982), found that the Ds-receptor agonist, bromocriptine, had therapeutic effects on psychiatric and somatic alcohol-withdrawal symptoms, as well as suppressing elevated prolactin levels, in alcoholic patients, proposed the hypothesis that chronic alcohol consumption may be, at least partly, associated with a dysfunction of hypothalamic DA transmission. We recently found that with bromocriptine blocked pretreatment alcohol-induced increases in plasma prolactin levels in normal males (unpublished data). Thus, the present findings indicate one possible mechanism responsible for increased prolactin levels; alcohol might inhibit the release of hypothalamic DA to the hypophyseal portal system, thus resulting in these elevated levels. We observed that when one subject had toxic physical symptoms (discomfort, nausea, and vomiting), his prolactin levels, as well as ACTH and cortisol levels, were remarkably increased. Since all these hormones are stress parameters, the toxic symptoms were probably stressful for this subject. In the present study, decreased testosterone levels were not accompanied by significant changes in LH levels, although, in the first experiment, we observed a slight tendency for these latter levels to increase. These findings suggest that the major effect of alcohol on plasma testosterone in humans is exerted at a peripheral (testis) rather than central
63
(hypothalamus-pituitary) site. This suggestion is supported by previous findings, which showed that acute alcohol administration to normal subjects produced not only decreases in plasma testosterone levels, but also increases in plasma LH levels (Mendelson et al., 1977; Gordon et al., 1978) and that decreased testosterone levels were accompanied by increased LH levels in alcoholics (Lester and Van Thiel, 1977; Van Thiel et al., 1978). The present finding of a slight tendency to increased LH levels may be explained thus: the magnitude andlor continuation of the reduction in testosterone levels might not be sufficient to stimulate LH secretion via a longloop feedback mechanism. Decreased plasma testosterone levels seem to be associated with the acute toxic effects of alcohol on the peripheral mechanism which regulates the and/or biotransformation of biosynthesis testosterone, as indicated by previous studies (Van Thiel, 1984). Hyperactivity of the HPA system has been demonstrated during the withdrawal period in some groups of alcoholics, as indicated by convincing evidence of hypercortisolism (Burov et al., 1986; Holsboer et al., 1987; Risher-Flowers et al., 1988; Adinoff et al., 1991), abnormal suppression of plasma cortisol after dexamethasone treatment (Burov et al., 1986) and the blunted ACTH response to the CRH challenge test (Holsboer et al., 1987). However, it remains unclear whether the hypercortisolism was a result of the long-term activation effects of alcohol on the HPA system or of non-specific stress effects induced by severe alcoholwithdrawal symptoms, or both. Many studies have investigated whether alcohol has a direct activation effect on the HPA system in normal subjects; however, the results seem to be conflicting. In these studies, alcohol was administered at doses which resulted in blood ethanol levels in excess of 1.0 mg/ml (Fazekas, 1966; Jenkins and Connolly, 1968; Merry and Marks, 1969; Bellet et al., 1970; Farmer and Fabre, 1975; Ylikahri et al., 1978). Some of these studies found that the elevation of plasma cortisol showed parallel changes with the blood ethanol curve (Fazekas, 1966; Jenkins and Con-
nolly, 1968; Belle et al., 1970), whereas others reported that cortisol began to increase during the period of descending blood ethanol levels and continued to be obviously elevated even when blood ethanol levels were undetectable (Farmer and Fabre, 1975; Ylikahri et al., 1978). Other investigators (Merry and Marks, 1969) reported that increases in cortisol levels induced by acute administration were observed only in non-drinkers who had symptoms of alcohol intolerance (e.g., discomfort, vomiting and unconsciousness), but not in heavy drinkers or in alcoholic patients. It has also been shown that acute alcohol administration did not produce any elevation in ACTH or cortisol levels in normal subjects (Jeffcoate et al., 1980; Elias et al., 1982), while Hasselbalch et al. (1982) found that 15 male heavy drinkers with a daily alcohol intake of 100 g or more over a period of at least 10 years did not show any evidence of activation of the HPA system. In the present study, we did not find any increases in plasma ACTH or cortisol levels at any time points after acute or repeated administration of alcohol, even when the dose produced blood ethanol levels in excess of 1.0 mg/ml. Increased ACTH and cortisol levels, as well as prolactin levels, was observed in only 1 subject, when he had gastrointestinal symptoms and the increases were remarkable. Hypercortisolism was observed in alcoholwithdrawal patients characterized predominantly by anxiety and dysphoria (Burov et al., 1986). Thus, the present data suggest that alcohol by itself does not have a direct action in increasing either ACTH secretion from the pituitary or cortisol secretion from the adrenal glands, and that the elevation of cortisol may be associated with emotional andlor somatic disorders that arise during alcohol consumption and alcoholwithdrawal. The discrepancies between the present and some previous findings might be due, at least in part, to differences in the tolerance of subjects to acute alcohol intoxication. Further studies of the alcohol-withdrawal stage in normal subjects and alcoholic patients, which are now underway, may provide some additional explanations of the relationship between alcohol and HPA activity.
64
In conclusion, in normal subjects, both acute and repeated alcohol ingestion affected levels of plasma hormones associated with the functioning of the HPG system, but not those associated with the functioning of the HPA system. Thus, excessive alcohol consumption over a long period may have more toxic effects on the functioning of the HPG system than on the functioning of the HPA system. References Adinoff, B., Risher-Flowers, D., De Jong, J., Ravitz, B., Bone, G.H.A., Nutt, D.J., Roehrich, L., Martin, P.R. and Linnoila, M. (1991) Disturbances of hypothalamicpituitary-adrenal axis functioning during ethanol withdrawal in six men. Am. J. Psychiatr. 148, 1023 - 1025. Bellet, S., Roman, L., DeCastro, 0. and Herrera, M. (1970) Effect of acute ethanol intake on plasma 11 hydroxycorticosteroid levels. Metabolism 19, 664 - 667. Borg, V. and Weinholdt, T. (1982) Bromocriptine in the treatment of the alcohol-withdrawal syndrome. Acta Psychiatr. &and. 65, 101- 111. Burov, Y.V., Treskov, V.G., Vedernikova, N.N. and Shevelyova, O.S. (1986) Types of alcohol withdrawal syndrome and dexamethasone suppression test. Drug Alcohol Depend. 1’7, 81-88. Castilla-Garcia, A., Santolaria-Fernandez, F.J., GonzalezReimers, C.E., Batista-Lopez, N., Gonzalez-Garcia, C., Jorge-Hernandez, J.A. and Hernandez-Nieto, L. (1987) Alcohol-induced hypogonadism: reversal after ethanol withdrawal. Drug Alcohol Depend. 20, 255 - 260. Elias, A.N., Meshkinpour, H., Valenta, L.J. and Grossman, M.K. (1982) Pseudo-Cushing’s syndrome: the role of alcohol. J. Clin. Gastroenterol. 4, 137 - 139. Ellingboe, J., Mendelson, J.H., Kuehnle, J.C., Skupny, A.S.T. and Miller, K.D. (1980) Effect of acute ethanol ingestion on integrated plasma prolactin levels in normal men. Pharmacol. Biochem. Behav. 12, 297-301. Farmer, R.W. and Fabre, L.F.Jr. (1975) Some endocrine aspects of alcoholism. In: Biochemical Pharmacology of Ethanol: Advances in Experimental Medicine and Biology Series. (Majchrowicz, E., ed.), vol. 56, pp. 277-289. Plenum Press, New York. Fazekas, LG. (1966) Hydrocortisone content of human blood and alcohol content of blood and urine, after wine consumption. Q. J. Stud. Alcohol. 27, 439-446. Findling, J.W. and Tyrrell, J.B. (1991) Anterior pituitary gland. In: Basic and Clinical Endocrinology: Third Edition (Greespan, F.S., ed.), pp. 79- 132, Prentice-Hall International Inc., New York. Gordon, G.G., Altman, K., Southren, A.L., Rubin, E. and Lieber, C.S. (1976) Effect of alcohol (ethanol) administration on sex-hormone metabolism in normal men. New Engl. J. Med. 295, 793-797. Gordon, G.G., Southren, A.L. and Lieber, C.S. (1978) The
effects of alcoholic liver disease and alcohol ingestion on sex hormone levels. Alcohol.: Clin. Exp. Res. 2, 259 - 263. Hasselbalch, H., Selmer, J., Sestoft, L. and Kehlet, H. (1982) Hypothalamic-pituitary-adrenocortical function in chronic alcoholism. Clin. Endocrinol. 16, 73 - 76. Holsboer, F., von Bardeleben, U., Buller, R., Heuser, I. and Steiger, A. (1987) Stimulation response to corticotropinreleasing hormone (CRH) in patients with depression, alcoholism and panic disorder. Horm. Metab. Res. 16, 80-88. Jeffcoate, W.J., Platts, P., Ridout, M., Hastings, A.G., MacDonald, I. and Selby, C. (1980) Endocrine effects of ethanol infusion in normal subjects: modification by naloxone. Pharmacol. Biochem. Behav. 13, 145 - 148. Jenkins, J.S. and Connolly, J. (1968) Adrenocortical response to ethanol in man. Br. Med. J. 2, 804-805. Lester, R. and Van Thiel, D.H. (1977) Gonadal function in alcoholic men. Adv. Exp. Biol. Med. 85A, 399-414. Mendelson, J.H., Mello, N.K. and Ellingboe, J. (1977) Effects of acute alcohol intake on pituitary-gonadal hormones in normal human males. J. Pharmacol. Exp. Ther. 202, 676 - 682. Merry, J. and Marks, V. (1969) Plasma-hydrocortisone response to ethanol in chronic alcoholics. Lancet i, 921-923. Miller, F., Barasch, A., Sacks, M., Levitan, J. and Ashcroft, L. (1986) Serum prolactin correlates with depressive mood during alcohol withdrawal. Drug Alcohol Depend. 17, 331-338. North, R.H. and Walter, R.M. (1984) The effects of alcohol on the endocrine system. Med. Clin. North Am. 68, 133 - 146. Risher-Flowers, D., Adinoff, B., Ravitz, B., Bone, G.H.A. Martin, P.R., Nutt, D. and Linnoila, M. (1988) Circadian rhythms of cortisol during alcohol withdrawal. Adv. Alcohol Subst. Abuse 7, 37-41. Van Thiel, D.H., Gavaler, J.S., Lester, R., Loriaux, D.L. and Braunstein, G.D. (1975) Plasma estrone, prolactin, neurophysin, and sex steroid-binding globulin in chronic alcoholic men. Metabolism 24, 1015 - 1019. Van Thiel, D.H., Lester, R. and Vaitukaitis, J. (1978) Evidence for a defect in pituitary secretion of luteinizing hormone in chronic alcoholic men. J. Clin. Endcrinol. Metab. 47, 499-507. Van Thiel, D.H. (1984) Ethyl alcohol and gonadal function. Hosp. Pratt. Novemeber 152- 158. Yamano, H., Fukuda, K. and Matsumoto, F. (1968) Studies on blood alcohol-development of gas chromatographic method and relationship between serum alcohol levels and whole blood level. Jpn. J. Stud. Alcohol 3, 144- 149. Ylikahri, R.H., Huttunen, M.O., Harkonen, M., Leino, T., Helenius, T., Liewendahl, K and Karonen, S. (1978) Acute effects of alcohol on anterior pituitary secretion of the tropic hormones. J. Clin. Endocrinol. Metab. 46, 715 - 720. Ylikahri, R.H., Huttunen, M.O. and Harkonen, M. (1980) Hormonal changes during alcohol intoxication and withdrawal. Pharmacol. Biochem. Behav. 13, 131- 137.
Elsevier Scientific Publishers
31(1992) 57-64 Ireland Ltd.
57
Effects of acute and repeated alcohol ingestion on hypothalamic-pituitary-gonadal and hypothalamic-pituitaryadrenal functioning in normal males Yoshishige
Ida, Shusaku Tsujimaru, Katsura Nakamaura, Ishou Shirao, Hirotaka Mukasa, Hideaki Egami and Yoichi Nakazawa Department
of Neuropsychiatry,
Kurum.e
University School of Medicine,
Kurum
880 (Japan)
(Accepted April 2Oth, 1992)
We investigated the effects of acute and repeated alcohol ingestion on plasma levels of hormones associated with the functioning of the hypothalamic-pituitary-gonadal (I-IPG) and hypothalamic-pituitary-adrenal (HPA) systems in normal males. In the first experiment, 7 normal male subjects were given ethanol (1.3 g/kg) in the form of a 43% alcohol solution of whiskey and water over a 30-min period (from 19:00 h to 19:30 h); blood samples were collected 30 min and immediately before the beginning of alcohol ingestion and then at intervals of 30 min for 180 min. Blood ethanol levels rose sharply and reached their maximum at 60 min, remaining above 1.0 mg/ml until 180 min. Prolactin levels increased, reaching a peak at 60 min, gradually returning to the initial value at 180 min. Decreased testosterone levels were observed only at 30 min. Luteinizing hormone (LH), adrenocorticotrophic hormone (ACTH) and cortisol levels did not show any increases. In the second experiment, 9 normal males were given the same dose of alcohol, but this was given on 7 consecutive evenings and the hormonal changes were examined on the 1st and 7th days, only at 30 and 60 min after alcohol ingestion began (during the period that blood ethanol levels were ascending to their peak). The results on the 1st day reconfirmed the findings in the first experiment and on the 7th day, the last alcohol ingestion produced increases in prolactin levels and decreases in testosterone levels at 30 and 60 min, but did not change other hormone levels. The present findings indicate that (1) acute alcohol ingestion produces increases in plasma prolactin levels and decreases in plasma testosterone levels; (2) these changes in hormone levels occur during the period when blood ethanol levels are ascending, but hormone levels return to their respective initial values even when the level of blood ethanol remains high (above 1.0 mg/ml); (3) increases in prolactin levels appear to be induced by the pharmacological action of alcohol, but not by non-specific factors such as stress; (4) alcohol may reduce testosterone levels by acting on peripheral sites, but not by suppressing LH secretion; (5) alcohol at doses sufficient to affect plasma prolactin and testosterone levels does not produce any increases in plasma ACTH and cortisol levels; (6) repeated alcohol ingestion over 7 consecutive evenings did not lead to the development of any tolerance with regard to these hormonal changes. The present study thus suggests that acute and repeated alcohol ingestion in normal males affects the plasma levels of hormones associated with the functioning of the HPG system, but not those associated with the functioning of the HPA system. Key words: alcohol; prolactin;
luteinizing hormone; testosterone;
Introduction It is well established that long-term alcohol consumption induces endocrine abnormalities, especially in the hypothalamic-pituitary-gonadal Correspondence to: Yoshishige Ida, Department of Neuropsychiatry, Kurume University School of Medicine, Kurume 830, Japan. 0376-8716/92/$05.00 0 1992 Elsevier Scientific Publishers Printed and Published in Ireland
adrenocorticotrophic
hormone; cortisol
(HPG) and hypothalamic-pituitary-adrenal(HPA) systems (Ylikahri et al., 1980; Noth and Walter, 1984). Hypogonadism in alcoholics is thought to be partly due to the dysfunction of the HPG system as characterized by decreased testosterone levels, which are accompanied by increased LH levels (Lester and Van Thiel, 1977; Van Thiel et al., 1978; Castilla-Garcia et al., 1987). Prolactin has a minor inhibitory effect on Ireland Ltd.
58
the HPG system (Findling and Tyrrell, 1991) and its elevation in alcoholics may be partly associated with clinical manifestations of hypogonadism (Van Thiel et al., 1975; Lester and Van Thiel, 1977). The hypothalamic neural mechanism that results in increased prolactin levels and its relationship with psychiatric and somatic alcohol-withdrawal symptoms have also been investigated (Borg and Weinholdt, 1982; Miller et al., 1986). Another line of investigation has found hyperactivity of the HPA system during the withdrawal period in some groups of alcoholics, as indicated by findings of hypercortisolism (Burov et al., 1986; Holsboer et al., 1987; Risher-Flowers et al., 1988; Adinoff et al., 1991), negative dexamethasone suppression tests (Burov et al., 1986), and blunted responses of ACTH to CRH challenge test (Holsboer et al., 1987). The hypercortisolism has been suggested to be related to anxiety and dysphoria (Burov et al., 1986) or to the degree of severity of the withdrawal syndrome (Adinoff et al., 1991). However, to our knowledge, there have been few reports of simultaneous investigations into dysfunctions of the HPG and HPA systems induced by long-term alcohol consumption, and therefore, it is not known which of these systems is more vulnerable to the toxic effects of alcohol. Many studies have shown that acute and short-term alcohol ingestion in normal subjects causes similar hormonal changes to those observed in alcoholics, i.e., decreased testosterone (Gordon et al., 1976; Mendelson et al., 1977; Gordon et al., 1978), increased prolactin (Ellingboe et al., 1980) and increased cortisol (Fazekas 1966; Jenkins and Connolly, 1968 Merry and Marks, 1969; Bellet et al., 1970; Farmer and Fabre, 1975; Ylikahri et al., 1978), although these changes are not so continuous or severe. However, some of these findings in normal subjects are conflicting and are currently the subject of discussion. Accordingly, the present study was undertaken to investigate the effects of acute and repeated alcohol ingestion on plasma levels of hormones associated with the functioning of the HPG and HPA systems in normal male subjects. In the first experiment,
we examined the effects of acute alcohol ingestion on plasma prolactin, LH, testosterone, ACTH and cortisol levels by concomitant measurement of these hormone levels and blood ethanol levels. In the second experiment, we examined the effects of repeated alcohol ingestion (over 7 consecutive evenings) on these hormone levels. Subjects and Methods Subjects Adult male volunteers gave their informed consent to participate in these studies. The subjects in the first experiment were seven healthy men, aged 26- 37 years and weighing 62 - 75 kg, and those in the second experiment were 9 healthy men, aged 22 -27 years and weighing 57 - 68 kg. None of the subjects had any past or current history of alcohol or drug abuse or mental or physical illness. They were ‘social drinkers’ whose weekly consumption of alcohol did not exceed 80 g; they had not consumed alcohol (ethanol) for at least 3 days prior to the beginning of the study. The weight of the volunteers remained unchanged during the study. Procedures Taking into account the circadian rhythm of the endocrine systems, the present study was performed in the evening, when plasma hormone levels are relatively low and stable (Findling and Tyrrell, 1991). In the first experiment, after they had fasted for 6 h, all subjects were given ethanol (1.3 g/kg body wt.), in the form of a 43% alcohol solution of whiskey and water, over a 30-min period from 19:00 h to 19:30 h. An indwelling intravenous catheter (inserted into an antecubital vein at 18:00 h) was kept open with heparinized saline solution. Blood samples were collected 30 min and immediately before the beginning of alcohol ingestion and then at 30-min intervals for 180 min. In the second experiment, alcohol at the same dose and using the same time schedule was given on 7 consecutive evenings. On the first and seventh days, blood samples were collected at 18:30 h,
59
19:00 h, 19:30 h and 20:00 h. During the experimental period, all subjects stayed in our university hospital and their mental and physical conditions were carefully monitored by two medical physicians. Assays Blood ethanol levels were determined using a gas chromatographic method (Yamano et al., 1968); the detection limit of ethanol was 0.1 mg/ml. Plasma hormone levels were determined commercial radioimmunoassay kits with (Mitsubishi-Yuka Biochemical Laboratory Inc., Tokyo). The detection limits were: prolactin, 0.5 ng/ml; LH, 0.2 mIU/ml; testosterone, 12.5 pgltube; ACTH, 3.2 pg/ml; cortisol, 0.23 pgldl. The intra-assay and inter-assay coefficients of variation were: prolactin, 4% and 2%, respectively; LH, 4% and 7%; testosterone, 8% and 6%; ACTH, 5% and 6%; cortisol, 4% and 6%. Statistical analysis All statistical comparisons were made between values at 0 min and values at each time point, using Student’s paired t-test. Results The results of the first experiment are shown in Figs. 1, 2 and 3. After alcohol ingestion began, blood ethanol levels rose sharply, reaching a peak (1.27 + 0.11 (S.E.M.) mglml) at 60 min, and remaining at above 1.0 mg/ml until 180 min. Plasma prolactin levels, which showed significant elevation from 30 to 150 min after the beginning of alcohol ingestion, reached a maximum at 60 min and gradually returned to the normal initial value at 180 min (Fig. 1). Plasma LH levels showed a slight tendency to increase from 30 to 60 min, but did not show significant changes at any time points when compared with the initial value (Fig. 2). Plasma testosterone levels decreased significantly at 30 min after alcohol ingestion began, but did not show a significant difference from the initial value at 60 min (Fig. 2). Plasma ACTH and cortisol levels showed a slight tendency to decrease, and ACTH levels at 120 and 150 min and cor-
tisol levels at 150 and 180 min showed significant differences from their respective initial values (Fig. 3). All subjects displayed a moderate degree of intoxication after alcohol ingestion during the experiment, but none had complaints of dysphoria or gastrointestinal symptoms such as nausea and vomiting. However, about 20 min after the last blood samples were collected, one subject indicated that he felt discomfort and nausea and he vomited. We collected an additional sample from him to examine the effect of non-specific physical stress. Some of his plasma hormone levels were remarkably elevated, compared with levels at 180 min (values at 180 min vs. values after vomiting: prolactin 4.4 vs. 13.5 (ng/ml); LH 3.3 vs. 6.4 (mIU/ml); testosterone 5.1 vs 4.4 (ng/ml); ACTH 24 vs. 58 (pg/ml) and cortisol4.1 vs. 20.2 (lLg/dl)). The results of the second experiment are shown in Figs. 4, 5, and 6. On the first and seventh experimental days, blood ethanol levels rose sharply, reaching levels above 1.0 mg/ml. Significant increases in plasma prolactin levels were observed at 30 and 60 min after alcohol ingestion began on the first and last days (Fig. 4). The first and last alcohol ingestion had no significant effect on plasma LH levels when compared with the initial values (Fig. 5). On the first day, plasma testosterone levels were significantly decreased at 60 min after alcohol ingestion began and, on the seventh day, these levels were significantly decreased at both 30 and 60 min (Fig. 5). Plasma ACTH and cortisol levels were not changed by acute or repeated alcohol ingestion (Fig. 6). During the ‘I-day experiment, none of subjects were dysphoric or had gastrointestinal symptoms of alcohol ingestion. Discussion Both experiments revealed that acute alcohol ingestion produced increases in prolactin levels and decreases in testosterone levels, while no increases were shown in LH, ACTH and cortisol levels. The later decreases in ACTH and cortisol levels observed 2 h and more after alcohol
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ingestion in the first experiment were probably a reflection of the circadian rhythm of HPA activity, in which plasma ACTH and cortisol levels are highest early in the morning and lowest late in the evening (Findling and Tyrrell, 1991). In the second experiment, the hormonal changes induced by acute alcohol ingestion were also observed after the last ingestion on the 7th day. The present findings suggest that alcohol affects plasma levels of hormones associated with the functioning of the HPG system rather than the HPA system. The findings also suggest that repeated alcohol ingestion over a 7-day period does not lead to the development of tolerance with regard to either the increase of prolactin levels or the decrease of testosterone levels. In the first experiment, prolactin levels reached their maximum at maximum blood ethanol levels, but returned to the initial value even when blood ethanol remained at levels above 1.0 mg/ml. A decrease of testosterone levels was observed at only one time point during the ascending period of the blood ethanol curve. In the second experiment, however, we found that this decrease continued until 60 min, when blood ethanol levels probably reached maximum. These results are virtually in agreement with previous findings, which showed that after ingestion of alcohol at 1.0 g/kg over a 15 minperiod, plasma prolactin and testosterone levels began to change during the ascending period of the blood ethanol curve, being most elevated and depressed, respectively, at peak blood ethanol levels above 1.0 mglml (Mendelson et al., 1977; Ellingboe et al., 1980). In other studies, no changes in either prolactin or testosterone levels were observed after acute ingestion of alcohol at 1.5 glkg over a 3-h period (Ylikahri et al., 1978; Ylikahri et al., 1980). Taken together with these studies, the present findings suggest that changes in plasma prolactin and testosterone levels may occur following rapid and sufficient elevation of blood ethanol levels during a short period of alcohol consumption, probably within 30 min; however, such hormonal changes may recover even when blood ethanol is sustained at high levels. Prolactin secretion is predominantly under
the inhibitory control of the tuberoinfundibular dopamine (TIF-DA) system in the hypothalamus and although prolactin does not appear to play a physiologic role in the regulation of gonadal function, hyperprolactinemia leads to hypogonadism (Findling and Tyrrel, 1991). The exact mechanisms of prolactin inhibition of gonadal functions are unclear, but the principal one appears to be an inhibitory effect on LH-RH secretion from the hypothalamus, produced by activation of the TIF-DA system via a feedback mechanism (Findling and Tyrrel, 1991). Therefore, the increased prolactin levels observed in alcoholics could be partly associated with hypogonadism (Van Thiel et al., 1975; Lester and Van Thiel, 1977). The authors of a previous study (Borg and Weinholdt, 1982), found that the Ds-receptor agonist, bromocriptine, had therapeutic effects on psychiatric and somatic alcohol-withdrawal symptoms, as well as suppressing elevated prolactin levels, in alcoholic patients, proposed the hypothesis that chronic alcohol consumption may be, at least partly, associated with a dysfunction of hypothalamic DA transmission. We recently found that with bromocriptine blocked pretreatment alcohol-induced increases in plasma prolactin levels in normal males (unpublished data). Thus, the present findings indicate one possible mechanism responsible for increased prolactin levels; alcohol might inhibit the release of hypothalamic DA to the hypophyseal portal system, thus resulting in these elevated levels. We observed that when one subject had toxic physical symptoms (discomfort, nausea, and vomiting), his prolactin levels, as well as ACTH and cortisol levels, were remarkably increased. Since all these hormones are stress parameters, the toxic symptoms were probably stressful for this subject. In the present study, decreased testosterone levels were not accompanied by significant changes in LH levels, although, in the first experiment, we observed a slight tendency for these latter levels to increase. These findings suggest that the major effect of alcohol on plasma testosterone in humans is exerted at a peripheral (testis) rather than central
63
(hypothalamus-pituitary) site. This suggestion is supported by previous findings, which showed that acute alcohol administration to normal subjects produced not only decreases in plasma testosterone levels, but also increases in plasma LH levels (Mendelson et al., 1977; Gordon et al., 1978) and that decreased testosterone levels were accompanied by increased LH levels in alcoholics (Lester and Van Thiel, 1977; Van Thiel et al., 1978). The present finding of a slight tendency to increased LH levels may be explained thus: the magnitude andlor continuation of the reduction in testosterone levels might not be sufficient to stimulate LH secretion via a longloop feedback mechanism. Decreased plasma testosterone levels seem to be associated with the acute toxic effects of alcohol on the peripheral mechanism which regulates the and/or biotransformation of biosynthesis testosterone, as indicated by previous studies (Van Thiel, 1984). Hyperactivity of the HPA system has been demonstrated during the withdrawal period in some groups of alcoholics, as indicated by convincing evidence of hypercortisolism (Burov et al., 1986; Holsboer et al., 1987; Risher-Flowers et al., 1988; Adinoff et al., 1991), abnormal suppression of plasma cortisol after dexamethasone treatment (Burov et al., 1986) and the blunted ACTH response to the CRH challenge test (Holsboer et al., 1987). However, it remains unclear whether the hypercortisolism was a result of the long-term activation effects of alcohol on the HPA system or of non-specific stress effects induced by severe alcoholwithdrawal symptoms, or both. Many studies have investigated whether alcohol has a direct activation effect on the HPA system in normal subjects; however, the results seem to be conflicting. In these studies, alcohol was administered at doses which resulted in blood ethanol levels in excess of 1.0 mg/ml (Fazekas, 1966; Jenkins and Connolly, 1968; Merry and Marks, 1969; Bellet et al., 1970; Farmer and Fabre, 1975; Ylikahri et al., 1978). Some of these studies found that the elevation of plasma cortisol showed parallel changes with the blood ethanol curve (Fazekas, 1966; Jenkins and Con-
nolly, 1968; Belle et al., 1970), whereas others reported that cortisol began to increase during the period of descending blood ethanol levels and continued to be obviously elevated even when blood ethanol levels were undetectable (Farmer and Fabre, 1975; Ylikahri et al., 1978). Other investigators (Merry and Marks, 1969) reported that increases in cortisol levels induced by acute administration were observed only in non-drinkers who had symptoms of alcohol intolerance (e.g., discomfort, vomiting and unconsciousness), but not in heavy drinkers or in alcoholic patients. It has also been shown that acute alcohol administration did not produce any elevation in ACTH or cortisol levels in normal subjects (Jeffcoate et al., 1980; Elias et al., 1982), while Hasselbalch et al. (1982) found that 15 male heavy drinkers with a daily alcohol intake of 100 g or more over a period of at least 10 years did not show any evidence of activation of the HPA system. In the present study, we did not find any increases in plasma ACTH or cortisol levels at any time points after acute or repeated administration of alcohol, even when the dose produced blood ethanol levels in excess of 1.0 mg/ml. Increased ACTH and cortisol levels, as well as prolactin levels, was observed in only 1 subject, when he had gastrointestinal symptoms and the increases were remarkable. Hypercortisolism was observed in alcoholwithdrawal patients characterized predominantly by anxiety and dysphoria (Burov et al., 1986). Thus, the present data suggest that alcohol by itself does not have a direct action in increasing either ACTH secretion from the pituitary or cortisol secretion from the adrenal glands, and that the elevation of cortisol may be associated with emotional andlor somatic disorders that arise during alcohol consumption and alcoholwithdrawal. The discrepancies between the present and some previous findings might be due, at least in part, to differences in the tolerance of subjects to acute alcohol intoxication. Further studies of the alcohol-withdrawal stage in normal subjects and alcoholic patients, which are now underway, may provide some additional explanations of the relationship between alcohol and HPA activity.
64
In conclusion, in normal subjects, both acute and repeated alcohol ingestion affected levels of plasma hormones associated with the functioning of the HPG system, but not those associated with the functioning of the HPA system. Thus, excessive alcohol consumption over a long period may have more toxic effects on the functioning of the HPG system than on the functioning of the HPA system. References Adinoff, B., Risher-Flowers, D., De Jong, J., Ravitz, B., Bone, G.H.A., Nutt, D.J., Roehrich, L., Martin, P.R. and Linnoila, M. (1991) Disturbances of hypothalamicpituitary-adrenal axis functioning during ethanol withdrawal in six men. Am. J. Psychiatr. 148, 1023 - 1025. Bellet, S., Roman, L., DeCastro, 0. and Herrera, M. (1970) Effect of acute ethanol intake on plasma 11 hydroxycorticosteroid levels. Metabolism 19, 664 - 667. Borg, V. and Weinholdt, T. (1982) Bromocriptine in the treatment of the alcohol-withdrawal syndrome. Acta Psychiatr. &and. 65, 101- 111. Burov, Y.V., Treskov, V.G., Vedernikova, N.N. and Shevelyova, O.S. (1986) Types of alcohol withdrawal syndrome and dexamethasone suppression test. Drug Alcohol Depend. 1’7, 81-88. Castilla-Garcia, A., Santolaria-Fernandez, F.J., GonzalezReimers, C.E., Batista-Lopez, N., Gonzalez-Garcia, C., Jorge-Hernandez, J.A. and Hernandez-Nieto, L. (1987) Alcohol-induced hypogonadism: reversal after ethanol withdrawal. Drug Alcohol Depend. 20, 255 - 260. Elias, A.N., Meshkinpour, H., Valenta, L.J. and Grossman, M.K. (1982) Pseudo-Cushing’s syndrome: the role of alcohol. J. Clin. Gastroenterol. 4, 137 - 139. Ellingboe, J., Mendelson, J.H., Kuehnle, J.C., Skupny, A.S.T. and Miller, K.D. (1980) Effect of acute ethanol ingestion on integrated plasma prolactin levels in normal men. Pharmacol. Biochem. Behav. 12, 297-301. Farmer, R.W. and Fabre, L.F.Jr. (1975) Some endocrine aspects of alcoholism. In: Biochemical Pharmacology of Ethanol: Advances in Experimental Medicine and Biology Series. (Majchrowicz, E., ed.), vol. 56, pp. 277-289. Plenum Press, New York. Fazekas, LG. (1966) Hydrocortisone content of human blood and alcohol content of blood and urine, after wine consumption. Q. J. Stud. Alcohol. 27, 439-446. Findling, J.W. and Tyrrell, J.B. (1991) Anterior pituitary gland. In: Basic and Clinical Endocrinology: Third Edition (Greespan, F.S., ed.), pp. 79- 132, Prentice-Hall International Inc., New York. Gordon, G.G., Altman, K., Southren, A.L., Rubin, E. and Lieber, C.S. (1976) Effect of alcohol (ethanol) administration on sex-hormone metabolism in normal men. New Engl. J. Med. 295, 793-797. Gordon, G.G., Southren, A.L. and Lieber, C.S. (1978) The
effects of alcoholic liver disease and alcohol ingestion on sex hormone levels. Alcohol.: Clin. Exp. Res. 2, 259 - 263. Hasselbalch, H., Selmer, J., Sestoft, L. and Kehlet, H. (1982) Hypothalamic-pituitary-adrenocortical function in chronic alcoholism. Clin. Endocrinol. 16, 73 - 76. Holsboer, F., von Bardeleben, U., Buller, R., Heuser, I. and Steiger, A. (1987) Stimulation response to corticotropinreleasing hormone (CRH) in patients with depression, alcoholism and panic disorder. Horm. Metab. Res. 16, 80-88. Jeffcoate, W.J., Platts, P., Ridout, M., Hastings, A.G., MacDonald, I. and Selby, C. (1980) Endocrine effects of ethanol infusion in normal subjects: modification by naloxone. Pharmacol. Biochem. Behav. 13, 145 - 148. Jenkins, J.S. and Connolly, J. (1968) Adrenocortical response to ethanol in man. Br. Med. J. 2, 804-805. Lester, R. and Van Thiel, D.H. (1977) Gonadal function in alcoholic men. Adv. Exp. Biol. Med. 85A, 399-414. Mendelson, J.H., Mello, N.K. and Ellingboe, J. (1977) Effects of acute alcohol intake on pituitary-gonadal hormones in normal human males. J. Pharmacol. Exp. Ther. 202, 676 - 682. Merry, J. and Marks, V. (1969) Plasma-hydrocortisone response to ethanol in chronic alcoholics. Lancet i, 921-923. Miller, F., Barasch, A., Sacks, M., Levitan, J. and Ashcroft, L. (1986) Serum prolactin correlates with depressive mood during alcohol withdrawal. Drug Alcohol Depend. 17, 331-338. North, R.H. and Walter, R.M. (1984) The effects of alcohol on the endocrine system. Med. Clin. North Am. 68, 133 - 146. Risher-Flowers, D., Adinoff, B., Ravitz, B., Bone, G.H.A. Martin, P.R., Nutt, D. and Linnoila, M. (1988) Circadian rhythms of cortisol during alcohol withdrawal. Adv. Alcohol Subst. Abuse 7, 37-41. Van Thiel, D.H., Gavaler, J.S., Lester, R., Loriaux, D.L. and Braunstein, G.D. (1975) Plasma estrone, prolactin, neurophysin, and sex steroid-binding globulin in chronic alcoholic men. Metabolism 24, 1015 - 1019. Van Thiel, D.H., Lester, R. and Vaitukaitis, J. (1978) Evidence for a defect in pituitary secretion of luteinizing hormone in chronic alcoholic men. J. Clin. Endcrinol. Metab. 47, 499-507. Van Thiel, D.H. (1984) Ethyl alcohol and gonadal function. Hosp. Pratt. Novemeber 152- 158. Yamano, H., Fukuda, K. and Matsumoto, F. (1968) Studies on blood alcohol-development of gas chromatographic method and relationship between serum alcohol levels and whole blood level. Jpn. J. Stud. Alcohol 3, 144- 149. Ylikahri, R.H., Huttunen, M.O., Harkonen, M., Leino, T., Helenius, T., Liewendahl, K and Karonen, S. (1978) Acute effects of alcohol on anterior pituitary secretion of the tropic hormones. J. Clin. Endocrinol. Metab. 46, 715 - 720. Ylikahri, R.H., Huttunen, M.O. and Harkonen, M. (1980) Hormonal changes during alcohol intoxication and withdrawal. Pharmacol. Biochem. Behav. 13, 131- 137.
