Alcohol intake alters melatonin secretion both in healthy volunteers and in alcoholics in a variety of different situations (while drinking, during or after withdrawal, and with neurological complications). This alteration may reduce secretion or affect its circadian rhythm, thus causing daytime secretion in some alcoholics. We sought to determine if daytime melatonin secretion is caused directly by acute alcohol consumption or if it instead indicates a change in circadian synchronization. Because alcohol consumption as it occurs in alcoholics (continuous consumption of large amounts) has never been examined in healthy volunteers, we exposed 11 healthy volunteers to 256 g of alcohol over 24 h to study the circadian profiles of melatonin secretion. Our results demonstrate a lack of daytime secretion in our subjects. This suggests that the disordered circadian melatonin secretion seen in alcoholics indicates a shift in melatonin secretion rather than an acute effect of alcohol on this secretion, or alternatively, that it is a direct effect of chronic rather than acute exposure to high blood alcohol levels.
(Received 4 January 2006; first review notified 14 February 2006; in revised form 24 March 2006; accepted 30 March 2006)
The disturbance of circadian rhythms (Danel et al., 2001) may explain in part some mental disorders secondary to alcohol consumption. Examination of this hypothesis requires a study of the changes in melatonin secretion that may be due to alcohol consumption.
Two primary findings emerge from the studies of melatonin and alcohol consumption in healthy volunteers and in chronic alcohol-dependent drinkers in different situations (review in Danel and Touitou, 2004). The first is that both acute and chronic alcohol consumption inhibit melatonin secretion. Experimental studies in healthy volunteers (Ekman et al., 1993; Rojdmark et al., 1993), in the general population (Stevens et al., 2000), and in alcohol-dependent individuals in the general population (Touitou et al., 1985; Wetterberg et al., 1992) show this, as do studies during treatment of alcohol-dependent subjects, both during withdrawal (Schmitz et al., 1996) and with Wernicke's syndrome (Wikner et al., 1995).
The second major finding is that the nycthemeral rhythm of melatonin secretion is disrupted in some alcohol-dependent individuals. Melatonin is normally secreted only at night but daytime secretion has been observed during the first 24 h of withdrawal or when continued alcohol intoxication occurs in alcohol-dependent subjects (Majumdar and Miles, 1987; Murialdo et al., 1991; Fonzi et al., 1992, 1994; Mukai et al., 1998). It is not clear, however, whether daytime secretion of the hormone is caused directly by the acute alcohol consumption or instead it indicates a change in the alcoholic's circadian synchronization. Studies conducted in healthy volunteers might answer this question, but no such studies are reported. Trials among healthy volunteers have generally involved acute administration of alcohol at a time of day (evening) and in amounts (from 10 to 100 g) usually associated with social drinking (Ekman et al., 1993; Rojdmark et al., 1993; Stevens et al., 2000). Alcohol consumption in heavy drinkers, on the other hand, often occurs over a much longer portion of the day and involves higher quantities. To increase our understanding of the action of alcohol on melatonin secretion and in particular to determine whether alcohol enhances melatonin secretion during daylight hours, we conducted a blinded crossover study over a 26 h (circadian) period. In one session alcohol was administered regularly and repeatedly, and in the other, a placebo was administered. Healthy volunteers thus acted as their own controls, and we controlled for masking effects in both sessions. The total dose administered represented an amount generally consumed daily by alcoholic subjects, i.e. 256 g per day (equivalent to ∼2.5 l of 12% wine, 700 ml of 40% whisky, or 6 l of 4.5% beer) administered at regular intervals throughout the session. Serum melatonin secretions were measured throughout the circadian cycle at 29 time points of the 26 h alcohol consumption session and the 26 h control session.
SUBJECTS AND METHODS
Eleven healthy male volunteers between the ages of 18 and 30 years (23.3 + 2.9 years) were included after they provided their informed written consent. None had a current or past diagnosis of alcohol, tobacco, or other substance abuse or dependency. No subjects were taking medication or working rotating shifts, and none had flown on transmeridian flights in the past 2 months. All were synchronized with diurnal activity and nocturnal rest. None had a current diagnosis of delayed or advanced phase or hypernycthemeral syndrome. Horne and Ostberg (1976) scores ranged from 39 to 59 (mean 49.5 + 6.8). This criterion excluded subjects who were clearly ‘morning’ or ‘evening’ types. No subject had a current or past history of depressive disorder or psychosis. The Montgomery and Asberg (1979) depression rating scale scores, because they were less than 18, ruled out any current depressive disorder. The subjects had no physical abnormalities at the time of examination and had had no infection or other disease for at least 1 month before the sessions. Body mass indices ranged from 20 to 25. Routine blood counts and blood chemistry were within normal ranges, and HIV and hepatitis B and C tests were negative.
The study was approved by the Lille (France) Committee on human experimentation and was consistent with the standards and ethical principles for research on biological rhythms on humans (Touitou et al., 2004). Melatonin circadian rhythm was studied during a single-blinded, randomized, crossover study that compared a 26 h alcohol session with a 26 h placebo session. During the alcohol session, 256 g of ethanol were administered between 10:00 on day 1 and 12:00 on day 2 (Table 1) to produce and maintain blood alcohol concentrations (BAC) between 0.5 and 0.7 g/l throughout the session. To obtain a significant BAC at the beginning of the data collection period (12:00), 20 g of ethanol, mixed with fruit juice, was administered orally at 10:00, 11:00, and 12:00; followed by 10 g/h from 13:00 to 21:00 and from 07:00 to 11:00 on the second day. Fruit juice was administered alone during the placebo session. In addition, 7 g/h of alcohol (Curethyl*, AJC Pharma, Chateauneuf, France) in saline solution was administered intravenously overnight (between 22:00 and 06:00) during the alcohol session, and saline solution only in the control session, to allow the subjects to sleep and maintain a sufficient BAC. All sessions took place between November and April. The sessions for each subject were between 2 and 5 weeks apart. The subjects were admitted to the Clinical Investigation Center at 08:00. During the observation period from 10:00 on day 1 to 15:00 on day 2 they remained in bed, reading and watching television. They ate standardized meals at 08:00, 12:00, and 19:00 on day 1 and at 08:00 and 12:00 on day 2. They left the center at 15:00 on day 2. The lights were switched off between 22:00 and 06:00. Blood samples were collected to measure BAC at 5 time points (12:00, 18:00, 24:00, 06:00, and 12:00) and melatonin concentrations at 29 time points (12:00, 15:00, 18:00 and then every 30 min between 18:00 and 04:00, 05:00, 06:00, 07:00, 08:00, 11:00, 15:00). During the blood collections between 22:00 and 06:00, the room was illuminated with light at an average intensity of 50 lux.
|Amount of alcohol administered (g)||60||90||56||50|
|Route of administration||Oral||Oral||Intravenous||Oral|
|Amount of alcohol administered (g)||60||90||56||50|
|Route of administration||Oral||Oral||Intravenous||Oral|
Melatonin concentrations were measured with a commercial enzyme-linked immunosorbent assay (ELISA) (Boeringer Mannheim, France) in a fully automated analyser (ES 700). To avoid inter-assay analytical variability, all assays were performed in a single large batch at the end of the protocol. All samples were frozen at −20°C until assay. Intra-assay coefficients of variation for melatonin (65 pg/ml) were 6.1%.
We used ANOVA with mixed linear models for paired comparisons to study the differences between the mean curves under two conditions (alcohol, without alcohol). The fixed effects were condition (2 levels), time (29 levels), and time–condition interaction. The subject effect was considered to be random, and we chose a first-order autoregressive covariance pattern to take into account dependency between repeated measurements. This model was chosen according to AIC criteria (Akaike, 1974). Comparisons of the differences at each time point used a Bonferroni correction. A P-value < 0.002 was therefore considered to be significant.
Blood alcohol concentrations
Mean BAC in the alcohol sessions were 0.54 g/l at 12:00, 0.78 g/l at 18:00, 0.62 g/l at 00:00, 0.37 g/l at 06:00, and 0.29 g/l at 12:00 on day 2. These were consistent with the study protocol.
Melatonin. Groupwise analysis (Figure 1)
The melatonin curves during the alcohol and alcohol-free sessions were nearly identical. While the curve during the alcohol session tended to show a delay in the onset of melatoninsecretion compared with the alcohol-free session, analysis of the data showed no statistically significant effect by alcohol on melatonin secretion (P = 0.45). The mean value of the secretion peak was 128 pg/ml ±68 in the alcohol-free session and 120 pg/ml ±57 in the alcohol session. Mean melatonin secretion over 24 h was 50.8 pg/ml ±27 and 45 pg/ml ±20, respectively.
Melatonin. Individual analysis (Figure 2)
Strictly nocturnal secretion of melatonin was seen in all subjects both during the alcohol-free session and during the alcohol session. Plasma concentrations were entirely consistent in the subjects between the two sessions. Some subjects appeared to show a delay in the onset of melatonin secretion or in the time when the half-peak value was reached (subjects 1, 2, 3, 4, 6, and 10). Thus, the time at which melatonin secretion began thus changed for 6 of the 11 subjects. This difference was consistent in direction for all subjects, always a delay, either in the time that nocturnal melatonin secretion started or in the time that the half-peak value was reached.
Our data show clearly that all subjects in this study secreted melatonin only at night. Circadian melatonin secretion was observed in both sessions, with nocturnal secretion and no circulating melatonin during the day.
Our results thus shed new light on the observations in the literature about diurnal melatonin secretion in alcoholics.
Majumdar and Miles (1987) were the first to report disturbances in the time of melatonin secretion in alcohol-dependent patients. These authors examined melatonin secretion during the afternoon in 28 male alcohol-dependent patients, all of whom had consumed more than 100 g/d of alcohol for more than 7 years. Melatonin was detectable (concentrations <5 ng/l) in 13 subjects during the afternoon. This key article describing diurnal melatonin secretion was followed by three publications from an Italian team. Two reported melatonin measurements in 10 alcohol-dependent patients, first during alcohol consumption and then after 2 weeks of abstinence. These were compared with measurements in healthy age-matched volunteer controls (Murialdo et al., 1991; Fonzi et al., 1992). The results showed that urinary melatonin concentrations during alcohol consumption were significantly higher in the alcohol-dependent patients than in the controls (316.2 ± 36 pmol/24 h in the patients and 147 ± 34 pmol/24 h in the controls). This difference was due principally to high urinary melatonin levels in the daytime fraction. Conversely, the two groups did not differ significantly for the night-time fraction. The ratio of night/day fractions was less than 1 in 6 of the 10 patients and greater (generally far greater) than 1 in the other 4. After 14 days of abstinence, this ratio exceeded 1 in all patients and control subjects.
Fonzi et al. (1994) demonstrated in 10 alcohol-dependent subjects before and after withdrawal that serum melatonin concentrations were higher during alcohol consumption than after withdrawal and greater than those in control subjects. They also reported the disappearance of the circadian rhythm of melatonin secretion during acute withdrawal. Similar observations were made in two patients with delirium tremens (Mukai et al., 1998), for whom night-time and daytime serum melatonin concentrations were equivalent (samples taken every 4 h).
Our results show clearly that alcohol had no effect on diurnal melatonin secretion. A hypothesis is thus necessary to attempt to explain the reports from the literature showing a progressive delay in the secretion peak that eventually leads to diurnal secretion. Although previous work indicates that alcohol inhibits melatonin secretion (Danel and Touitou, 2004), this experiment fail to show such inhibition. The absence of this finding may be related to blood alcohol level. The level at which ethanol may inhibit melatonin secretion remains to be determined. One recent publication (Kuhlwein et al., 2003) reports a delay in the melatonin secretion of alcohol-dependent patients who had very recently stopped drinking, in the same direction as that we observed in 6 of our 11 subjects. That study, which sought to examine the relations between sleep disorders in alcohol-dependent patients soon after withdrawal and melatonin and cortisol concentrations, observed a delay in the nocturnal melatonin peak in 11 alcohol-dependent patients soon after withdrawal, compared with 10 age-matched controls. The delay was correlated between with sleep latency periods. However, they did not test for daytime secretion of melatonin.
Now that we have shown that alcohol itself does not directly cause daytime melatonin secretion, we can hypothesize that disturbances in the timing of melatonin secretion in the literature are due to a shift in the circadian clock of melatonin secretion and appear to indicate possible internal desynchronization during chronic alcohol consumption. The ethanol level at which this occurs remains to be determined. Another possible explanation is that the daytime melatonin secretion seen in alcoholics is due to chronically high blood alcohol levels that cause daytime melatonin secretion via mechanisms that have nothing to do with circadian timing. In such a case, the melatonin daytime secretion seen in alcoholics would be a cause rather than a consequence of desynchronization.
Supported by the Institut national de la Santé et de la Recherche médicale and the Centre Régional et Universitaire de Lille.
1Service d'Addictologie, Centre Hospitalier Universitaire, Lille 59037 Lille Cedex, France
2Service de Biochimie médicale et de Biologie moléculaire, Faculté de Médecine Pitié Salpêtrière 75013, Paris, France