Alcohol & Alcoholism Vol. 41, No. 4, pp. 386–390, 2006
Advance Access publication 5 May 2006
doi:10.1093/alcalc/agl036
ALCOHOL CONSUMPTION DOES NOT AFFECT MELATONIN CIRCADIAN
SYNCHRONIZATION IN HEALTHY MEN
THIERRY DANEL1,2* and YVAN TOUITOU2
1
Service d’Addictologie, Centre Hospitalier Universitaire, Lille 59037 Lille Cedex, France and 2Service de Biochimie médicale et de Biologie moléculaire,
Faculté de Médecine Pitié Salpêtrière 75013, Paris, France
(Received 4 January 2006; first review notified 14 February 2006; in revised form 24 March 2006; accepted 30 March 2006;
advance access publication 5 May 2006)
Abstract — 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.
INTRODUCTION
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.
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;
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
*Author to whom correspondence should be addressed at: Thierry Danel,
Service d’Addictologie, CHRU de Lille, 59037 Lille cedex, France.
Tel: +33 3 20 44 41 78; Fax: +33 3 20 44 57 78; E-mail: [email protected]
386
Ó The Author 2006. Published by Oxford University Press on behalf of the Medical Council on Alcohol. All rights reserved
MELATONIN AND ALCOHOL
from 20 to 25. Routine blood counts and blood chemistry were
within normal ranges, and HIV and hepatitis B and C tests
were negative.
Protocol
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.
387
Statistical analysis
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.
RESULTS
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 melatonin
secretion 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
Hormone assays
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%.
Table 1 Study protocol
10:00–12:00 13:00–21:00 22:00–6:00 7:00–11:00
Amount of alcohol
administered (g)
Frequency (g/h)
Route of administration
60
90
56
50
20
Oral
10
Oral
7
Intravenous
10
Oral
Fig. 1. Groupwise analysis. Circadian melatonin profile (pg/ml) in 11 healthy
adult volunteers during one session without alcohol consumption (open
circles) and one session with consumption of 256 g of alcohol divided
regularly over 24 h (closed circles). Each subject acted as his own control.
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T. DANEL and Y. TOUITOU
Fig. 2. Circadian melatonin profile (pg/ml) in 11 healthy adult volunteers during: one session without alcohol consumption (open circles) and one session with
consumption of 256 g of alcohol divided regularly over 24 h (closed circles). Each subject acted as his own control.
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 AND ALCOHOL
melatonin secretion started or in the time that the half-peak
value was reached.
DISCUSSION
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 alcoholdependent 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
389
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.
Acknowledgements — Supported by the Institut national de la Santé et de la
Recherche médicale and the Centre Régional et Universitaire de Lille.
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