snencan Sleep Disorders Asovation and Slep Research Socey Melatonin: A Sleep-Promoting Hormone Irina V. Zhdanova, Harry J. Lynch and Richard J. Wurtman Massachusetts Institute of Technology, Cambridge, Massachusetts, U.S.A ‘Summary: This review discusses the issue of a dual effect of melatonin on sleep: acute sleep promotion that typically occurs within one hour of administration, and the ability to alter the phase of an underlying ircadian pacemaker after a repeated melatonin treatment. The authors suggest that both mechanisms are at work, that they fare complementary, and that they may manifest jointly or separately. The review provides some basic information fon melatonin, an overview of the literature, and the authors’ experience in studying the acute effects of melatonin treatment in humans of different age groups. This review also illustrates the authors’ cautious attitude toward ‘melatonin treatment that induces supraphysiologic circulating levels of the hormone. Key Words: Melatonin— ‘Sleep—Human-=Circadian ‘The unbridled exuberance that has greeted the abun- dantly publicized claims about melatonin’s positive ef- fects has overshadowed the acquisition of knowledge about what the pineal hormone really does. Many questions remain about melatonin’s effects in humans. This paper assembles some basic information about the pineal hormone’s role in humans. It specifically reviews the available data on melatonin’s acute hyp- notic effect and considers the relation between this ef- fect and melatonin’s ability to alter the phase of an underlying circadian pacemaker. Our view is that mel- atonin directly affects rest-activity mechanisms, pro- moting the rest phase in humans, and that its ability to suppress the activity of the circadian pacemaker, thus shifting its phase, is part of its net effect. ‘The identification of melatonin as a pineal hormone was a twentieth century event. It began with studies performed by McCord and Allen (1) in 1915, who showed that mammalian pineal tissue contains a sub- stance that causes the skin of tadpoles to blanch. In 1958, Lerner et al. (2) isolated and identified the pineal principal responsible for the blanching effect as mel- atonin (N-acetyl-5-methoxytriptamine). In 1961, Axelrod and Weisbach (3) demonstrated that pineal tissue contains all the enzymes needed to synthesize melatonin from its circulating amino acid precursor, tryptophan. Their discoveries established that the pineal gland converts tryptophan to serotonin (SHT), and that pineal SHT becomes melatonin ‘Accepted for publication July 1997. ‘Address for correspondence and reprint requests to trina V. Zhda- nova, M.D., Ph.D., Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, M.LT. Clinical Research Center, E18-439, 50 Ames Street, Cambridge, MA 02139, US.A. through a two-step process that involves serotonin’s N- acetylation, catalyzed by N-acetyltransferease (NAT), and then its methylation, catalyzed by hydroxyindole- (O-methyltransferase (HIOMT). Soon thereafter, Wurt- man et al. (4) showed that melatonin does function in mammals as a hormone. Initially, the only way one could estimate the rate of melatonin production in a pineal gland was to infer this rate from the enzyme activity measured in vitro (5). In the 1960s, using this approach, it was shown that in rats the rate of mela- tonin synthesis is low when animals are exposed to light, and high in darkness (6), and that information about ambient lighting conditions reaches the pineal via a pathway involving the eyes, central neural tracts, and the pineal’s sympathetic innervation (7). Subse- quent development of melatonin assay methods has made it possible to confirm these basic tenets of pineal physiology and further to establish that melatonin, a lipid soluble hormone, is released directly into the blood stream and the cerebrospinal fluid as it is syn- thesized. Inactivation of melatonin occurs in the liver, where it is converted to 6-hydroxymelatonin by the P- 450-dependent microsomal mixed-function oxidase enzyme system. Most of this 6-hydroxymelatonin is excreted in the urine and feces as a sulfate conjugate (6-sulfatoxymelatonin) (8). The advent of melatonin assay methods allowed pineal function to be studied in humans for the first time. Lynch et al. (9) demonstrated in 1975 that in humans, as in rats, melatonin is secreted at night but not to a great extent in daytime, so that nocturnal cir- culating melatonin levels in young adults are 10-40 times higher than those found at midday. This rhyth- 399900 mic pattern of melatonin secretion persists in people maintained in constant darkness (10); however, the abrupt imposition of bright light can suppress it (11). The human fetus and newborn infant do not produce melatonin but rely on hormone supplied by the pla- cental blood and, postnatally, by the mother’s milk. After infants are 9-12 weeks old, rhythmic melatonin production increases rapidly (12), with highest noctur- nal melatonin levels attained in children under five years of age. Thereafter, melatonin levels decrease with physiologic aging (13). Marked interindividual variations in nocturnal melatonin levels are observed in all age groups; however, the circulating melatonin pattern for a given individual tends to remain surpris- ingly constant from day to day. MELATONIN AND SLEEP Numerous indirect and direct observations link mel- atonin secretion and sleep. The earliest observations on melatonin secretion in humans documented the concurrence of melatonin release from the pineal gland and the habitual hours of sleep (9). Reports from var- ious laboratories confirmed this temporal relationship and correlated the daily onset of melatonin secretion with the onset of nocturnal sleepiness (14,15) or the opening of the so-called “sleep gate” (16). A relevant observation in human babies is that consolidation of nocturnal sleep and stabilization of the sleep-wake cy- cle occur when the infant is about 3 months old, an age coincident with the normal onset of rhythmic mel- atonin secretion (12). Both melatonin secretion and sleep efficiency tend to decline with age. These phe- nomena may be related (17,18). Though such corre- lations do not prove a causal relationship between mel- atonin secretion and sleep, they are consistent with and tend to substantiate direct experimental results. The very earliest report of a soporific effect of mel- atonin treatment appeared in the pioneering work of Lerner and Case (19). Initial studies specifically ad- dressing melatonin’s effects on sleep were conducted at a time when sensitive methods for measuring cir- culating melatonin levels were not available. In the absence of knowledge of normal melatonin levels, ini- tial studies employed enormous doses of the hormone. Intravenous administration of 50 or 100 mg of mela- tonin reportedly induced sleepiness and significantly reduced latency to sleep onset, without altering normal sleep architecture (20,21). The daily oral administra- tion of 1 g of melatonin for 6 days to healthy subjects increased the duration of their stage 2 sleep, decreased the duration of stage 4 sleep, and increased the number of rapid eye movements (REMs) during REM sleep (22). Oral administration of a divided 240-mg dose of melatonin to healthy volunteers induced fatigue, sig- Sleep, Vol.20, No. 10, 1997 I. V, ZHDANOVA ET AL. nificantly increased reaction time, and diminished sub- jects’ alertness (23). An oral 50-mg melatonin dose ‘was reported to increase self-rated fatigue during a 3- hour period following its ingestion if given at 9:00 a.m. but not if given at 7:00 p.m.; latency to sleep onset tended to decrease after both morning and eve- ning treatment; however the magnitude of this effect, compared with that produced by placebo, did not achieve statistical significance in the volunteers stud- ied (24). A group of investigators, led by Vollrath (25), reported that morning intranasal administration of a 1.7-mg melatonin dose promoted sleep onset in 7 of 10 subjects, whereas only one of the subjects on pla- cebo fell asleep. A well-documented effect of mela- tonin on polysomnographically recorded overnight sleep was obtained by using an 80-mg oral dose of melatonin in situational insomnia induced by exposing subjects to recorded traffic noise (26). In that study, treatment of 20 healthy subjects with melatonin at 9: 00 p.m. significantly reduced sleep latency, the number of nocturnal awakenings, the duration of stage I sleep, and the mean REM interval. Melatonin treatment also increased sleep efficiency and the duration of stage 2 sleep. Other sleep parameters were not altered. The effects of lower but still pharmacologic doses of melatonin (1-6 mg) were examined next in different laboratories, and most of the studies also found the treatment to induce fatigue, sleepiness, or increases in sleep propensity. A I-mg or higher dose administered at 10:00 a.m. significantly reduced latency to sleep on- set, increased sleep efficiency, increased the relative duration of stage 2 sleep, and decreased that of slow- wave sleep (27). Similarly, a 5-mg oral dose of mel- atonin given at 6:00 p.m. increased subjectively as- sessed fatigue within 90 minutes, as well as the theta band of subjects’ electroencephalogram spectrum while they were awake (28,29). An extensive study conducted in Israel assessed polysomnographically re- corded sleep propensity after a 5-mg dose of melatonin was administered to young adults at noon, 200, or 9:00 p.m. (30). The hormone significantly increased sleep propensity at all the time points studied and in- creased the duration of stage 2 sleep. In the discussion of this study, the authors suggested that melatonin ex- ents a delayed effect on sleep, the development of which requires 1-2 hours. However, a further exami- nation of this phenomenon in the same laboratory led by Lavie (31) showed that the administration of the hormone 30 or 120 minutes prior to a nap significantly reduced sleep latency and increased total sleep time independent of the dose or time of its administration. ‘Though most of the studies that employed phar- macological doses of melatonin described changes in subjective and/or objective sleep parameters, there were also studies with negative or indeterminate find-MELATONIN AND SLEEP PROMOTION ings (32-34). Two complementary studies tested the effects of 1- and 5-mg oral doses of melatonin on sleep in normal volunteers (32) and in patients with insom- nia (33). Normal volunteers displayed no consistent changes; however, for insomniacs, the authors reported that “although a trend in the induction of sleep with increasing dosages of melatonin was observed, no sta- tistically significant difference was found in this pa- rameter or in sleep duration, sleep efficiency, slow- wave sleep, or percent of movement time”. The pa- tients did not report any change in daytime sleepiness; however, “surprisingly, although they reported less sleep than usual when taking melatonin, subjects felt that the quality of the sleep had improved (p = 0.03)”. The only remarkable effect of melatonin treatment de- scribed in both of these studies was an increase in latency to REM sleep among normal subjects receiv- ing the 5-mg dose and among insomniacs receiving the 1-mg but not the 5-mg dose. Such an effect on REM sleep has not been described by other research- ers. The negative results in these two studies regarding sleep onset latency may relate, in part, to the experi- mental design used by the Mendelson group (32,33), i.e. only a short 15-minute interval was allowed to elapse between administration of the hormone and ex- perimental bedtime. The sleep promoting effects of melatonin have typically been observed when the hor- mone was administered at least 30 minutes prior to bedtime, an interval we find necessary to achieve a substantial increase in circulating melatonin levels af- ter its oral administration. Because circulating mela- tonin levels induced by the particular melatonin prep- aration used in these studies were not measured, it is not clear when and to what extent serum levels in- creased. The availability of new, sensitive assay methods that allow accurate measurement of circulating melatonin concentrations in the picogram-per-milliliter range has allowed the demonstration that melatonin doses in ex- cess of 1 mg, which had been shown to induce sopo- rific effects in humans, elevate circulating melatonin levels to very high, supraphysiologic levels. Thus, it was unclear whether or not the observed effects on sleep represented side effects of such pharmacologic doses of the hormone. A program of studies to explore the dose dependen- cy of melatonin’s sleep-promoting effect was initiated in our laboratory in the early 1990s. In the first study, one of four melatonin doses (10, 20, 40, or 80 mg) was administered at noon to young healthy volunteers. Each treatment significantly decreased the number of correct responses in computerized performance tests and increased reaction time, self-reported fatigue, con- fusion, and sleepiness. However, in this dose range, no dose-response relationships were discernible (35). We 901 next tested a still lower range of doses, now admin- istering 0.1, 0.3, 1, or 10 mg of melatonin at noon to 20 young healthy volunteers. Measurements included serum melatonin levels, subjectively assessed sleepi- ness and mood, and performance using computerized visual and auditory tests, prior to and for 6 hours after the treatment (36). Sleep onset and duration were also studied, beginning 1.75 hours after the administration of melatonin or placebo, using a test involving invol- untary muscle relaxation. Serum melatonin concentra- tions were found to be roughly proportional to the dose of melatonin ingested; peak amplitudes observed after the 0.1- and 0.3-mg doses yielded mean serum mela- tonin levels of 48 and 121 pg/ml, respectively, values within the physiological nocturnal range of human se- rum melatonin levels. Sleep onset latencies after in- gestion of any of the doses tested were significantly shorter than those following ingestion of placebo. However, the magnitude of the effect induced by the 0.1-mg dose was discernibly smaller than that after any of the higher doses, suggesting dose dependency. Sim- ilar tendencies were detected using a profile of mood states test (POMS) and the Stanford sleepiness test. On the basis of these two dose-dependency studies, we concluded that treatments that result in pharmacolog- ical levels of circulating melatonin (i.e. serum levels cover 200 pg/ml) induce sleepiness and promote sleep onset without clear dose dependency; however, treat- ments that result in physiological concentrations with- in the normal nocturnal range (i.e. 50-200 pg/ml) do demonstrate dose dependency of the soporific effect. Thus, we hereafter refer to doses of melatonin that produce serum levels of the hormone within the nor- ‘mal nocturnal range as physiologic doses. ‘These findings formed the basis for a new line of research into the physiologic role of melatonin in sleep. A series of experiments was initiated that in- volved polysomnographic recording of subjects’ sleep, as well as mood and performance testing, after inges- tion of physiologic doses of melatonin. One study de- termined that acute administration of a physiologic (0.3-mg) or a low pharmacologic (1.0-mg) dose of mel- atonin in the evening produced sedativelike effects, compared with placebo, similar to those we had ob- served when such doses were administered at noon- time. Another question that we addressed was whether the effects of such doses varied depending on how ‘many hours prior to habitual bedtime they were ad- ministered. Recognizing that individual variations ex- ist in sensitivity to melatonin, we tested the same group of six subjects repeatedly, each subject receiving the hormone or placebo at 6:00, 8:00, and 9:00 p.m., with at least 5 days elapsing between treatments. The sleep-promoting effects of melatonin were tested be- fore, during, and after a 2-hour experimental nap that Sleep, Vol 20, No. 10, 1997