Josephine Arendt

An action spectrum for melatonin suppression: evidence for a novel non‐rod, non‐cone photoreceptor system in humans

1 Non‐image forming, irradiance‐dependent responses mediated by the human eye include synchronisation of the circadian axis and suppression of pineal melatonin production. The retinal photopigment(s) transducing these light responses in humans have not been characterised. 2 Using the ability of light to suppress nocturnal melatonin production, we aimed to investigate its spectral sensitivity and produce an action spectrum. Melatonin suppression was quantified in 22 volunteers in 215 light exposure trials using monochromatic light (30 min pulse administered at circadian time (CT) 16‐18) of different wavelengths (λmax 424, 456, 472, 496, 520 and 548 nm) and irradiances (0.7‐65.0 μW cm−2). 3 At each wavelength, suppression of plasma melatonin increased with increasing irradiance. Irradiance‐response curves (IRCs) were fitted and the generated half‐maximal responses (IR50) were corrected for lens filtering and used to construct an action spectrum. 4 The resulting action spectrum showed unique short‐wavelength sensitivity very different from the classical scotopic and photopic visual systems. The lack of fit (r2 < 0.1) of our action spectrum with the published rod and cone absorption spectra precluded these photoreceptors from having a major role. Cryptochromes 1 and 2 also had a poor fit to the data. Fitting a series of Dartnall nomograms generated for rhodopsin‐based photopigments over the λmax range 420‐480 nm showed that rhodopsin templates between λmax 457 and 462 nm fitted the data well (r2≥ 0.73). Of these, the best fit was to the rhodopsin template with λmax 459 nm (r2= 0.74). 5 Our data strongly support a primary role for a novel short‐wavelength photopigment in light‐induced melatonin suppression and provide the first direct evidence of a non‐rod, non‐cone photoreceptive system in humans.

Comparison between subjective and actigraphic measurement of sleep and sleep rhythms

Sleep is often assessed in circadian rhythm studies and long‐term monitoring is required to detect any changes in sleep over time. The present study aims to investigate the ability of the two most commonly employed methods, actigraphy and sleep logs, to identify circadian sleep/wake disorders and measure changes in sleep patterns over time. In addition, the study assesses whether sleep measured by both methods shows the same relationship with an established circadian phase marker, urinary 6‐sulphatoxymelatonin. A total of 49 registered blind subjects with different types of circadian rhythms were studied daily for at least four weeks. Grouped analysis of all study days for all subjects was performed for all sleep parameters (1062–1150 days data per sleep parameter). Good correlations were observed when comparing the measurement of sleep timing and duration (sleep onset, sleep offset, night sleep duration, day‐time nap duration). However, the methods were poorly correlated in their assessment of transitions between sleep and wake states (sleep latency, number and duration of night awakenings, number of day‐time naps). There were also large and inconsistent differences in the measurement of the absolute sleep parameters. Overall, actigraphs recorded a shorter sleep latency, advanced onset time, increased number and duration of night awakenings, delayed offset, increased night sleep duration and increased number and duration of naps compared with the subjective sleep logs. Despite this, there was good agreement between the methods for measuring changes in sleep patterns over time. In particular, the methods agreed when assessing changes in sleep in relation to a circadian phase marker (the 6‐sulphatoxymelatonin (aMT6s) rhythm) in both entrained (n= 30) and free‐running (n= 4) subjects.

Melatonin and human rhythms.

Melatonin signals time of day and time of year in mammals by virtue of its pattern of secretion, which defines ‘biological night.’ It is supremely important for research on the physiology and pathology of the human biological clock. Light suppresses melatonin secretion at night using pathways involved in circadian photoreception. The melatonin rhythm (as evidenced by its profile in plasma, saliva, or its major metabolite, 6‐sulphatoxymelatonin [aMT6s] in urine) is the best peripheral index of the timing of the human circadian pacemaker. Light suppression and phase‐shifting of the melatonin 24 h profile enables the characterization of human circadian photoreception, and circulating concentrations of the hormone are used to investigate the general properties of the human circadian system in health and disease. Suppression of melatonin by light at night has been invoked as a possible influence on major disease risk as there is increasing evidence for its oncostatic effects. Exogenous melatonin acts as a ‘chronobiotic.’ Acutely, it increases sleep propensity during ‘biological day.’ These properties have led to successful treatments for serveal circadian rhythm disorders. Endogenous melatonin acts to reinforce the functioning of the human circadian system, probably in many ways. The future holds much promise for melatonin as a research tool and as a therapy for various conditions.

Efficacy of melatonin treatment in jet lag, shift work, and blindness.

Melatonin has chronobiotic properties in humans. It is able to phase shift strongly endogenous rhythms, such as core temperature and its own endogenous rhythm, together with the sleep-wake cycle. Its ability to synchronize free-running rhythms has not been fully investigated in humans. There is evidence for synchronization of the sleep-wake cycle, but the available data suggest that it is less effective with regard to endogenous melatonin and core temperature rhythms. When suitably timed, most studies indicate that fast release preparations are able to hasten adaptation to phase shift in both field and simulation studies of jet lag and shift work. Both subjective and objective measures support this statement. However, not all studies have been successful. Careful evaluation of the effects on work-related performance is required. When used to alleviate the non-24-h sleep-wake disorder in blind subjects, again most studies report a successful outcome using behavioral measures, albeit in a small number of individuals. The pres suggest, however, that although leep-wake can be stabilized to 24 h, entrainment of other rhythms is exceptionally rare.

Radioimmunoassay for 6-Sulphatoxymelatonin in Urine Using an Iodinated Tracer

A radioimmunoassay (RIA) for urinary 6-sulphatoxymelatonin (aMT6s) using an 125I-aMT6s tracer is described. Iodinated aMT6s used as the label was synthesised by direct iodination of aMT6s using 1,3,4,6 tetrachloro 3,6 diphenylglycouril as the oxidant. The assay shows low cross reactivity with related compounds. Serial dilutions of 1:250 and 1:125 diluted urine gave parallel displacement curves. Comparison of the new RIA using 125I-aMT6s with the RIA using 3H-aMT6s label gave good correlation, as did comparison with a gas chromatography mass spectroscopy (GCMS) for total free and conjugated 6-hydroxymelatonin.

Melatonin: Characteristics, Concerns, and Prospects

Melatonin is of great importance to the investigation of human biological rhythms. Its rhythm in plasma or saliva provides the best available measure of the timing of the internal circadian clock. Its major metabolite 6-sulphatoxymelatonin is robust and easily measured in urine. It thus enables long-term monitoring of human rhythms in real-life situations where rhythms may be disturbed, and in clinical situations where invasive procedures are difficult. Melatonin is not only a “hand of the clock”; endogenous melatonin actsto reinforce the functioning of the human circadian system, probably in many ways. Most is known about its relationship to sleep and the decline in core body temperature and alertness at night. Current perspectives also include a possible influence on major disease risk, arising from circadian rhythm disruption. Melatonin clearly has the ability to induce sleepiness and lower core body temperature during “biological day” and to change the timing of human rhythms when treatment is appropriately timed. It can entrain free-running rhythms and maintain entrainment in most blind and some sighted people. Used therapeutically it has proved a successful treatment for circadian rhythm disorder, particularly the non-24-h sleep wake disorder of the blind. Numerous other clinical applications are under investigation. There are, however, areas of controversy, large gaps in knowledge, and insufficient standardization of experimental conditions and analysis for general conclusions to be drawn with regard to most situations. The future holds much promise for melatonin as a therapeutic treatment. Most interesting, however, will be the dissection of its effects on human genes.

Melatonin-induced temperature suppression and its acute phase-shifting effects correlate in a dose-dependent manner in humans.

Melatonin is able to phase-shift the endogenous circadian clock and can induce acute temperature suppression. It is possible that there is a direct relationship between these phenomena. In a double-blind, placebo-controlled crossover study, 6 healthy volunteers maintained a regular sleep/wake cycle in a normal environment. From dusk until 24:00 h on days (D) 1-4 subjects remained in dim artificial lighting (< 50 lux) and darkness (< 1 lux) from 24:00-08:00 h. At 17:00 h on D3 either melatonin (0.05 mg, 0.5 mg or 5 mg) or placebo was administered. Melatonin treatment induced acute, dose-dependent temperature suppression and decrements in alertness and performance efficiency. On the night of D3, earlier sleep onset, offset and better sleep quality were associated with increasing doses of melatonin. The following day, a significant dose-dependent phase-advance in the plasma melatonin onset time and temperature nadir (D4-5) was observed with a trend for the alertness rhythm to phase-advance. A significant dose-response relationship existed between the dose of oral melatonin, the magnitude of temperature suppression and the degree of advance phase shift in the endogenous melatonin and temperature rhythms, suggesting that acute changes in body temperature by melatonin may be a primary event in phase-shifting mechanisms.

The 3111 Clock gene polymorphism is not associated with sleep and circadian rhythmicity in phenotypically characterized human subjects

Mutations in clock genes are associated with abnormal circadian parameters, including sleep. An association has been reported previously between a polymorphism (3111C), situated in the 3′‐untranslated region (3′‐UTR) of the circadian gene Clock and evening preference. In the present study, this polymorphism was assessed in: (1) 105 control subjects with defined diurnal preference, (2) 26 blind subjects with free‐running circadian rhythms and characterized with regard to circadian period (τ) and (3) 16 delayed sleep phase syndrome patients. The control group was chosen from a larger population (n = 484) by Horne‐Östberg questionnaire analysis, from which three subgroups were selected (evening, intermediate and morning preference). Data from sleep diaries completed by 90% of these subjects showed a strong correlation between preferred and estimated timings of sleep and wake. The mean timings of activities for the evening group were at least 2 h later than the morning group. Genetic analysis showed that, in contrast with the previously published finding, there was no association between 3111C and eveningness. Neither was there an association between 3111C and τ, nor a significant difference in 3111C frequency between the normal and delayed sleep phase syndrome groups. To assess the effect of this polymorphism on messenger RNA (mRNA) translatability, luciferase reporter gene constructs containing the two Clock polymorphic variants in their 3′‐UTR were transfected into COS‐1 cells and luciferase activity measured. No significant difference was observed between the two variants. These results do not support Clock 3111C as a marker for diurnal preference, τ, or delayed sleep phase syndrome in humans.

The effects of chronic, small doses of melatonin given in the late afternoon on fatigue in man: A preliminary study

In a double-blind cross-over study, melatonin (2 mg) or placebo, was administered daily for 4 weeks to 12 volunteers (10 men and 2 women) at 17.00 h during February and March. Self-rated fatigue (tiredness) was significantly increased in the evening during melatonin treatment. No other consistent effects on sleep ratings or mood parameters were observed and the dose was well tolerated.

Can Melatonin Improve Shift Workers' Tolerance of the Night Shift? Some Preliminary Findings

The pineal hormone melatonin is potentially useful in the treatment of disorders, especially sleep disorders, associated with circadian rhythm disturbance. We have examined its effects on sleep, mood, and behaviour in a double-blind, placebo-controlled study of a small group of police officers working spans of seven successive night shifts. Compared to placebo, and to no treatment, melatonin (5 mg) taken at the desired bedtime improved problems related to sleep and increased alertness during working hours, especially during the early morning. In letter-target performance tests visual search speed and accuracy were either unchanged or slightly improved. Memory scanning speed and perception of mental load were adversely affected. This preliminary study suggests that melatonin has beneficial effects on sleep and alertness, but that its effects on performance need careful evaluation.