Machine learning estimation of human body time using metabolomic profiling.

Circadian rhythms influence physiology, metabolism, and molecular processes in the human body. Estimation of individual body time (circadian phase) is therefore highly relevant for individual optimization of behavior (sleep, meals, sports), diagnostic sampling, medical treatment, and for treatment of circadian rhythm disorders. Here, we provide a partial least squares regression (PLSR) machine learning approach that uses plasma-derived metabolomics data in one or more samples to estimate dim light melatonin onset (DLMO) as a proxy for circadian phase of the human body. For this purpose, our protocol was aimed to stay close to real-life conditions. We found that a metabolomics approach optimized for either women or men under entrained conditions performed equally well or better than existing approaches using more labor-intensive RNA sequencing-based methods. Although estimation of circadian body time using blood-targeted metabolomics requires further validation in shift work and other real-world conditions, it currently may offer a robust, feasible technique with relatively high accuracy to aid personalized optimization of behavior and clinical treatment after appropriate validation in patient populations.

Method to determine whether sleep phenotypes are driven by endogenous circadian rhythms or environmental light by combining longitudinal data and personalised mathematical models.

Sleep timing varies between individuals and can be altered in mental and physical health conditions. Sleep and circadian sleep phenotypes, including circadian rhythm sleep-wake disorders, may be driven by endogenous physiological processes, exogeneous environmental light exposure along with social constraints and behavioural factors. Identifying the relative contributions of these driving factors to different phenotypes is essential for the design of personalised interventions. The timing of the human sleep-wake cycle has been modelled as an interaction of a relaxation oscillator (the sleep homeostat), a stable limit cycle oscillator with a near 24-hour period (the circadian process), man-made light exposure and the natural light-dark cycle generated by the Earth's rotation. However, these models have rarely been used to quantitatively describe sleep at the individual level. Here, we present a new Homeostatic-Circadian-Light model (HCL) which is simpler, more transparent and more computationally efficient than other available models and is designed to run using longitudinal sleep and light exposure data from wearable sensors. We carry out a systematic sensitivity analysis for all model parameters and discuss parameter identifiability. We demonstrate that individual sleep phenotypes in each of 34 older participants (65-83y) can be described by feeding individual participant light exposure patterns into the model and fitting two parameters that capture individual average sleep duration and timing. The fitted parameters describe endogenous drivers of sleep phenotypes. We then quantify exogenous drivers using a novel metric which encodes the circadian phase dependence of the response to light. Combining endogenous and exogeneous drivers better explains individual mean mid-sleep (adjusted R-squared 0.64) than either driver on its own (adjusted R-squared 0.08 and 0.17 respectively). Critically, our model and analysis highlights that different people exhibiting the same sleep phenotype may have different driving factors and opens the door to personalised interventions to regularize sleep-wake timing that are readily implementable with current digital health technology.

S-cone contribution to the acute melatonin suppression response in humans.

Light influences diverse aspects of human physiology and behaviour including neuroendocrine function, the circadian system and sleep. A role for melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) in driving such effects is well established. However, rod and/or cone signals routed through ipRGCs could also influence "non-visual" spectral sensitivity. In humans, this has been most extensively studied for acute, light-dependent, suppression of nocturnal melatonin production. Of the published action spectra for melatonin suppression, one demonstrates a spectral sensitivity consistent with that expected for melanopsin while our own (using briefer 30 minute light exposures) displays very high sensitivity to short wavelength light, suggesting a contribution of S-cones. To clarify that possibility, six healthy young male participants were each exposed to 30 minutes of five irradiances of 415 nm monochromatic light (1-40 µW/cm2 ) across different nights. These data were then combined with the original action spectrum. The aggregated data are incompatible with the involvement of any single-opsin and multi-opsin models based on the original action spectrum (including Circadian Stimulus) fail to predict the responses to 415 nm stimuli. Instead, the extended action spectrum can be most simply approximated by an ~2:1 combination of melanopsin and S-cone signals. Such a model also better describes the magnitude of melatonin suppression observed in other studies using an equivalent 30 minute mono- or polychromatic light paradigm but not those using longer (90 minute) light exposures. In sum, these data provide evidence for an initial S-cone contribution to melatonin suppression that rapidly decays under extended light exposure.

Effect of acute total sleep deprivation on plasma melatonin, cortisol and metabolite rhythms in females.

Disruption to sleep and circadian rhythms can impact on metabolism. The study aimed to investigate the effect of acute sleep deprivation on plasma melatonin, cortisol and metabolites, to increase understanding of the metabolic pathways involved in sleep/wake regulation processes. Twelve healthy young female participants remained in controlled laboratory conditions for ~92 hr with respect to posture, meals and environmental light (18:00-23:00 hr and 07:00-09:00 hr <8 lux; 23:00-07:00 hr 0 lux (sleep opportunity) or <8 lux (continuous wakefulness); 09:00-18:00 hr ~90 lux). Regular blood samples were collected for 70 hr for plasma melatonin and cortisol, and targeted liquid chromatography-mass spectrometry metabolomics. Timepoints between 00:00 and 06:00 hr for day 1 (baseline sleep), day 2 (sleep deprivation) and day 3 (recovery sleep) were analysed. Cosinor analysis and MetaCycle analysis were performed for detection of rhythmicity. Night-time melatonin levels were significantly increased during sleep deprivation and returned to baseline levels during recovery sleep. No significant differences were observed in cortisol levels. Of 130 plasma metabolites quantified, 41 metabolites were significantly altered across the study nights, with the majority decreasing during sleep deprivation, most notably phosphatidylcholines. In cosinor analysis, 58 metabolites maintained their rhythmicity across the study days, with the majority showing a phase advance during acute sleep deprivation. This observation differs to that previously reported for males. Our study is the first of metabolic profiling in females during sleep deprivation and recovery sleep, and offers a novel view of human sleep/wake regulation and sex differences.

Machine-learning-derived sleep-wake staging from around-the-ear electroencephalogram outperforms manual scoring and actigraphy.

Quantification of sleep is important for the diagnosis of sleep disorders and sleep research. However, the only widely accepted method to obtain sleep staging is by visual analysis of polysomnography (PSG), which is expensive and time consuming. Here, we investigate automated sleep scoring based on a low-cost, mobile electroencephalogram (EEG) platform consisting of a lightweight EEG amplifier combined with flex-printed cEEGrid electrodes placed around the ear, which can be implemented as a fully self-applicable sleep system. However, cEEGrid signals have different amplitude characteristics to normal scalp PSG signals, which might be challenging for visual scoring. Therefore, this study evaluates the potential of automatic scoring of cEEGrid signals using a machine learning classifier ("random forests") and compares its performance with manual scoring of standard PSG. In addition, the automatic scoring of cEEGrid signals is compared with manual annotation of the cEEGrid recording and with simultaneous actigraphy. Acceptable recordings were obtained in 15 healthy volunteers (aged 35 ± 14.3 years) during an extended nocturnal sleep opportunity, which induced disrupted sleep with a large inter-individual variation in sleep parameters. The results demonstrate that machine-learning-based scoring of around-the-ear EEG outperforms actigraphy with respect to sleep onset and total sleep time assessments. The automated scoring outperforms human scoring of cEEGrid by standard criteria. The accuracy of machine-learning-based automated scoring of cEEGrid sleep recordings compared with manual scoring of standard PSG was satisfactory. The findings show that cEEGrid recordings combined with machine-learning-based scoring holds promise for large-scale sleep studies.

Investigation of metabolites for estimating blood deposition time.

Trace deposition timing reflects a novel concept in forensic molecular biology involving the use of rhythmic biomarkers for estimating the time within a 24-h day/night cycle a human biological sample was left at the crime scene, which in principle allows verifying a sample donor's alibi. Previously, we introduced two circadian hormones for trace deposition timing and recently demonstrated that messenger RNA (mRNA) biomarkers significantly improve time prediction accuracy. Here, we investigate the suitability of metabolites measured using a targeted metabolomics approach, for trace deposition timing. Analysis of 171 plasma metabolites collected around the clock at 2-h intervals for 36 h from 12 male participants under controlled laboratory conditions identified 56 metabolites showing statistically significant oscillations, with peak times falling into three day/night time categories: morning/noon, afternoon/evening and night/early morning. Time prediction modelling identified 10 independently contributing metabolite biomarkers, which together achieved prediction accuracies expressed as AUC of 0.81, 0.86 and 0.90 for these three time categories respectively. Combining metabolites with previously established hormone and mRNA biomarkers in time prediction modelling resulted in an improved prediction accuracy reaching AUCs of 0.85, 0.89 and 0.96 respectively. The additional impact of metabolite biomarkers, however, was rather minor as the previously established model with melatonin, cortisol and three mRNA biomarkers achieved AUC values of 0.88, 0.88 and 0.95 for the same three time categories respectively. Nevertheless, the selected metabolites could become practically useful in scenarios where RNA marker information is unavailable such as due to RNA degradation. This is the first metabolomics study investigating circulating metabolites for trace deposition timing, and more work is needed to fully establish their usefulness for this forensic purpose.

Effect of sleep deprivation on the human metabolome.

Sleep restriction and circadian clock disruption are associated with metabolic disorders such as obesity, insulin resistance, and diabetes. The metabolic pathways involved in human sleep, however, have yet to be investigated with the use of a metabolomics approach. Here we have used untargeted and targeted liquid chromatography (LC)/MS metabolomics to examine the effect of acute sleep deprivation on plasma metabolite rhythms. Twelve healthy young male subjects remained in controlled laboratory conditions with respect to environmental light, sleep, meals, and posture during a 24-h wake/sleep cycle, followed by 24 h of wakefulness. Two-hourly plasma samples collected over the 48 h period were analyzed by LC/MS. Principal component analysis revealed a clear time of day variation with a significant cosine fit during the wake/sleep cycle and during 24 h of wakefulness in untargeted and targeted analysis. Of 171 metabolites quantified, daily rhythms were observed in the majority (n = 109), with 78 of these maintaining their rhythmicity during 24 h of wakefulness, most with reduced amplitude (n = 66). During sleep deprivation, 27 metabolites (tryptophan, serotonin, taurine, 8 acylcarnitines, 13 glycerophospholipids, and 3 sphingolipids) exhibited significantly increased levels compared with during sleep. The increased levels of serotonin, tryptophan, and taurine may explain the antidepressive effect of acute sleep deprivation and deserve further study. This report, to our knowledge the first of metabolic profiling during sleep and sleep deprivation and characterization of 24 h rhythms under these conditions, offers a novel view of human sleep/wake regulation.

A Protocol for Evaluating Digital Technology for Monitoring Sleep and Circadian Rhythms in Older People and People Living with Dementia in the Community.

Sleep and circadian rhythm disturbance are predictors of poor physical and mental health, including dementia. Long-term digital technology-enabled monitoring of sleep and circadian rhythms in the community has great potential for early diagnosis, monitoring of disease progression, and assessing the effectiveness of interventions. Before novel digital technology-based monitoring can be implemented at scale, its performance and acceptability need to be evaluated and compared to gold-standard methodology in relevant populations. Here, we describe our protocol for the evaluation of novel sleep and circadian technology which we have applied in cognitively intact older adults and are currently using in people living with dementia (PLWD). In this protocol, we test a range of technologies simultaneously at home (7-14 days) and subsequently in a clinical research facility in which gold standard methodology for assessing sleep and circadian physiology is implemented. We emphasize the importance of assessing both nocturnal and diurnal sleep (naps), valid markers of circadian physiology, and that evaluation of technology is best achieved in protocols in which sleep is mildly disturbed and in populations that are relevant to the intended use-case. We provide details on the design, implementation, challenges, and advantages of this protocol, along with examples of datasets.

Human phase response curve to intermittent blue light using a commercially available device.

Light shifts the timing of the circadian clock according to a phase response curve (PRC). To date, all human light PRCs have been to long durations of bright white light. However, melanopsin, the primary photopigment for the circadian system, is most sensitive to short wavelength blue light. Therefore, to optimise light treatment it is important to generate a blue light PRC.We used a small, commercially available blue LED light box, screen size 11.2 × 6.6 cm at ∼50 cm, ∼200 µW cm(−2), ∼185 lux. Subjects participated in two 5 day laboratory sessions 1 week apart. Each session consisted of circadian phase assessments to obtain melatonin profiles before and after 3 days of free-running through an ultradian light­dark cycle (2.5 h wake in dim light, 1.5 h sleep in the dark), forced desynchrony protocol. During one session subjects received intermittent blue light (three 30 min pulses over 2 h) once a day for the 3 days of free-running, and in the other session (control) they remained in dim room light, counterbalanced. The time of blue light was varied among subjects to cover the entire 24 h day. For each individual, the phase shift to blue light was corrected for the free-run determined during the control session. The blue light PRC had a broad advance region starting in the morning and extending through the afternoon. The delay region started a few hours before bedtime and extended through the night. This is the first PRC to be constructed to blue light and to a stimulus that could be used in the real world.

Effects of the selective orexin-2 receptor antagonist JNJ-48816274 on sleep initiated in the circadian wake maintenance zone: a randomised trial.

The effects of orexinergic peptides are diverse and are mediated by orexin-1 and orexin-2 receptors. Antagonists that target both receptors have been shown to promote sleep initiation and maintenance. Here, we investigated the role of the orexin-2 receptor in sleep regulation in a randomised, double-blind, placebo-controlled, three-period crossover clinical trial using two doses (20 and 50 mg) of a highly selective orexin-2 receptor antagonist (2-SORA) (JNJ-48816274). We used a phase advance model of sleep disruption where sleep initiation is scheduled in the circadian wake maintenance zone. We assessed objective and subjective sleep parameters, pharmacokinetic profiles and residual effects on cognitive performance in 18 healthy male participants without sleep disorders. The phase advance model alone (placebo condition) resulted in disruption of sleep at the beginning of the sleep period compared to baseline sleep (scheduled at habitual time). Compared to placebo, both doses of JNJ-48816274 significantly increased total sleep time, REM sleep duration and sleep efficiency, and reduced latency to persistent sleep, sleep onset latency, and REM latency. All night EEG spectral power density for both NREM and REM sleep were unaffected by either dose. Participants reported significantly better quality of sleep and feeling more refreshed upon awakening following JNJ-48816274 compared to placebo. No significant residual effects on objective performance measures were observed and the compound was well tolerated. In conclusion, the selective orexin-2 receptor antagonist JNJ-48816274 rapidly induced sleep when sleep was scheduled earlier in the circadian cycle and improved self-reported sleep quality without impact on waking performance.

Age-related changes in acute and phase-advancing responses to monochromatic light.

Reduced sensitivity to short-wavelength (blue) light with age has been shown for light-induced melatonin suppression. The current research aimed to determine if a similar age-related reduction occurs in subjective alertness, mood, and circadian phase-advancing responses. Young (n = 11, 23.0 +/- 2.9 years) and older (n = 15, 65.8 +/- 5.0 years) healthy males participated in laboratory sessions that included a 2-h intermittent monochromatic light exposure, individually timed to begin 8.5 h after their dim light melatonin onset (DLMO) determined in a prior visit. In separate sessions, pupil-dilated subjects were exposed to short-wavelength blue (lambda max 456 nm) and medium-wavelength green (lambda max 548 nm) light matched for photon density (6 x 1013 photons/cm2/sec). Subjective alertness, sleepiness, and mood were verbally assessed every 15 to 30 min before, during, and up to 5 h after the light exposure. The magnitude of phase advance was assessed as the difference in plasma melatonin rhythm phase markers before and after light exposure. Following blue light exposure, responses in older men were significantly diminished compared with young men for subjective alertness (p < 0.0001), sleepiness (p < 0.0001), and mood (p < 0.05) during and after light exposure. There was no significant effect of age on these parameters following green light exposure. The phase advances to both blue and green light were larger in the young than older subjects, but did not reach statistical significance. In general, phase advances to blue light were slightly larger than to green light in both young and old, but did not reach statistical significance. The current results add to previous findings demonstrating reduced responsiveness to the acute effects of blue light in older people (melatonin suppression, alertness). However, under the study paradigm, the phase-advancing response to light does not appear to be significantly impaired with age.

Living Without Temporal Cues: A Case Study.

Isolation from external time cues allows endogenous circadian rhythmicity to be demonstrated. In this study, also filmed as a television documentary, we assessed rhythmic changes in a healthy man time isolated in a bunker for 9 days/nights. During this period the lighting conditions were varied between: (1) self-selected light/dark cycle, (2) constant dim light, and (3) light/dark cycle with early wake up. A range of variables was assessed and related to the sleep-wake cycle, psychomotor and physical performance and clock-time estimation. This case study using modern non-invasive monitoring techniques emphasizes how different physiological circadian rhythms persist in temporal isolation under constant dim light conditions with different waveforms, free-running with a period (τ) between 24 and 25 h. In addition, a significant correlation between time estimation and mid-sleep time, a proxy for circadian phase, was demonstrated.

Phase advancing the human circadian clock with blue-enriched polychromatic light.

BACKGROUND: Previous studies have shown that the human circadian system is maximally sensitive to short-wavelength (blue) light. Whether this sensitivity can be utilized to increase the size of phase shifts using light boxes and protocols designed for practical settings is not known. We assessed whether bright polychromatic lamps enriched in the short-wavelength portion of the visible light spectrum could produce larger phase advances than standard bright white lamps. METHODS: Twenty-two healthy young adults received either a bright white or bright blue-enriched 2-h phase advancing light pulse upon awakening on each of four treatment days. On the first treatment day the light pulse began 8h after the dim light melatonin onset (DLMO), on average about 2h before baseline wake time. On each subsequent day, light treatment began 1h earlier than the previous day, and the sleep schedule was also advanced. RESULTS: Phase advances of the DLMO for the blue-enriched (92+/-78 min, n=12) and white groups (76+/-45 min, n=10) were not significantly different. CONCLUSION: Bright blue-enriched polychromatic light is no more effective than standard bright light therapy for phase advancing circadian rhythms at commonly used therapeutic light levels.

Circadian regulation in human white adipose tissue revealed by transcriptome and metabolic network analysis.

Studying circadian rhythms in most human tissues is hampered by difficulty in collecting serial samples. Here we reveal circadian rhythms in the transcriptome and metabolic pathways of human white adipose tissue. Subcutaneous adipose tissue was taken from seven healthy males under highly controlled 'constant routine' conditions. Five biopsies per participant were taken at six-hourly intervals for microarray analysis and in silico integrative metabolic modelling. We identified 837 transcripts exhibiting circadian expression profiles (2% of 41619 transcript targeting probes on the array), with clear separation of transcripts peaking in the morning (258 probes) and evening (579 probes). There was only partial overlap of our rhythmic transcripts with published animal adipose and human blood transcriptome data. Morning-peaking transcripts associated with regulation of gene expression, nitrogen compound metabolism, and nucleic acid biology; evening-peaking transcripts associated with organic acid metabolism, cofactor metabolism and redox activity. In silico pathway analysis further indicated circadian regulation of lipid and nucleic acid metabolism; it also predicted circadian variation in key metabolic pathways such as the citric acid cycle and branched chain amino acid degradation. In summary, in vivo circadian rhythms exist in multiple adipose metabolic pathways, including those involved in lipid metabolism, and core aspects of cellular biochemistry.

Effect of Single and Combined Monochromatic Light on the Human Pupillary Light Response.

The pupillary light reflex (PLR) is a neurological reflex driven by rods, cones, and melanopsin-containing retinal ganglion cells. Our aim was to achieve a more precise picture of the effects of 5-min duration monochromatic light stimuli, alone or in combination, on the human PLR, to determine its spectral sensitivity and to assess the importance of photon flux. Using pupillometry, the PLR was assessed in 13 participants (6 women) aged 27.2 ± 5.41 years (mean ± SD) during 5-min light stimuli of purple (437 nm), blue (479 nm), red (627 nm), and combinations of red+purple or red+blue light. In addition, nine 5-min, photon-matched light stimuli, ranging in 10 nm increments peaking between 420 and 500 nm were tested in 15 participants (8 women) aged 25.7 ± 8.90 years. Maximum pupil constriction, time to achieve this, constriction velocity, area under the curve (AUC) at short (0-60 s), and longer duration (240-300 s) light exposures, and 6-s post-illumination pupillary response (6-s PIPR) were assessed. Photoreceptor activation was estimated by mathematical modeling. The velocity of constriction was significantly faster with blue monochromatic light than with red or purple light. Within the blue light spectrum (between 420 and 500 nm), the velocity of constriction was significantly faster with the 480 nm light stimulus, while the slowest pupil constriction was observed with 430 nm light. Maximum pupil constriction was achieved with 470 nm light, and the greatest AUC0-60 and AUC240-300 was observed with 490 and 460 nm light, respectively. The 6-s PIPR was maximum after 490 nm light stimulus. Both the transient (AUC0-60) and sustained (AUC240-300) response was significantly correlated with melanopic activation. Higher photon fluxes for both purple and blue light produced greater amplitude sustained pupillary constriction. The findings confirm human PLR dependence on wavelength, monochromatic or bichromatic light and photon flux under 5-min duration light stimuli. Since the most rapid and high amplitude PLR occurred within the 460-490 nm light range (alone or combined), our results suggest that color discrimination should be studied under total or partial substitution of this blue light range (460-490 nm) by shorter wavelengths (~440 nm). Thus for nocturnal lighting, replacement of blue light with purple light might be a plausible solution to preserve color discrimination while minimizing melanopic activation.

Light-induced melatonin suppression in humans with polychromatic and monochromatic light.

The relative contribution of rods, cones, and melanopsin to non-image-forming (NIF) responses under light conditions differing in irradiance, duration, and spectral composition remains to be determined in humans. NIF responses to a polychromatic light source may be very different to that predicted from the published human action spectra data, which have utilized narrow band monochromatic light and demonstrated short wavelength sensitivity. To test the hypothesis that only melanopsin is driving NIF responses in humans, monochromatic blue light (lambda(max) 479 nm) was matched with polychromatic white light for total melanopsin-stimulating photons at three light intensities. The ability of these light conditions to suppress nocturnal melatonin production was assessed. A within-subject crossover design was used to investigate the suppressive effect of nocturnal light on melatonin production in a group of diurnally active young male subjects aged 18-35 yrs (24.9+/-3.8 yrs; mean+/-SD; n=11). A 30 min light pulse, individually timed to occur on the rising phase of the melatonin rhythm, was administered between 23:30 and 01:30 h. Regularly timed blood samples were taken for measurement of plasma melatonin. Repeated measures two-way ANOVA, with irradiance and light condition as factors, was used for statistical analysis (n=9 analyzed). There was a significant effect of both light intensity (p<0.001) and light condition (p<0.01). Polychromatic light was more effective at suppressing nocturnal melatonin than monochromatic blue light matched for melanopsin stimulation, implying that the melatonin suppression response is not solely driven by melanopsin. The findings suggest a stimulatory effect of the additional wavelengths of light present in the polychromatic light, which could be mediated via the stimulation of cone photopigments and/or melanopsin regeneration. The results of this study may be relevant to designing the spectral composition of polychromatic lights for use in the home and workplace, as well as in the treatment of circadian rhythm disorders.

Modulation of Plasma Metabolite Biomarkers of the MAPK Pathway with MEK Inhibitor RO4987655: Pharmacodynamic and Predictive Potential in Metastatic Melanoma.

MAPK pathway activation is frequently observed in human malignancies, including melanoma, and is associated with sensitivity to MEK inhibition and changes in cellular metabolism. Using quantitative mass spectrometry-based metabolomics, we identified in preclinical models 21 plasma metabolites including amino acids, propionylcarnitine, phosphatidylcholines, and sphingomyelins that were significantly altered in two B-RAF-mutant melanoma xenografts and that were reversed following a single dose of the potent and selective MEK inhibitor RO4987655. Treatment of non-tumor-bearing animals and mice bearing the PTEN-null U87MG human glioblastoma xenograft elicited plasma changes only in amino acids and propionylcarnitine. In patients with advanced melanoma treated with RO4987655, on-treatment changes of amino acids were observed in patients with disease progression and not in responders. In contrast, changes in phosphatidylcholines and sphingomyelins were observed in responders. Furthermore, pretreatment levels of seven lipids identified in the preclinical screen were statistically significantly able to predict objective responses to RO4987655. The RO4987655 treatment-related changes were greater than baseline physiological variability in nontreated individuals. This study provides evidence of a translational exo-metabolomic plasma readout predictive of clinical efficacy together with pharmacodynamic utility following treatment with a signal transduction inhibitor. Mol Cancer Ther; 16(10); 2315-23. ©2017 AACR.

Advancing human circadian rhythms with afternoon melatonin and morning intermittent bright light.

CONTEXT: Both light and melatonin can be used to phase shift the human circadian clock, but the phase-advancing effect of the combination has not been extensively investigated. OBJECTIVE: The objective of the study was to determine whether phase advances induced by morning intermittent bright light and a gradually advancing sleep schedule could be increased with afternoon melatonin. PARTICIPANTS: Healthy adults (25 males, 19 females, between the ages of 19 and 45 yr) participated in the study. DESIGN: There were 3 d of a gradually advancing sleep/dark period (wake time 1 h earlier each morning), bright light on awakening [four 30-min bright-light pulses (approximately 5000 lux) alternating with 30 min room light < 60 lux] and afternoon melatonin, either 0.5 or 3.0 mg melatonin timed to induce maximal phase advances, or matching placebo. The dim light melatonin onset was measured before and after the treatment to determine the phase advance. RESULTS: There were significantly larger phase advances with 0.5 mg (2.5 h, n = 16) and 3.0 mg melatonin (2.6 h, n = 13), compared with placebo (1.7 h, n = 15), but there was no difference between the two melatonin doses. Subjects did not experience jet lag-type symptoms during the 3-d treatment CONCLUSIONS: Afternoon melatonin, morning intermittent bright light, and a gradually advancing sleep schedule advanced circadian rhythms almost 1 h/d and thus produced very little circadian misalignment. This treatment could be used in any situation in which people need to phase advance their circadian clock, such as before eastward jet travel or for delayed sleep phase syndrome.

Alerting effects of light are sensitive to very short wavelengths.

In humans a range of non-image-forming (NIF) light responses (melatonin suppression, phase shifting and alertness) are short wavelength sensitive (440-480 nm). The aim of the current study was to assess the acute effect of three different short wavelength light pulses (420, 440 and 470 nm) and 600 nm light on subjective alertness. Healthy male subjects (n = 12, aged 27 +/- 4 years, mean +/- S.D.) were studied in 39, 4-day laboratory study sessions. The subjects were maintained in dim light (<8 lx) and on day 3 they were exposed to a single 4-h light pulse (07:15-11:15 h). Four monochromatic wavelengths were administered at two photon densities: 420 and 440 nm at 2.3 x 10(13)photons/cm(2)/s and 440, 470 and 600 nm at 6.2 x 10(13)photons/cm(2)/s. Subjective mood and alertness were assessed at 30 min intervals during the light exposure, using four 9-point VAS scales. Mixed model regression analysis was used to compare alertness and mood ratings during the 470 nm light to those recorded with the other four light conditions. There was a significant effect of duration of light exposure (p < 0.001) on alertness but no significant effect of subject. Compared to 470 nm light, alertness levels were significantly higher in 420 nm light and significantly lower in the 600 nm light (p < 0.05). These data (420 nm>470 nm>600 nm) suggest that subjective alertness may be maximally sensitive to very short wavelength light.