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This work was supported in part by the Netherlands Forensic Institute, Netherlands Genomics Initiative/Netherlands Organization for Scientific Research within the framework of the Forensic Genomics Consortium Netherlands, the 6th Framework project EUCLOCK (018741), and UK Biotechnology and Biological Sciences Research Council Grant BB/I019405/1. Additional funding was received from the Cancer Research UK Cancer Therapeutics Unit award (Ref: C2739/A22897) and a Cancer Therapeutics Centre award (Ref: C309/A25144 to FR), the NWO-STW Perspective Program grant ‘OnTime’ (project 12185 to TW and RAH). 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. Publisher PDF

Time of day variation in metabolites in healthy volunteers and in patients treated with the MEK inhibitor.

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, Young females were kept in controlled laboratory conditions comprising baseline sleep, sleep deprivation and recovery sleep and 1–2 hr blood sampling for 70 hr. Night‐time melatonin increased during sleep deprivation, returning to baseline levels during recovery sleep, whilst no significant changes were observed in cortisol. Of 130 plasma metabolites quantified by targeted metabolomics, 41 were altered across the nights (00:00–06:00 hr) and 58 maintained their rhythmicity across the three study days.

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.

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.

This study was supported by a UK Biotechnology and Biological Sciences Research Council (BBSRC) Grant (BB/I019405/1) to DJS, grant 727.011.001 from the Netherlands Organization for Scientific Research (NWO) Forensic Science Program to MK and by Erasmus MC University Medical Centre Rotterdam. DJS is a Royal Society Wolfson Research Merit Award holder. RAH and IH were funded by the Dutch applied research foundation (STW Perspectief Program ‘OnTime’ project 12185). 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. Publisher PDF

Common cold sufferers frequently report sleep disruption during the symptomatic period of infections. We examined the effects of treatment with a topical aromatic pharmaceutical ointment (Vicks VapoRub®), on associated sleep disturbances. The effects of Vicks VapoRub® versus placebo (petrolatum ointment) on subjective and objective measured sleep parameters were assessed in an exploratory study of 100 common cold patients, in a randomized, single blind, controlled, two-arm, parallel design study. The primary efficacy variable was subjective sleep quality measured with the SQSQ (Subjective Quality of Sleep Questionnaire). Additional measures included, ease of falling asleep and depth of sleep (measured with a post-sleep Visual Analog Scale), total sleep time, sleep onset latency, activity score, percentage of sleep, sleep efficiency (measured with actigraphy and SQSQ) and sleep quality index measured with a modified Karolinska Sleep Diary (KSD). The primary endpoint, “How was the quality of your sleep last night?” showed a statistically significant difference in change from baseline in favour of VapoRub treatment (p = 0.0392) versus placebo. Positive effects of VapoRub versus placebo were also observed for “How refreshed did you feel upon waking up?” (p = 0.0122) (SQSQ), “Did you get enough sleep?” (p = 0.0036) (KSD), “How was it to get up?” (p = 0.0120) (KSD) and “Do you feel well-rested?” (p = 0.0125) (KSD). No statistically significant changes from baseline versus placebo were detected in the Actiwatch endpoints. Vicks VapoRub® when applied before retiring to bed can reduce subjective sleep disturbances during a common cold. The results of this exploratory study support the belief among patients that the use of VapoRub improves subjective sleep quality during common cold which was associated with more refreshing sleep.

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.

Since there is less movement during sleep than during wake, the recording of body movements by actigraphy has been used to indirectly evaluate the sleep-wake cycle. In general, most actigraphic devices are placed on the wrist and their measures are based on acceleration detection. Here, we propose an alternative way of measuring actigraphy at the level of the arm for joint evaluation of activity and body position. This method analyzes the tilt of three axes, scoring activity as the cumulative change of degrees per minute with respect to the previous sampling, and measuring arm tilt for the body position inference. In this study, subjects (N = 13) went about their daily routine for 7 days, kept daily sleep logs, wore three ambulatory monitoring devices and collected sequential saliva samples during evenings for the measurement of dim light melatonin onset (DLMO). These devices measured motor activity (arm activity, AA) and body position (P) using the tilt sensing of the arm, with acceleration (wrist acceleration, WA) and skin temperature at wrist level (WT). Cosinor, Fourier and non-parametric rhythmic analyses were performed for the different variables, and the results were compared by the ANOVA test. Linear correlations were also performed between actimetry methods (AA and WA) and WT. The AA and WA suitability for circadian phase prediction and for evaluating the sleep-wake cycle was assessed by comparison with the DLMO and sleep logs, respectively. All correlations between rhythmic parameters obtained from AA and WA were highly significant. Only parameters related to activity levels, such as mesor, RA (relative amplitude), VL5 and VM10 (value for the 5 and 10 consecutive hours of minimum and maximum activity, respectively) showed significant differences between AA and WA records. However, when a correlation analysis was performed on the phase markers acrophase, mid-time for the 10 consecutive hours of highest (M10) and mid-time for the five consecutive hours of lowest activity (L5) with DLMO, all of them showed a significant correlation for AA (R = 0.607, p = 0.028; R = 0.582, p = 0.037; R = 0.620, p = 0.031, respectively), while for WA, only acrophase did (R = 0.621, p = 0.031). Regarding sleep detection, WA showed higher specificity than AA (0.95 ± 0.01 versus 0.86 ± 0.02), while the agreement rate and sensitivity were higher for AA (0.76 ± 0.02 versus 0.66 ± 0.02 and 0.71 ± 0.03 versus 0.53 ± 0.03, respectively). Cohen's kappa coefficient also presented the highest values for AA (0.49 ± 0.04) and AP (0.64 ± 0.04), followed by WT (0.45 ± 0.06) and WA (0.37 ± 0.04). The findings demonstrate that this alternative actigraphy method (AA), based on tilt sensing of the arm, can be used to reliably evaluate the activity and sleep-wake rhythm, since it presents a higher agreement rate and sensitivity for detecting sleep, at the same time allows the detection of body position and improves circadian phase assessment compared to the classical actigraphic method based on wrist acceleration.

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.

The increased prevalence of circadian disruptions due to abnormal coupling between internal and external time makes the detection of circadian phase in humans by ambulatory recordings a compelling need. Here, we propose an accurate practical procedure to estimate circadian phase with the least possible burden for the subject, that is, without the restraints of a constant routine protocol or laboratory techniques such as melatonin quantification, both of which are standard procedures. In this validation study, subjects (N = 13) wore ambulatory monitoring devices, kept daily sleep diaries and went about their daily routine for 10 days. The devices measured skin temperature at wrist level (WT), motor activity and body position on the arm, and light exposure by means of a sensor placed on the chest. Dim light melatonin onset (DLMO) was used to compare and evaluate the accuracy of the ambulatory variables in assessing circadian phase. An evening increase in WT: WTOnset (WTOn) and "WT increase onset" (WTiO) was found to anticipate the evening increase in melatonin, while decreases in motor activity (Activity Offset or AcOff), body position (Position Offset (POff)), integrative TAP (a combination of WT, activity and body position) (TAPOffset or TAPOff) and an increase in declared sleep propensity were phase delayed with respect to DLMO. The phase markers obtained from subjective sleep (R = 0.811), WT (R = 0.756) and the composite variable TAP (R = 0.720) were highly and significantly correlated with DLMO. The findings strongly support a new method to calculate circadian phase based on WT (WTiO) that accurately predicts and shows a temporal association with DLMO. WTiO is especially recommended due to its simplicity and applicability to clinical use under conditions where knowing endogenous circadian phase is important, such as in cancer chronotherapy and light therapy.

This study investigated the impact of sleep deprivation on the human circadian system. Plasma melatonin and cortisol levels and leukocyte expression levels of 12 genes were examined over 48 h (sleep vs. no-sleep nights) in 12 young males (mean ± SD: 23 ± 5 yrs). During one night of total sleep deprivation, BMAL1 expression was suppressed, the heat shock gene HSPA1B expression was induced, and the amplitude of the melatonin rhythm increased, whereas other high-amplitude clock gene rhythms (e.g., PER1-3, REV-ERBα) remained unaffected. These data suggest that the core clock mechanism in peripheral oscillators is compromised during acute sleep deprivation.

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.

Intrinsically photosensitive retinal ganglion cells (ipRGCs), whose photopigment melanopsin has a peak of sensitivity in the short wavelength range of the spectrum, constitute a common light input pathway to the olivary pretectal nucleus (OPN), the pupillary light reflex (PLR) regulatory centre, and to the suprachiasmatic nuclei (SCN), the major pacemaker of the circadian system. Thus, evaluating PLR under short wavelength light (λmax ≤ 500 nm) and creating an integrated PLR parameter, as a possible tool to indirectly assess the status of the circadian system, becomes of interest. Nine monochromatic, photon-matched light stimuli (300 s), in 10 nm increments from λmax 420 to 500 nm were administered to 15 healthy young participants (8 females), analyzing: i) the PLR; ii) wrist temperature (WT) and motor activity rhythms (WA), iii) light exposure (L) pattern and iv) diurnal preference (Horne-Östberg), sleep quality (Pittsburgh) and daytime sleepiness (Epworth). Linear correlations between the different PLR parameters and circadian status index obtained from WT, WA and L recordings and scores from questionnaires were calculated. In summary, we found markers of robust circadian rhythms, namely high stability, reduced fragmentation, high amplitude, phase advance and low internal desynchronization, were correlated with a reduced PLR to 460-490 nm wavelengths. Integrated circadian (CSI) and PLR (cp-PLR) parameters are proposed, that also showed an inverse correlation. These results demonstrate, for the first time, the existence of a close relationship between the circadian system robustness and the pupillary reflex response, two non-visual functions primarily under melanopsin-ipRGC input.

Summary Photoreceptive, melanopsin-expressing retinal ganglion cells (mRGCs) encode ambient light (irradiance) for the circadian clock, the pupillomotor system, and other influential behavioral/physiological responses. mRGCs are activated both by their intrinsic phototransduction cascade and by the rods and cones. However, the individual contribution of each photoreceptor class to irradiance responses remains unclear. We address this deficit using mice expressing human red cone opsin, in which rod-, cone-, and melanopsin-dependent responses can be identified by their distinct spectral sensitivity. Our data reveal an unexpectedly important role for rods. These photoreceptors define circadian responses at very dim “scotopic” light levels but also at irradiances at which pattern vision relies heavily on cones. By contrast, cone input to irradiance responses dissipates following light adaptation to the extent that these receptors make a very limited contribution to circadian and pupillary light responses under these conditions. Our data provide new insight into retinal circuitry upstream of mRGCs and optimal stimuli for eliciting irradiance responses., Highlights ► Red cone knockin (Opn1mwR) mice reveal rod, cone, and melanopsin phases to NIF vision ► Rods drive circadian responses to very low irradiances (scotopic threshold) ► Circadian responses to light in the photopic range can be rod driven ► Light adaptation limits the influence of cones on NIF vision

Conflicting evidence exists as to whether there are differences between males and females in circadian timing. The aim of the current study was to assess whether sex differences are present in the circadian regulation of melatonin and cortisol in plasma and urine matrices during a constant routine protocol. Thirty-two healthy individuals (16 females taking the oral contraceptive pill (OCP)), aged 23.8 ± 3.7 (mean ± SD) years, participated. Blood (hourly) and urine (4-hourly) samples were collected for measurement of plasma melatonin and cortisol, and urinary 6-sulfatoxymelatonin (aMT6s) and cortisol, respectively. Data from 28 individuals (14 females) showed no significant differences in the timing of plasma and urinary circadian phase markers between sexes. Females, however, exhibited significantly greater levels of plasma melatonin and cortisol than males (AUC melatonin: 937 ± 104 (mean ± SEM) vs. 642 ± 47 pg/ml.h; AUC cortisol: 13581 ± 1313 vs. 7340 ± 368 mmol/L.h). Females also exhibited a significantly higher amplitude rhythm in both hormones (melatonin: 43.8 ± 5.8 vs. 29.9 ± 2.3 pg/ml; cortisol: 241.7 ± 23.1 vs. 161.8 ± 15.9 mmol/L). Males excreted significantly more urinary cortisol than females during the CR (519.5 ± 63.8 vs. 349.2 ± 39.3 mol) but aMT6s levels did not differ between sexes. It was not possible to distinguish whether the elevated plasma melatonin and cortisol levels observed in females resulted from innate sex differences or the OCP affecting the synthetic and metabolic pathways of these hormones. The fact that the sex differences observed in total plasma concentrations for melatonin and cortisol were not reproduced in the urinary markers challenges their use as a proxy for plasma levels in circadian research, especially in OCP users.

This study was supported in part by the Netherlands Organization for Scientific Research (NWO) Forensic Science Program Grant 27.011.001, the European Union 6th Framework project EUCLOCK (018741), the UK Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/I019405/1, and Erasmus MC University Medical Center Rotterdam. D.J.S. is a Royal Society Wolfson Research Merit Award holder. The identification and investigation of novel clock-controlled genes (CCGs) has been conducted thus far mainly in model organisms such as nocturnal rodents, with limited information in humans. Here, we aimed to characterize daily and circadian expression rhythms of CCGs in human peripheral blood during a sleep/sleep deprivation (S/SD) study and a constant routine (CR) study. Blood expression levels of 9 candidate CCGs (SREBF1, TRIB1, USF1, THRA1, SIRT1, STAT3, CAPRIN1, MKNK2, and ROCK2), were measured across 48 h in 12 participants in the S/SD study and across 33 h in 12 participants in the CR study. Statistically significant rhythms in expression were observed for STAT3, SREBF1, TRIB1, and THRA1 in samples from both the S/SD and the CR studies, indicating that their rhythmicity is driven by the endogenous clock. The MKNK2 gene was significantly rhythmic in the S/SD but not the CR study, which implies its exogenously driven rhythmic expression. In addition, we confirmed the circadian expression of PER1, PER3, and REV-ERBα in the CR study samples, while BMAL1 and HSPA1B were not significantly rhythmic in the CR samples; all 5 genes previously showed significant expression in the S/SD study samples. Overall, our results demonstrate that rhythmic expression patterns of clock and selected clock-controlled genes in human blood cells are in part determined by exogenous factors (sleep and fasting state) and in part by the endogenous circadian timing system. Knowledge of the exogenous and endogenous regulation of gene expression rhythms is needed prior to the selection of potential candidate marker genes for future applications in medical and forensic settings. Postprint

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 light–dark 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.

Photoreception in the mammalian retina is not restricted to rods and cones but extends to a small number of intrinsically photosensitive retinal ganglion cells expressing the photopigment melanopsin. These mRGCs are especially important contributors to circadian entrainment, the pupil light reflex, and other so-called nonimage-forming (NIF) responses. The spectral sensitivity of melanopsin phototransduction has been addressed in several species by comparing responses to a range of monochromatic stimuli. The resultant action spectra match the predicted profile of an opsin:vitamin A–based photopigment (nomogram) with a peak sensitivity (λmax) around 480 nm. It would be most useful to be able to use this spectral sensitivity function to predict melanopsin’s sensitivity to broad-spectrum, including “white,” lights. However, evidence that melanopsin is a bistable pigment with an intrinsic light-dependent bleach recovery mechanism raises the possibility of a more complex relationship between spectral quality and photoreceptor response. Here, we set out to empirically determine whether simply weighting optical power at each wavelength according to the 480-nm nomogram and integrating across the spectrum could predict melanopsin sensitivity to a variety of polychromatic stimuli. We show that pupillomotor and circadian responses of mice relying solely on melanopsin for their photosensitivity ( rd/rd cl) can indeed be accurately predicted using this methodology. Our data therefore suggest that the 480-nm nomogram may be employed as the basis for a new photometric measure of light intensity (which we term “melanopic”) relevant for melanopsin photoreception. They further show that measuring light in these terms predicts the melanopsin response to light of divergent spectral composition much more reliably than other methods for quantifying irradiance or illuminance currently in widespread use.

The short-wavelength (blue) light sensitivity of human circadian, neurobehavioral, neuroendocrine, and neurophysiological responses is attributed to melanopsin. Whether melanopsin is the sole factor in determining the efficacy of a polychromatic light source in driving nonvisual responses, however, remains to be established. Monochromatic (λ(max) 437, 479, and 532 nm administered singly and in combination with 479 nm light) and polychromatic (color temperature: 4000 K and 17000 K) light stimuli were photon matched for their predicted ability to stimulate melanopsin, and their capacity to affect nocturnal melatonin levels, auditory reaction time, and subjective alertness and mood was assessed. Young, healthy male participants aged 18-35 yrs (23.6 ± 3.6 yrs [mean ± SD]; n=12) participated in 12 overnight sessions that included an individually timed 30-min nocturnal light stimulus on the rising limb of the melatonin profile. At regular intervals before, during, and after the light stimulus, subjective mood and alertness were verbally assessed, blood samples were taken for analysis of plasma melatonin levels, and an auditory reaction time task (psychomotor vigilance task; PVT) was performed. Proc GLM (general linear model) repeated-measures ANOVA (analysis of variance) revealed significantly lower melatonin suppression with the polychromatic light conditions (4000 and 17000 K) compared to the "melanopsin photon-matched" monochromatic light conditions (p .05). In contrast, subjective alertness was significantly lower under the 479 nm monochromatic light condition compared to the 437 and 532 nm monochromatic and both polychromatic light conditions. The alerting responses more reflected the total photon content of the light stimulus. The demonstration that the melatonin suppression response to polychromatic light was significantly lower than predicted by the melanopsin photosensitivity function suggests this function is not the sole consideration when trying to predict the efficacy of broadband lighting. The different spectral sensitivity of subjective alertness and melatonin suppression responses may imply a differential involvement of the cone photopigments. An analysis of the photon densities in specific wavelength bands for the polychromatic lights used in this and the authors' previous study suggests the spectral composition of a polychromatic light source, and particularly the very short-wavelength content, may be critical in determining response magnitude for the neuroendocrine and neurobehavioral effects of nocturnal light.

Ageing is associated with increased disturbances in the timing, duration, and quality of sleep. These disruptions may reflect changes in the circadian timing system and/or the sleep homeostat which are both necessary to produce consolidated sleep at an appropriate time. In addition, it is possible that age-related alterations in the detection and transmission of the photic signal responsible for synchronizing the circadian clock may play a role. Ageing is accompanied by many changes within the eye including alterations in pupil size, lens transmission, and number of photoreceptors. The observed increase in ocular lens density with age will diminish the transmission of short wavelength blue light to which the circadian system has been shown to be most sensitive, and may contribute, in part, to the observed increase in sleep disturbances in older people. We were the first group to test the hypothesis that non-visual responses to blue light would be impaired in older individuals. Our research has demonstrated that whilst acute non-visual effects of blue light are impaired with age, the light resetting effect appears unaltered. Future research should work towards optimizing the light environment for older people to promote good quality sleep and daytime functioning.

In our modern society, we are exposed to different artificial light sources that could potentially lead to disturbances of circadian rhythms and, hence, represent a risk for health and welfare. Investigating the acute impact of light on clock-gene expression may thus help us to better understand the mechanisms underlying disorders rooted in the circadian system. Here, we show an overall significant reduction in PER2 expression in oral mucosa with aging in the morning, noon, and afternoon. In the afternoon, 10 h after exposure to early morning blue light, PER2 was significantly elevated in the young compared to green light exposure and to older participants. Our findings demonstrate that human buccal samples are a valuable tool for studying clock-gene rhythms and the response of PER2 to light. Additionally, our results indicate that the influence of light on clock-gene expression in humans is altered with age.

The timing of clock gene expression in human leukocytes was investigated following a phase-advancing light stimulus to determine whether the response is wavelength- and/or age-dependent. PERIOD3 (PER3) clock gene expression in leukocytes and plasma melatonin were analyzed before and after monochromatic blue and green light exposure. Significant phase advances were observed in the peak timing of both PER3 expression and melatonin following blue but not green light. The amplitude of the PER3 rhythm at baseline was significantly reduced with age. However, age did not affect the response of the PER3 rhythm to light. (Author correspondence: d.skene@surrey.ac.uk)

Exogenous melatonin is increasingly used for its phase shifting and soporific effects. We generated a three pulse phase response curve (PRC) to exogenous melatonin (3 mg) by administering it to free-running subjects. Young healthy subjects (n = 27) participated in two 5 day laboratory sessions, each preceded by at least a week of habitual, but fixed sleep. Each 5 day laboratory session started and ended with a phase assessment to measure the circadian rhythm of endogenous melatonin in dim light using 30 min saliva samples. In between were three days in an ultradian dim light (< 150 lux)‐dark cycle (LD 2.5 : 1.5) during which each subject took one pill per day at the same clock time (3 mg melatonin or placebo, double blind, counterbalanced). Each individual’s phase shift to exogenous melatonin was corrected by subtracting their phase shift to placebo (a free-run). The resulting PRC has a phase advance portion peaking about 5 h before the dim light melatonin onset, in the afternoon. The phase delay portion peaks about 11 h after the dim light melatonin onset, shortly after the usual time of morning awakening. A dead zone of minimal phase shifts occurred around the first half of habitual sleep. The fitted maximum advance and delay shifts were 1.8 h and 1.3 h, respectively. This new PRC will aid in determining the optimal time to administer exogenous melatonin to achieve desired phase shifts and demonstrates that using exogenous melatonin as a sleep aid at night has minimal phase shifting effects.

Understanding how metabolite levels change over the 24 hour day is of crucial importance for clinical and epidemiological studies. Additionally, the association between sleep deprivation and metabolic disorders such as diabetes and obesity requires investigation into the links between sleep and metabolism. Here, we characterise time-of-day variation and the effects of sleep deprivation on urinary metabolite profiles. Healthy male participants (n = 15) completed an in-laboratory study comprising one 24 h sleep/wake cycle prior to 24 h of continual wakefulness under highly controlled environmental conditions. Urine samples were collected over set 2–8 h intervals and analysed by 1H NMR spectroscopy. Significant changes were observed with respect to both time of day and sleep deprivation. Of 32 identified metabolites, 7 (22%) exhibited cosine rhythmicity over at least one 24 h period; 5 exhibiting a cosine rhythm on both days. Eight metabolites significantly increased during sleep deprivation compared with sleep (taurine, formate, citrate, 3-indoxyl sulfate, carnitine, 3-hydroxyisobutyrate, TMAO and acetate) and 8 significantly decreased (dimethylamine, 4-DTA, creatinine, ascorbate, 2-hydroxyisobutyrate, allantoin, 4-DEA, 4-hydroxyphenylacetate). These data indicate that sampling time, the presence or absence of sleep and the response to sleep deprivation are highly relevant when identifying biomarkers in urinary metabolic profiling studies. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Com-mons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0

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 (p0.001) 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 (p0.05). These data (420 nm470 nm600 nm) suggest that subjective alertness may be maximally sensitive to very short wavelength light.

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.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.Healthy adults (25 males, 19 females, between the ages of 19 and 45 yr) participated in the study.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 light60 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.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 treatmentAfternoon 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.

Night shift work and rapid transmeridian travel result in a misalignment between circadian rhythms and the new times for sleep, wake, and work, which has health and safety implications for both the individual involved and the general public. Entrainment to the new sleep/wake schedule requires circadian rhythms to be phase-shifted, but this is often slow or impeded. The authors show superimposed light and melatonin PRCs to explain how to appropriately time these zeitgebers to promote circadian adaptation. They review studies in which bright light and melatonin were administered to try to counteract jet lag or to produce circadian adaptation to night work. They demonstrate how jet lag could be prevented entirely if rhythms are shifted before the flight using their preflight plan and discuss the combination of interventions that they now recommend for night shift workers.

The aim of this study was to determine whether circadian phase from salivary melatonin profiles is the same when measured in phase assessments 1 wk apart. Eleven healthy young men and women maintained a fixed, home sleep-wake schedule, in bed, in the dark 23:00-07:00 hr on weekdays. On Friday and Saturday nights they were permitted to wake up and go to bed up to 1 hr later, and on Saturdays and Sundays they could nap between 13:30 and 16:30 hr. The study was run in the summer. Subjects wore sunglasses when outside during the day, and went outside for at least 15 min between 08:00 and 09:00 hr each morning. They maintained this schedule for 15 days before the first assessment and the 6 days in between the two assessments. During the assessments subjects remained awake overnight in

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.

A recent proof-of-concept pilot study proposed using microRNA (miRNA) markers for time of death determination. The markers - miRNA-142-5p and miRNA-541, were reported to show considerable expression differences in vitreous humor between individuals who died during the day or night. Here, we investigated whether these miRNA markers show the same diurnal expression pattern in blood, which would make them useful for estimating bloodstain deposition time to allow molecular alibi testing for forensic casework. We analyzed venous blood samples collected from 12 healthy individuals every 4 h during the 24 h day/night period under controlled sleep-laboratory conditions. MiRNA-142-5p normalized against miRNA-222 showed no statistically significant expression differences between blood samples collected during daytime and nighttime (one-way ANOVA p = 0.81), and also no statistically significant rhythmicity during the 24 h day/night period (cosine fit for all individuals p > 0.05, averaged data p = 0.932). MicroRNA-541 amplification in blood was above the 34-cycle threshold applied in the study, indicating too low quantities for obtaining reliable data. Overall, we conclude that the two miRNA markers previously suggested for time of death determination in vitreous humor are not suitable for estimating the deposition time of forensic bloodstains. Future studies may find out if miRNA markers with significant diurnal expression patterns can be identified and how useful they would be for forensic trace deposition timing. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

The sleep/wake cycle is accompanied by changes in circulating numbers of immune cells. The goal of this study was to provide an in-depth characterization of diurnal rhythms in different blood cell populations and to investigate the effect of acute sleep deprivation on the immune system, as an indicator of the body's acute stress response.Observational within-subject design.Home environment and Clinical Research Centre.15 healthy male participants aged 23.7 ± 5.4 (standard deviation) yr.Total sleep deprivation.Diurnal rhythms of several blood cell populations were assessed under a normal sleep/wake cycle followed by 29 hr of extended wakefulness. The effect of condition (sleep versus sleep deprivation) on peak time and amplitude was investigated. Interindividual variation of, and the level of correlation between, the different cell populations was assessed. Comprehensive nonlinear curve fitting showed significant diurnal rhythms for all blood cell types investigated, with CD4 (naïve) cells exhibiting the most robust rhythms independent of condition. For those participants exhibiting significant diurnal rhythms in blood cell populations, only the amplitude of the granulocyte rhythm was significantly reduced by sleep deprivation. Granulocytes were the most diverse population, being most strongly affected by condition, and showed the lowest correlations with any other given cell type while exhibiting the largest interindividual variation in abundance.Granulocyte levels and diurnal rhythmicity are directly affected by acute sleep deprivation; these changes mirror the body's immediate immune response upon exposure to stress.

Context: Phase response curves (PRCs) to melatonin exist, but none compare different doses of melatonin using the same protocol. Objective: The aim was to generate a PRC to 0.5 mg of oral melatonin and compare it to our previously published 3.0 mg PRC generated using the same protocol. Design and Setting: The study included two 5-d sessions in the laboratory, each preceded by 7–9 d of fixed sleep times. Each session started and ended with a phase assessment to measure the dim light melatonin onset (DLMO). In between were 3 d in an ultradian dim 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 (λ max 456 nm) and medium-wavelength green (λmax 548 nm) light matched for photon density (6 × 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.

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±78min, n =12) and white groups (76±45min, 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.

Melatonin synthesis from the pineal gland is regulated by the circadian pacemaker located in the suprachiasmatic nuclei and by ocular light exposure. Melatonin has a circadian rhythm that peaks during the night in normally entrained individuals. In the absence of light and other synchronizing signals, the rhythm of melatonin production persists with an elevation that occurs during the subjective, as opposed to the actual, night. There is a relatively direct anatomic pathway between the suprachiasmatic nuclei and the pineal gland, and comparatively few exogenous factors are known to affect melatonin concentrations (see Table 1 from Arendt, 2005, for a summary of these factors).1 As a result, the rhythm of melatonin production has been shown to reflect both the phase and, if collected over more than 1 cycle, the period of the endogenous circadian oscillator, thus providing a reliable means to estimate the timing of the internal circadian clock located in the suprachiasmatic nuclei. The circadian melatonin rhythm is currently the most commonly used circadian phase marker in humans.2–4 A variety of methods for sampling and analyzing melatonin have been described, but there are no established guidelines on when and how these various methods should be used. The multitude of assessment methods described can be confusing to those seeking to assess circadian rhythms in their own patients. Furthermore, the variety of melatonin phase markers reported in the literature increases the difficulty of comparing results from different studies, and the lack of comparable normative values impedes evaluation of results from clinical populations. This workgroup was formed following an Associated Professional Sleep Societies workshop on the use of melatonin as a circadian phase marker. Our goal was to achieve consensus for collecting and analyzing melatonin, thereby assisting clinicians and researchers in determining which method of measuring melatonin is most appropriate for their particular needs, as well as facilitating the comparison of data among researchers and clinicians. We present here a consensus statement on the preferred uses of urine, saliva, and blood samples and discuss the advantages and disadvantages of each approach. Also discussed are factors that may affect the measurement of melatonin and the different circadian phase markers that might be derived from these methods.

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

Exogenous melatonin is increasingly used for its phase shifting and soporific effects. We generated a three pulse phase response curve (PRC) to exogenous melatonin (3 mg) by administering it to free-running subjects. Young healthy subjects (n = 27) participated in two 5 day laboratory sessions, each preceded by at least a week of habitual, but fixed sleep. Each 5 day laboratory session started and ended with a phase assessment to measure the circadian rhythm of endogenous melatonin in dim light using 30 min saliva samples. In between were three days in an ultradian dim light (< 150 lux)–dark cycle (LD 2.5 : 1.5) during which each subject took one pill per day at the same clock time (3 mg melatonin or placebo, double blind, counterbalanced). Each individual's phase shift to exogenous melatonin was corrected by subtracting their phase shift to placebo (a free-run). The resulting PRC has a phase advance portion peaking about 5 h before the dim light melatonin onset, in the afternoon. The phase delay portion peaks about 11 h after the dim light melatonin onset, shortly after the usual time of morning awakening. A dead zone of minimal phase shifts occurred around the first half of habitual sleep. The fitted maximum advance and delay shifts were 1.8 h and 1.3 h, respectively. This new PRC will aid in determining the optimal time to administer exogenous melatonin to achieve desired phase shifts and demonstrates that using exogenous melatonin as a sleep aid at night has minimal phase shifting effects.

The aim of this study was to determine whether circadian phase from salivary melatonin profiles is the same when measured in phase assessments 1 wk apart. Eleven healthy young men and women maintained a fixed, home sleep-wake schedule, in bed, in the dark 23:00-07:00 hr on weekdays. On Friday and Saturday nights they were permitted to wake up and go to bed up to 1 hr later, and on Saturdays and Sundays they could nap between 13:30 and 16:30 hr. The study was run in the summer. Subjects wore sunglasses when outside during the day, and went outside for at least 15 min between 08:00 and 09:00 hr each morning. They maintained this schedule for 15 days before the first assessment and the 6 days in between the two assessments. During the assessments subjects remained awake overnight in5 lux and gave saliva samples every 30 min. A recovery nap (13:00-17:00 hr) followed the first session. The dim light melatonin onset (DLMO), offset (DLMOff) and midpoint were used as phase markers. There was minimal change in their timing between the two phase assessments. The average absolute change in midpoint (the change in phase regardless of direction) was 20 min. There was a small, 30 min delay in the DLMO. Thus, circadian phase can be measured a week in advance of any phase shifting intervention and, as long as the prescribed sleep and morning light schedule is maintained, the phase at the start of treatment can be confidently estimated.

Introduction Since there is less movement during sleep than during wake, actigraphy permits to indirectly evaluate the sleep-wake cycle. In general, actigraphs are placed on the wrist and based on acceleration. In this validation study, we propose an alternative way of actigraphy at the level of the arm and expressed as change of degrees per minute. Its accuracy for phase prediction and for evaluating the sleep-wake cycle is assessed by comparing with the DLMO and sleep logs, respectively. Materials and methods In this study, subjects (N = 13) went about their daily routine for 7 days, kept daily sleep logs and wore ambulatory monitoring devices. These devices measured skin temperature at wrist level (WT), motor activity (Arm Activity, AA) and body position (P) on the arm and acceleration at wrist level (Wrist Acceleration, WA). Cosinor, non parametric and Fourier analysis were performed using AA and WA methods, and the results were compared by ANOVA test. Linear correlations were also performed between both methods and WT. DLMO was used to compare and correlate with estimated phase markers. Results Only mesor, RA (relative amplitude), VL5 and VM10 (value for the five and ten consecutive hours of lower and maximum activity, respectively) showed significant differences when estimated by both methods. The remaining parameters were not significantly different between both methods and all correlations between AA and WA were high. However, when phase markers were correlated with DLMO, acrophase, M10 and L5 (timing for the ten and five consecutive hours of highest and lowest activity, respectively) for AA showed a high correlation, while for WA, only acrophase did. Regarding sleep detection, WA showed higher specificity than AA, while the agreement rate and sensitivity were higher for AA. Conclusion An alternative actigraphy method (AA) can be used to evaluate more accurately sleep-wake and motor activity-rest rhythm than conventional actigraphy, since this alternative is more sensitive and presents higher agreement rate for detecting sleep. Besides, it estimates better the phase according to its correlation with the “gold standard” for phase detection: DLMO. Acknowledgements Study supported by Instituto de Salud Carlos III, RETICEF (RD12/0043/0011), MINECO (BFU 2010-21945-C02-01 and IPT-2011-0833-900000) with FEDER cofunding to JAM, and a research fellowship to MABC (FPU2009-1051). We thank Stockgrand Ltd. for melatonin assay reagents. DJ Skene is a Royal Society Wolfson Research Merit Award holder.

Introduction It is important to develop a system able to detect the endogenous circadian phase easily, since current techniques imply either expensive hormonal quantification or uncomfortable monitorization of central temperature by rectal probes. Materials and methods In this work, a new ambulatory circadian monitoring system (ACM) was validated. To this, activity and position, wrist temperature rhythm as well as light exposure pattern were recorded for 10 days in 13 healthy subjects, thanks to an actimeter on the arm, a temperature sensor on the ventral side of the wrist and a pendant luxometer. DLMO was determined in saliva, and it was used as “the gold standard” to evaluate the accuracy of ACM to assess the circadian phase. Besides, subjects completed a sleep log and Horne-Ostberg morningness–eveningness, Pittsburgh Sleep Quality Index and Epworth Sleepiness Scale questionnaires to evaluate diurnal preference and its influence in sleep quality. Results The peripheral wrist temperature increase in the evening (and its new indexes WTOnset or WTOn and WT increase onset or WTiO) anticipated that of melatonin; while the decrease in motor activity, position and the composite variable TAP (including wrist temperature, activity and position), and the increase in sleep probability occurred after the increase in melatonin levels. The score of morningness–eveningness and sleep quality questionnaires showed high correlations with DLMO, confirming that the later the DLMO occurs higher eveningness and worse sleep quality are reported. Conclusion This ambulatory method allows accurately detecting the circadian phase while subjects maintain their normal life style. The proposed new phase markers based on wrist temperature rhythm (named WTOn and WTiO) would be appropriate in Clinics when it is important to know the subject circadian phase, like in cancer chronotherapy or light therapy. Acknowledgements Study supported by Instituto de Salud Carlos III, RETICEF (RD12/0043/0011), MINECO (BFU 2010-21945-C02-01and IPT-2011-0833-900000) with FEDER cofounding to JAM, and a research fellowship to MA Bonmati-Carrion (FPU2009-1051). We also thank Stockgrand Ltd. (UK) for the melatonin assay reagents. DJ Skene is a Royal Society Wolfson Research Merit Award holder.