Victoria L. Revell

Effect of sleep deprivation on the human metabolome

Significance Sleep restriction and circadian clock disruption are associated with metabolic disorders including obesity and diabetes; this association can be studied by using the powerful tool of metabolomics. By using liquid chromatography/MS metabolomics, we have characterized plasma metabolites that were significantly affected by acute sleep deprivation (mainly lipids and acylcarnitines), all increasing during sleep deprivation. Observed increased levels of serotonin, tryptophan, and taurine may explain the antidepressive effect of sleep deprivation and deserve further study. Clear daily rhythms were observed in most metabolites, with 24 h wakefulness mainly reducing the amplitude of these rhythms. Our results further the understanding of sleep/wake regulation and the associated metabolic processes, and will be vital when using metabolic profiling to identify robust biomarkers for disease states and drug efficacy. 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.

How to Trick Mother Nature into Letting You Fly Around or Stay Up All Night

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.

A three pulse phase response curve to three milligrams of melatonin in humans

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 individuals 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.

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.

Human Phase Response Curves to Three Days of Daily Melatonin: 0.5 mg Versus 3.0 mg

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 (<150 lux)/dark cycle (light:dark, 2.5:1.5). PARTICIPANTS Healthy adults (16 men, 18 women) between the ages of 18 and 42 yr participated in the study. INTERVENTIONS During the ultradian days of the laboratory sessions, each participant took one pill per day at the same clock time (0.5 mg melatonin or placebo, double blind, counterbalanced). MAIN OUTCOME MEASURE Phase shifts to melatonin were derived by subtracting the phase shift to placebo. A PRC with time of pill administration relative to baseline DLMO and a PRC relative to midpoint of home sleep were generated. RESULTS Maximum advances occurred when 0.5 mg melatonin was taken in the afternoon, 2-4 h before the DLMO, or 9-11 h before sleep midpoint. The time for maximum phase delays was not as distinct, but a fitted curve peaked soon after wake time. CONCLUSIONS The optimal administration time for advances and delays is later for the lower dose of melatonin. When each dose of melatonin is given at its optimal time, both yield similarly sized advances and delays.

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.

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 (λmax 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.

The Physiological Period Length of the Human Circadian Clock In Vivo Is Directly Proportional to Period in Human Fibroblasts

Background Diurnal behavior in humans is governed by the period length of a circadian clock in the suprachiasmatic nuclei of the brain hypothalamus. Nevertheless, the cell-intrinsic mechanism of this clock is present in most cells of the body. We have shown previously that for individuals of extreme chronotype (“larks” and “owls”), clock properties measured in human fibroblasts correlated with extreme diurnal behavior. Methodology/Principal Findings In this study, we have measured circadian period in human primary fibroblasts taken from normal individuals and, for the first time, compared it directly with physiological period measured in vivo in the same subjects. Human physiological period length was estimated via the secretion pattern of the hormone melatonin in two different groups of sighted subjects and one group of totally blind subjects, each using different methods. Fibroblast period length was measured via cyclical expression of a lentivirally delivered circadian reporter. Within each group, a positive linear correlation was observed between circadian period length in physiology and in fibroblast gene expression. Interestingly, although blind individuals showed on average the same fibroblast clock properties as sighted ones, their physiological periods were significantly longer. Conclusions/Significance We conclude that the period of human circadian behaviour is mostly driven by cellular clock properties in normal individuals and can be approximated by measurement in peripheral cells such as fibroblasts. Based upon differences among sighted and blind subjects, we also speculate that period can be modified by prolonged unusual conditions such as the total light deprivation of blindness.

Short-wavelength sensitivity of the human circadian system to phase-advancing light.

The light-dark cycle is the most important environmental stimulus for entraining the human circadian system (reviewed in Czeisler and Wright, 1999; Mistlberger and Skene, 2004). In mice, all nonvisual light responses are maximally sensitive to wavelengths around 480 nm (Hattar et al., 2003), and in humans, a range of nonvisual light responses has been shown to short wavelengths (Brainard et al., 2001; Thapan et al., 2001; Wright and Lack, 2001; Lockley et al., 2003; Warman et al., 2003; Cajochen et al., 2004; Wright et al., 2004). However, the peak sensitivity of the human phase-shifting response has yet to be determined; in particular, the phase-shifting ability of wavelengths less than 460 nm needs to be assessed. The aim of the present study was to compare the ability of 3 short-wavelength monochromatic light pulses (420, 440, and 470 nm light) to phase advance the human circadian system in comparison to 600 nm light. Arepresentation of the protocol used is shown in Figure 1. The overall mean times of melatonin onset (Melon50%) and melatonin acrophase on night 1 (N1) were 23.3 0.4 h (n = 12) and 3.4 0.4 h (n = 12), respectively. The CT of light administration was 3.9 0.4 (n = 12). Figure 2 shows the mean phase shifts in all the phase markers under the 3 high-photon density light conditions. There was a significant difference in the phase advances observed in the SynOff (F = 4.24, df = 18, p = 0.03); phase advances with 470-nm light exposure (74.4 14.8 min) were larger than with 600 nm (–1.3 17.7 min; p < 0.05). The 440-nm light exposure gave an insignificant SynOff advance (–2.4 25.8 min; p = 0.07), but the error was too large to distinguish its lack of effect from the clear advance with 470 nm. The remaining markers also showed a greater advance at 470 nm compared to those seen using 440 nm or 600 nm, but the differences were not statistically significant. In general, phase shifts were small and variable, and there were essentially no phase shifts at 440 or 600 nm (Fig. 2). With low-photon density, there were, at best, paltry phase shifts, inconsistent in direction, and no significant differences with wavelength (data not shown). Our results indicate that the 470-nm light is more effective than the 600-nm and, perhaps, the 440-nm light in phase advancing the offset of the human melatonin rhythm. This is the 1st time that wavelengths shorter than 460 nm have been assessed for their phase-shifting ability. The results of the current study, combined with previous work (Wright et al., 2004), suggest that the phase-advancing response exhibits a similar spectral sensitivity to that observed for light-induced melatonin suppression (Brainard et al., 2001; Thapan et al., 2001), with a maximal sensitivity between 460 and 480 nm, and may involve the same proposed novel opsin-based photopigment (Hankins and Lucas, 2002). The clarity of the results from this study was likely limited by the small sample size and by the unequal impact of each subject on the data set. Furthermore, before final conclusions can be drawn, it will be necessary to complete a full-action spectrum using the same subjects for each wavelength tested. The melatonin onset and offset responded differentially to the morning light exposure, as was observed in our previous study (Warman et al., 2003), and this may reflect the putative morning and evening oscillators (Pittendrigh and Daan, 1976). However, the phase advances observed in the Meloff50% are smaller (approximately half) than those observed in our previous study (Warman et al., 2003). This is likely due to the intermittent rather than continuous exposure and the use of monochromatic light rather than a broader range of wavelengths (432-462 nm; Warman et al.,

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 (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.