The mammalian gastro-intestinal tract is a NOT a major extra-pineal source of melatonin.

In 1992, a paper reported that the melatonin content of the rat duodenum was 24 000 ± 2000 pg/g tissue (range: 4000-100 000 pg/g) while the pineal melatonin content was 580 000 ± 36 000 pg/g. The data has been used for the last 30 years to infer that the gut produces 400 hundred times more melatonin than the pineal gland and that it is the source of plasma melatonin during the daytime. No-one has ever challenged the statement. In this review, evidence is summarised from the literature that pinealectomy eliminates melatonin from the circulation and that studies to the contrary have relied upon poorly validated immunoassays that overstate the levels. Similarly studies that have reported increases in plasma melatonin following tryptophan administration failed to account for cross reactivity of tryptophan and its metabolites in immunoassays. The most extraordinary observation from the literature is that in those studies that have measured melatonin in the gut since 1992, the tissue content is vastly lower than the original report, even when the methodology used could be overestimating the melatonin content due to cross reactivity. Using the more contemporary results we can calculate that rather than a 400:1 ratio of duodenum: pineal melatonin, a ratio of 0.05-0.19: 1 is likely. The gut is not a major extra-pineal source of melatonin; indeed it may well not produce any.

The dim light melatonin onset across ages, methodologies, and sex and its relationship with morningness/eveningness.

The onset of melatonin secretion, the dim light melatonin onset (DLMO), is a tool for determining the phase of the circadian timing system. Although small studies have investigated the impacts of age and methods of calculating DLMO, there is no DLMO reference range. In the current study, the saliva DLMO from 3579 participants from 121 published studies and plasma DLMO from 818 healthy controls from 31 studies (aged 3-73 years) were analyzed. In a subset of 53 papers (1749 participants), individual saliva DLMO and Morningness Eveningness Questionaire (MEQ) scores were obtained from authors or mined from publications and a reference range was constructed. Saliva DLMO was earliest in children to 10 years of age and latest around 20 years of age and thereafter advanced with age by 30 min in the oldest participants. Melatonin assay methods and DLMO calculation methods had little effect on the determination of the DLMO. Saliva DLMO was correlated (p < 0.001) with the MEQ score; lower MEQ scores were associated with later DLMO. MEQ scores increased with age, reflecting a tendency toward morningness. An evaluation of 14 saliva DLMO studies of clinically diagnosed patients living with delayed sleep-wake phase disorder (mean ages 20 to 31 years) revealed mean saliva DLMO within the reference range albeit at the late extreme. Peak plasma melatonin levels from 179 studies of healthy participants revealed a high degree of variability within studies and age groups, but only a small decline between the 20 and 50 years and lowest levels after 70 years.

The role of circadian phase in sleep and performance during Antarctic winter expeditions.

The Antarctic environment presents an extreme variation in the natural light-dark cycle which can cause variability in the alignment of the circadian pacemaker with the timing of sleep, causing sleep disruption, and impaired mood and performance. This study assessed the incidence of circadian misalignment and the consequences for sleep, cognition, and psychological health in 51 over-wintering Antarctic expeditioners (45.6 ± 11.9 years) who completed daily sleep diaries, and monthly performance tests and psychological health questionnaires for 6 months. Circadian phase was assessed via monthly 48-h urine collections to assess the 6-sulphatoxymelatonin (aMT6s) rhythm. Although the average individual sleep duration was 7.2 ± 0.8 h, there was substantial sleep deficiency with 41.4% of sleep episodes <7 h and 19.1% <6 h. Circadian phase was highly variable and 34/50 expeditioners had sleep episodes that occurred at an abnormal circadian phase (acrophase outside of the sleep episode), accounting for 18.8% (295/1565) of sleep episodes. Expeditioners slept significantly less when misaligned (6.1 ± 1.3 h), compared with when aligned (7.3 ± 1.0 h; p < .0001). Performance and mood were worse when awake closer to the aMT6s peak and with increased time awake (all p < .0005). This research highlights the high incidence of circadian misalignment in Antarctic over-wintering expeditioners. Similar incidence has been observed in long-duration space flight, reinforcing the fidelity of Antarctica as a space analog. Circadian misalignment has considerable safety implications, and potentially longer term health risks for other circadian-controlled physiological systems. This increased risk highlights the need for preventative interventions, such as proactively planned lighting solutions, to ensure circadian alignment during long-duration Antarctic and space missions.

What do we really know about the safety and efficacy of melatonin for sleep disorders?

Melatonin is a hormonal product of the pineal gland, a fact that is often forgotten. Instead it is promoted as a dietary supplement that will overcome insomnia, as an antioxidant and as a prescription only drug in most countries outside the United States of America and Canada. The aim of this review is to step back and highlight what we know about melatonin following its discovery 60 years ago. What is the role of endogenous melatonin; what does melatonin do to sleep, body temperature, circadian rhythms, the cardiovascular system, reproductive system, endocrine system and metabolism when administered to healthy subjects? When used as a drug/dietary supplement, what safety studies have been conducted? Can we really say melatonin is safe when it has not been systematically studied and many studies show interactions with a wide range of physiological processes? Finally the results of studies investigating the efficacy of melatonin as a drug to alleviate insomnia are critically evaluated. In summary, melatonin is an endogenous pineal gland hormone with specific physiological functions in animals and humans, with its primary role in humans to maintain synchrony of sleep with the day/night cycle. When administered as a drug it affects a wide range of physiological systems and has clinically important drug interactions. With respect to efficacy for treating sleep disorders, melatonin can advance the time of sleep onset but the effect is modest and variable. In children with neurodevelopmental disabilities melatonin appears to have the greatest impact on sleep onset but little effect on sleep efficiency.

A Blue-Enriched, Increased Intensity Light Intervention to Improve Alertness and Performance in Rotating Night Shift Workers in an Operational Setting.

PURPOSE: This study examined the efficacy of a lighting intervention that increased both light intensity and short-wavelength (blue) light content to improve alertness, performance and mood in night shift workers in a chemical plant. PATIENTS AND METHODS: During rostered night shifts, 28 workers (46.0±10.8 years; 27 male) were exposed to two light conditions each for two consecutive nights (~19:00-07:00 h) in a counterbalanced repeated measures design: traditional-spectrum lighting set at pre-study levels (43 lux, 4000 K) versus higher intensity, blue-enriched lighting (106 lux, 17,000 K), equating to a 4.5-fold increase in melanopic illuminance (24 to 108 melanopic illuminance). Participants completed the Karolinska Sleepiness Scale, subjective mood ratings, and the Psychomotor Vigilance Task (PVT) every 2-4 hours during the night shift. RESULTS: A significant main effect of time indicated KSS, PVT mean reaction time, number of PVT lapses (reaction times > 500 ms) and subjective tension, misery and depression worsened over the course of the night shift (p<0.05). Percentage changes in KSS (p<0.05, partial η2=0.14) and PVT mean reaction time (p<0.05, partial η2=0.19) and lapses (p<0.05, partial η2=0.17) in the middle and end of night shift, expressed relative to start of shift, were significantly improved during the lighting intervention compared to the traditional lighting condition. Self-reported mood did not significantly differ between conditions (p>0.05). CONCLUSION: Our findings, showing improvements in alertness and performance with exposure to blue-enriched, increased intensity light, provide support for light to be used as a countermeasure for impaired alertness in night shift work settings.

Light-based methods for predicting circadian phase in delayed sleep-wake phase disorder.

Methods for predicting circadian phase have been developed for healthy individuals. It is unknown whether these methods generalize to clinical populations, such as delayed sleep-wake phase disorder (DSWPD), where circadian timing is associated with functional outcomes. This study evaluated two methods for predicting dim light melatonin onset (DLMO) in 154 DSWPD patients using ~ 7 days of sleep-wake and light data: a dynamic model and a statistical model. The dynamic model has been validated in healthy individuals under both laboratory and field conditions. The statistical model was developed for this dataset and used a multiple linear regression of light exposure during phase delay/advance portions of the phase response curve, as well as sleep timing and demographic variables. Both models performed comparably well in predicting DLMO. The dynamic model predicted DLMO with root mean square error of 68 min, with predictions accurate to within ± 1 h in 58% of participants and ± 2 h in 95%. The statistical model predicted DLMO with root mean square error of 57 min, with predictions accurate to within ± 1 h in 75% of participants and ± 2 h in 96%. We conclude that circadian phase prediction from light data is a viable technique for improving screening, diagnosis, and treatment of DSWPD.

Melatonin rich foods in our diet: food for thought or wishful thinking?

Melatonin continues to generate interest in the scientific community and the general public. In recent years, there has been growing interest in the possibility that melatonin present in human foods may have physiological effects. This has led to the promotion of "melatonin-rich" foods and "phyto-melatonin". The night time secretion of endogenous melatonin from the pineal gland provides a daily circadian signal which is detected by receptors in various tissues. In animals the changing circadian pattern of melatonin secretion across seasons is important to them to program their reproductive behaviours to ensure optimal reproductive success, while in humans it probably plays a prominent role in anchoring sleep to the night period. When melatonin is administered in non-physiological, milligram amounts to humans, the onset of sleep can be manipulated and in larger doses anti-oxidant properties may emerge. Melatonin-rich foods are considered in this context too, but the question remains whether the amounts of melatonin in the food can be expected to be high enough to realistically change sleep or have antioxidant properties. In this review, papers reporting the effects of ingestion of melatonin-rich food on plasma or saliva melatonin or its urinary metabolite are critically evaluated. Unfortunately many of the papers are compromised by poor experimental design and assay methodologies and uncritical evaluation of results. The conclusion drawn from this review is that it is wishful thinking to expect that the amount of melatonin in "melatonin-rich" foods will impact on sleep or have any other physiological impact.

Simulated shift work during pregnancy does not impair progeny metabolic outcomes in sheep.

KEY POINTS: Maternal shift work increases the risk of pregnancy complications, although its effects on progeny health after birth are not clear. We evaluated the impact of a simulated shift work protocol for one-third, two-thirds or all of pregnancy on the metabolic health of sheep progeny. Simulated shift work had no effect on growth, body size, body composition or glucose tolerance in pre-pubertal or young adult progeny. Glucose-stimulated insulin secretion was reduced in adult female progeny and insulin sensitivity was increased in adult female singleton progeny. The results of the present study do not support the hypothesis that maternal shift work exposure impairs metabolic health of progeny in altricial species. ABSTRACT: Disrupted maternal circadian rhythms, such as those experienced during shift work, are associated with impaired progeny metabolism in rodents. The effects of disrupted maternal circadian rhythms on progeny metabolism have not been assessed in altricial, non-litter bearing species. We therefore assessed postnatal growth from birth to adulthood, as well as body composition, glucose tolerance, insulin secretion and insulin sensitivity, in pre-pubertal and young adult progeny of sheep exposed to control conditions (CON: 10 males, 10 females) or to a simulated shift work (SSW) protocol for the first one-third (SSW0-7: 11 males, 9 females), the first two-thirds (SSW0-14: 8 males, 11 females) or all (SSW0-21: 8 males, 13 females) of pregnancy. Progeny growth did not differ between maternal treatments. In pre-pubertal progeny (12-14 weeks of age), adiposity, glucose tolerance and insulin secretion during an i.v. glucose tolerance test and insulin sensitivity did not differ between maternal treatments. Similarly, in young adult progeny (12-14 months of age), food intake, adiposity and glucose tolerance did not differ between maternal treatments. At this age, however, insulin secretion in response to a glucose bolus was 30% lower in female progeny in the combined SSW groups compared to control females (P = 0.031), and insulin sensitivity of SSW0-21 singleton females was 236% compared to that of CON singleton female progeny (P = 0.025). At least in this model, maternal SSW does not impair progeny metabolic health, with some evidence of greater insulin action in female young adult progeny.

A PERIOD3 variable number tandem repeat polymorphism modulates melatonin treatment response in delayed sleep-wake phase disorder.

We examined whether a polymorphism of the PERIOD3 gene (PER3; rs57875989) modulated the sleep-promoting effects of melatonin in Delayed Sleep-Wake Phase Disorder (DSWPD). One hundred and four individuals (53 males; 29.4 ±10.0 years) with DSWPD and a delayed dim light melatonin onset (DLMO) collected buccal swabs for genotyping (PER34/4 n = 43; PER3 5 allele [heterozygous and homozygous] n = 60). Participants were randomised to placebo or 0.5 mg melatonin taken 1 hour before desired bedtime (or ~1.45 hours before DLMO), with sleep attempted at desired bedtime (4 weeks; 5-7 nights/week). We assessed sleep (diary and actigraphy), Pittsburgh Sleep Quality Index (PSQI), Insomnia Severity Index (ISI), Patient-Reported Outcomes Measurement Information System (PROMIS: Sleep Disturbance, Sleep-Related Impairment), Sheehan Disability Scale (SDS) and Patient- and Clinician-Global Improvement (PGI-C, CGI-C). Melatonin treatment response on actigraphic sleep onset time did not differ between genotypes. For PER34/4 carriers, self-reported sleep onset time was advanced by a larger amount and sleep onset latency (SOL) was shorter in melatonin-treated patients compared to those receiving placebo (P = .008), while actigraphic sleep efficiency in the first third of the sleep episode (SE T1) did not differ. For PER3 5 carriers, actigraphic SOL and SE T1 showed a larger improvement with melatonin (P < .001). Melatonin improved ISI (P = .005), PROMIS sleep disturbance (P < .001) and sleep-related impairment (P = .017), SDS (P = .019), PGI-C (P = .028) and CGI-C (P = .016) in PER34/4 individuals only. Melatonin did not advance circadian phase. Overall, PER34/4 DSWPD patients have a greater response to melatonin treatment. PER3 genotyping may therefore improve DSWPD patient outcomes.

Measuring melatonin by immunoassay.

The pineal gland hormone melatonin continues to be of considerable interest to biomedical researchers. Of particular interest is the pattern of secretion of melatonin in relation to sleep timing as well as its potential role in certain diseases. Measuring melatonin in biological fluids such as blood and saliva presents particular methodological challenges since the production and secretion of the hormone are known to be extremely low during the light phase in almost all situations. Active secretion only occurs around the time of lights out in a wide range of species. The challenge then is to develop practical high-throughput assays that are sufficiently sensitive and accurate enough to detect levels of melatonin less than 1 pg/mL in biological fluids. Mass spectrometry assays have been developed that achieve the required sensitivity, but are really not practical or even widely available to most researchers. Melatonin radioimmunoassays and ELISA have been developed and are commercially available. But the quality of the results that are being published is very variable, partly not only because of poor experimental designs, but also because of poor assays. In this review, I discuss issues around the design of studies involving melatonin measurement. I then provide a critical assessment of 21 immunoassay kits marketed by 11 different companies with respect to validation, specificity and sensitivity. Technical managers of the companies were contacted in an attempt to obtain information not available online or in kit inserts. A search of the literature was also conducted to uncover papers that have reported the use of these assays, and where possible, both daytime and night-time plasma or saliva melatonin concentrations were extracted and tabulated. The results of the evaluations are disturbing, with many kits lacking any validation studies or using inadequate validation methods. Few assays have been properly assessed for specificity, while others report cross-reaction profiles that can be expected to result in over estimation of the melatonin levels. Some assays are not fit for purpose because they are not sensitive enough to determine plasma or saliva DLMO of 10 and 3 pg/mL, respectively. Finally, some assays produce unrealistically high daytime melatonin levels in humans and laboratory animals in the order of hundreds of pg/mL. In summary, this review provides a comprehensive and unique assessment of the current commercial melatonin immunoassays and their use in publications. It provides researchers new to the field with the information they need to design valid melatonin studies from both the perspective of experimental/clinical trial design and the best assay methodologies. It will also hopefully help journal editors and reviewers who may not be fully aware of the pitfalls of melatonin measurement make better informed decisions on publication acceptability.

Melatonin research in mice: a review.

There continues to be considerable interest in the physiological role(s) of the hormone melatonin and its potential as a drug more than 60 years after its discovery. While there is an emphasis on human studies, there is still a need for animal models in biology. Laboratory strains of mice remain at the forefront of much biomedical research for many reasons. However, in the case of melatonin research, mice represent a problem that is often not recognized by researchers entering from other fields of biomedicine. In this review, I have brought together the results of a large number of studies in which melatonin was measured in the pineal gland and blood of different mouse strains. The unequivocal conclusion to be drawn from the review is that the majority of laboratory mouse strains do not produce melatonin, whereas the CBA and C3H strains produce substantial amounts of melatonin during the late dark period. Of course, this has been known from studies conducted more than 30 years ago, but papers continue to be published that report high levels of melatonin in the pineal glands and blood and melatonin metabolites in the urine of melatonin deficient strains. Reasons for these erroneous results are discussed and center around the use of unvalidated, poorly documented immunoassays and inadequate sample preparation (i.e. direct versus extracted assays). Finally, I discuss the validity of using extraordinary high doses (as high as 200 mg/kg) of melatonin administered to melatonin deficient mice in experiments. It is hoped that the presentation of multiple sources of data in one place documenting the actual melatonin deficiency of the various strains will reduce the uncritical use of unreliable results to support the use of melatonin deficient strains in melatonin research.

Sleep regularity is associated with sleep-wake and circadian timing, and mediates daytime function in Delayed Sleep-Wake Phase Disorder.

BACKGROUND: In healthy populations, irregular sleep patterns are associated with delayed sleep and poor functional/mood outcomes. Currently, it is unknown whether irregular sleep contributes to poor functional/mood outcomes in individuals with Delayed Sleep-Wake Phase Disorder (DSWPD). METHODS: In 170 patients with DSWPD, we collected sleep-wake patterns, dim light melatonin onset (DLMO), and functional/mood outcomes. The Sleep Regularity Index (SRI) and other sleep timing metrics were computed. Correlations of SRI were computed with phase angle (difference between DLMO and desired bedtime), sleep timing and quality variables, daytime function, sleep-related daytime impairment, mood, and insomnia symptom severity. Path analyses assessed whether SRI or total sleep time mediated the associations between sleep onset time and phase angle with daytime functioning, sleep-related impairment, and mood outcomes. RESULTS: Higher SRI was associated with earlier sleep and longer total sleep time, but did not relate to sleep quality, daytime function, or mood outcomes. Path analysis showed that phase angle was directly associated with all outcome variables, whereas sleep onset time was not directly associated with any. SRI mediated the effects of sleep onset time and phase angle on daytime function. Total sleep time mediated the effects of sleep onset time and phase angle on sleep-related impairment. CONCLUSION: Individuals with DSWPD who have more delayed sleep and a greater phase angle also have more irregular sleep. This suggests that it is not delayed sleep timing per se that drives poor functional outcomes in DSWPD, but rather the timing of sleep relative to circadian phase and resultant irregular sleep patterns.

A critical review of melatonin assays: Past and present.

There has been increased interest in the measurement of melatonin in plasma and saliva recently either as a marker of circadian phase or to understand the physiological role of melatonin. For both situations, there is a need for a specific assay for melatonin that is sensitive enough to detect low concentrations (<2 pg/mL). Since the mid-1970s, there have been many assays developed to measure melatonin in blood and saliva. Radioimmunoassays and ELISA have predominated because of their relative simplicity and high throughput. In this review, I show that the early radioimmunoassays while providing valuable information about nocturnal melatonin levels in humans, generally produced inaccurate basal (daytime) levels. Mass spectrometry assays, however, have provided us with the target values that immunoassays need to achieve, that is, daytime plasma melatonin levels <1 pg/mL. There are now many contemporary commercial assays available utilising both RIA and ELISA technologies, but not all achieve the standards set by the mass spectrometry assays. The performance of these assays is reviewed. I conclude with recommendations on issues researchers need to consider when conducting melatonin studies, including the importance of time of day of collection, validation of assays, the potential causes of poor assay specificity at low levels, the advantages/disadvantages of using saliva vs plasma and extraction assays vs direct assays, kit manufacturers responsibilities and the reporting requirements when publishing melatonin studies.

Simulated shift work disrupts maternal circadian rhythms and metabolism, and increases gestation length in sheep.

KEY POINTS: Shift work impairs metabolic health, although its effects during pregnancy are not well understood We evaluated the effects of a simulated shift work protocol for one-third, two-thirds or all of pregnancy on maternal and pregnancy outcomes in sheep. Simulated shift work changed the timing of activity, disrupted hormonal and cellular rhythms, and impaired maternal glucose tolerance during early pregnancy. Gestation length was increased in twin pregnancies, whereas singleton lambs were lighter at a given gestational age if mothers were subjected to shift work conditions in the first one-third of pregnancy. Exposure to rotating night and day shifts, even if only in early pregnancy, may adversely affect maternal metabolic and pregnancy outcomes. ABSTRACT: Shift workers are at increased risk of developing type 2 diabetes and obesity; however, the impact during pregnancy on maternal metabolism is unknown. Using a large animal model, we assessed the impact of simulated shift work (SSW) exposure during pregnancy on maternal circadian rhythms, glucose tolerance and pregnancy outcomes. Following mating, ewes were randomly allocated to a control photoperiod (CON 12 h light, 12 h dark) or to SSW, where the timing of light exposure and food presentation was reversed twice each week for one-third, two-thirds or all of pregnancy. Maternal behaviour followed SSW cycles with increased activity during light exposure and feeding. Melatonin rhythms resynchronized within 2 days of the photoperiod shift, whereas peripheral circadian rhythms were arrhythmic. SSW impaired glucose tolerance (+29%, P = 0.019) and increased glucose-stimulated insulin secretion (+32%, P = 0.018) in ewes with a singleton fetus in early but not late gestation. SSW exposure did not alter rates of miscarriage or stillbirth, although it extended gestation length in twin pregnancies (+2.4 days, P = 0.032). Relative to gestational age, birth weight was lower in singleton progeny of SSW than CON ewes (-476 g, P = 0.016). These results have implications for the large number of women currently engaged in shift work, and further studies are required to determine progeny health impacts.