Diurnal Rhythm Of Melatonin Research Articles

Unanticipated daytime melatonin secretion on a simulated night shift schedule generates a distinctive 24-h melatonin rhythm with antiphasic daytime and nighttime peaks.

The daily rhythm of plasma melatonin concentrations is typically unimodal, with one broad peak during the circadian night and near-undetectable levels during the circadian day. Light at night acutely suppresses melatonin secretion and phase shifts its endogenous circadian rhythm. In contrast, exposure to darkness during the circadian day has not generally been reported to increase circulating melatonin concentrations acutely. Here, in a highly-controlled simulated night shift protocol with 12-h inverted behavioral/environmental cycles, we unexpectedly found that circulating melatonin levels were significantly increased during daytime sleep (p < .0001). This resulted in a secondary melatonin peak during the circadian day in addition to the primary peak during the circadian night, when sleep occurred during the circadian day following an overnight shift. This distinctive diurnal melatonin rhythm with antiphasic peaks could not be readily anticipated from the behavioral/environmental factors in the protocol (e.g., light exposure, posture, diet, activity) or from current mathematical model simulations of circadian pacemaker output. The observation, therefore, challenges our current understanding of underlying physiological mechanisms that regulate melatonin secretion. Interestingly, the increase in melatonin concentration observed during daytime sleep was positively correlated with the change in timing of melatonin nighttime peak (p = .002), but not with the degree of light-induced melatonin suppression during nighttime wakefulness (p = .92). Both the increase in daytime melatonin concentrations and the change in the timing of the nighttime peak became larger after repeated exposure to simulated night shifts (p = .002 and p = .006, respectively). Furthermore, we found that melatonin secretion during daytime sleep was positively associated with an increase in 24-h glucose and insulin levels during the night shift protocol (p = .014 and p = .027, respectively). Future studies are needed to elucidate the key factor(s) driving the unexpected daytime melatonin secretion and the melatonin rhythm with antiphasic peaks during shifted sleep/wake schedules, the underlying mechanisms of their relationship with glucose metabolism, and the relevance for diabetes risk among shift workers.

Seasonal effects on fertility in gilts and sows

The ancestral wild pig is a short day length seasonal breeder. The domestic pig appears to have retained some of this seasonality as evidenced by a reduction in fertility during the summer—autumn period. The most important aspect of this seasonality is a reduction in the number of mated sows that farrow. Many of these sows conceive and embryos develop normally for 20 - 25 days before pregnancy is terminated and the sow returns to oestrus (25 - 35 days after mating). In other species, transduction of photoperiodic information is achieved by release of melatonin during the dark period. In the pig, the pattern of melatonin secretion and the subsequent hypothalamo—pituitary—gonadal responses appear to be more complex. A relatively high light intensity is required for pigs to generate a distinct diurnal melatonin rhythm and they appear unable to respond appropriately to abrupt changes in photoperiod. Pigs on restricted feeding and maintained under long photoperiods (but not under short photoperiods) have higher concentrations of melatonin than do similarly maintained pigs fed ad libitum. Continuous release melatonin implants have a deleterious effect on farrowing rate, suggesting that the abnormally high melatonin concentrations observed in sows in summer—autumn play a role in the pathogenesis of seasonal infertility. Ad libitum feeding of sows during the first few weeks of pregnancy may prevent the increase in melatonin concentrations and so remove the seasonal influence on fertility. The pituitary response to different photoperiods is also somewhat confusing. Although there is some evidence of increased sensitivity to the negative feedback of ovarian steroids in the prepubertal gilts and weaned sows during summer—autumn, LH concentrations are increased in early pregnant sows. It is proposed that the failure of sows to maintain pregnancy in summer—autumn results from disruption of maternal recognition of pregnancy causing regression of the corpora lutea, loss of pregnancy and return of the sow to oestrus.

The Effects of Naturalistic Light on Diurnal Plasma Melatonin and Serum Cortisol Levels in Stroke Patients during Admission for Rehabilitation: A Randomized Controlled Trial.

Background: Stroke patients admitted for rehabilitation often lack sufficient daytime blue light exposure due to the absence of natural light and are often exposed to light at unnatural time points.We hypothesized that artificial light imitating daylight, termed naturalistic light, would stabilize the circadian rhythm of plasma melatonin and serum cortisol levels among long-term hospitalized stroke patients.Methods: A quasi-randomized controlled trial. Stroke patients in need of rehabilitation were randomized between May 1, 2014, and June 1, 2015 to either a rehabilitation unit equipped entirely with always on naturalistic lighting (IU), or to a rehabilitation unit with standard indoor lighting (CU). At both inclusion and discharge after a hospital stay of at least 2 weeks, plasma melatonin and serum cortisol levels were measured every 4 hours over a 24-hour period. Circadian rhythm was estimated using cosinor analysis, and variance between time-points.Results: A total of 43 were able to participate in the blood collection. Normal diurnal rhythm of melatonin was disrupted at both inclusion and discharge. In the IU group, melatonin plasma levels were increased at discharge compared to inclusion (n = 23; median diff, 2.9; IQR: -1.0 to 9.9, p = 0.030) and rhythmicity evolved (n = 23; p = 0.007). In the CU group, melatonin plasma levels were similar between discharge and inclusion and no rhythmicity evolved. Overall, both patient groups showed normal cortisol diurnal rhythms at both inclusion and discharge.Conclusions: This study is the first to demonstrate elevated melatonin plasma levels and evolved rhythmicity due to stimulation with naturalistic light.

Effects of MEK1/2 and MEK5 Pathway Disruption on Skeletal Phenotypes in Intact Female SCID Mice

Many receptor‐mediated cellular processes progress through mitogen‐activated protein kinase (MAPK) signaling pathways, which are initiated by activation of a surface receptor and ultimately lead to a change in cell physiology. Previous research has shown that the influence of melatonin on osteoblast activity is mediated in part through MAPK/ERK kinases (MEK) 1/2 and MEK5. Here we explore the possible effects of MEK 1/2 and MEK5 disruption on the skeletal structure in rodents with diurnal melatonin rhythms. To investigate the influence of MAPK signaling on bone mineral density and trabecular bone phenotypes, one group of SCID mice (SC; n = 4) was injected daily for a period of 30 days with a dual MEK1/2 and MEK5 inhibitor, SC‐1‐151. A second control group (n = 4) was injected daily with DMSO vehicle for the same period. At the end of the experimental period mice were euthanized, and tibiae were dissected free of soft tissues and fixed in formalin. Tibia were scanned at 9.61 μm3 voxel size for subsequent morphometric analyses. Regions of interest were identified on the scans and then analyzed for trabecular thickness (Tb.Th), trabecular spacing (Tb.Sp), bone volume fraction (BV/TV), and cortical bone mineral density (BMD) using ImageJ software. Resulting bone phenotypes were analyzed using Bayesian linear models. We found that the SC group showed credibly decreased BV/TV compared to the DMSO controls (Pr[SC &lt; DMSO] = 0.83). However, comparisons of Tb.Th, Tb.Sp, and BMD indicated minimal credible differences between the SC and DMSO treatment groups. Overall, our results suggest that, while previous research has shown that disruption of the MAPK signaling pathway may have a negative effect on osteoblast activity, these effects may result in only subtle changes for trabecular bone phenotypes and no change in cortical bone mineralization. This project provides preliminary results for a larger‐scale project investigating the role of melatonin, MEK1/2, and MEK5 in bone formation.Support or Funding InformationResearch support: University of Missouri School of Medicine, National Institute of Arthritis and Musculoskeletal and Skin DiseasesThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Changes in the diurnal rhythms of cortisol, melatonin, and testosterone after 2, 4, and 7 consecutive night shifts in male police officers

ABSTRACTNight work is associated with a large range of acute health problems and possibly also health consequences in the long run. Yet, only very few field studies specifically investigate the effects of consecutive night shift on key physiological regulatory systems. In this field study, we investigated the effects of consecutive night shifts on three hormones, melatonin, cortisol, and testosterone, among police officers at work. More specifically, the aim was to investigate how the diurnal rhythms of melatonin, cortisol, and testosterone responded to two, four, and seven consecutive night shifts and a corresponding number of days for recovery. The study was part of the “In the Middle of the Night” project and included 73 male police officers from five different police districts. The participants were exposed to three intervention conditions: “2+2”: two consecutive night shifts followed by two consecutive day recovery days; “4+4”: four consecutive night shifts followed by four consecutive recovery days; “7+7”: seven consecutive night shifts followed by seven consecutive recovery days. On the last day with night shift and the last recovery day in each intervention, the participants collected saliva samples every 4th hour when awake. The diurnal rhythms of melatonin, cortisol, and testosterone were all affected differently by an increasing number of consecutive night shifts: the amplitude of the melatonin rhythm was suppressed by 4.9% per day (95% CI 1.4–8.2% per day; p = 0.006). The diurnal rhythm of cortisol phase was delayed with an increasing number of night shifts by 33 min/day (95% CI 18–48 min per day; p ≤ 0.001), but did not show any changes in amplitude. For the diurnal rhythm of testosterone, there was no effect of the number of consecutive night shifts and the diurnal rhythm completely followed the sleep/wake cycle. We found that there were no differences in the rhythms of melatonin, cortisol, and testosterone after 2, 4, and 7 recovery days, respectively. In conclusion, we found signs of desynchronization in terms of suppressed amplitude of melatonin and phase delay of salivary cortisol as a consequence of the increasing number of consecutive night shifts among police officers at work. Lack of synchronization has been suggested as a possible mechanism linking night work to disease, but this remains to be determined.

Preventive effect of diurnal endogenous melatonin combined with exercise on the cartilage destruction in collagenase‐induced arthritic rats

Osteoarthritis (OA) is characterized by articular cartilage destruction, subsequent joint space narrowing, and pain. The purpose of the study is to determine effects of endogenous melatonin with exercise on the cartilage break‐down in OA rats. Rats, which were injected with type VII collagenase, were divided into two groups: OA + vehicle (Veh), and OA + exercise (Ex). Treadmill exercise (30min/day, 5 days/week) was initiated at 07:00 (Ex[7AM]), 14:00 (Ex[2PM]), and 22:00 (Ex[10PM]), depending on diurnal melatonin rhythm. Compared with the Veh, reduction was shown in the not joint thickness but width of Ex[2PM] as well as Ex[10PM] rats at 4 weeks (P&lt;0.05). MMP13 mRNA was significantly increased at 1 week (P&lt;0.05). MMP13 mRNA was the highest in the Ex[7AM] at 4 weeks, whereas its expression was the lowest in Ex[10PM] (P&lt;0.05). COL2A1 mRNA was increased in the Ex[10PM], whereas it was decreased in the Ex[7AM] as well as Ex[2PM] rats at 1 week (P&lt;0.05). Compared to unaffected side, the bone volume density of affected joint was increased in the Veh at 4 weeks (P&lt;0.05). Trabecular thickness was increased in the Veh (P&lt;0.05). However, there was no significance for both parameters in the intervention groups. These findings suggest that treadmill exercise has a prophylactic effect on articular cartilage destruction, and it is dependent on diurnal rhythm.

Adrenoceptor Expression and Diurnal Rhythms of Melatonin and Its Precursors in the Pineal Gland of Type 2 Diabetic Goto-Kakizaki Rats

A decrease in the nighttime release of the pineal hormone melatonin is associated with aging and chronic diseases in animals an humans. Melatonin has a protective role in type 2 diabetes; however, its synthesis itself is affected in the disease. The aim of this study was to detect crucially impaired steps in the pineal melatonin synthesis of type 2 diabetic Goto-Kakizaki (GK) rats. Therefore, plasma melatonin concentrations and the pineal content of melatonin and its precursors (tryptophan, 5-hydroxytryptophan, serotonin, and N-acetylserotonin) were quantified in GK rats compared with Wistar rats (each group 8 and 50 wk old) in a diurnal manner (four animals per group and per time point). Additionally, the expression of pineal adrenoceptor subtype mRNA was investigated. We found that in diabetic GK rats, 1) inhibitory alpha-2-adrenoceptors are significantly more strongly expressed than in Wistar rats, 2) the formation of 5-hydroxytryptophan is crucially impaired, and 3) the pineal gland protein content is significantly reduced compared with that in Wistar rats. This is the first time that melatonin synthesis is examined in a type 2 diabetic rat model in a diurnal manner. The present data unveil several reasons for a reduced melatonin secretion in diabetic animals and present an important link in the interaction between melatonin and insulin.

Development and validation of a high throughput direct radioimmunoassay for the quantitative determination of serum and plasma melatonin (N-acetyl-5-methoxytryptamine) in mice

A simple, sensitive and specific high throughput radioimmunoassay (RIA) for the quantitative determination of total melatonin (N-acetyl-5-methoxytryptamine) was developed. This method allows the analysis of melatonin in different biological fluids with small sample volumes (e.g. mice and rats), and wide working range. With the preparation of matrix-specific calibrators called "Equalizing Reagent" the influence of results due to different composition between standards and sample matrix was reduced. This reagent is produced by use of the respective biological liquid. Endogenous melatonin is removed by adsorption to activated charcoal. The melatonin-free biological liquid is then used to equalize the assay matrix of standards and untreated samples. Finally, all samples including the standards are digested by use of a protease to reduce non-specific binding, for example to albumin or albumin-like molecules. High-affinity specific antibodies were produced by immunization of rabbits with 5-methoxytryptamine-bovine serum albumin. The review of cross reactions to ten structurally similar compounds showed that the antibody has a high specificity for melatonin. This direct RIA uses a [(125)I]-melatonin tracer for the determination of melatonin. 5-methoxytryptamine was synthesized by direct iodination with [(125)I]-Bolton-Hunter-Reagent. The required acceptance criteria for validation parameters were fulfilled. The flexible standards cover a working range from 12 to 4000 pg/mL with a sample volume of 50 microL (e.g. working range from 3 to 1000 pg/mL with a sample volume of 200 microL). The limit of detection in mouse serum and mouse plasma was 9 pg/mL and 7 pg/mL, respectively. The recovery of melatonin in mouse serum was 108% and in mouse plasma 99%. The variation coefficients of the assay, within and between runs, ranged between 7 and 13% in mouse serum and between 5 and 8% in mouse plasma. Based on the determination of a 24-h profile of melatonin in mouse samples a characteristic diurnal rhythm of melatonin was observed. The wide working range makes it possible to analyse low and high melatonin concentrations. The validated direct RIA was compared with established sample preparation methods such as liquid-liquid- and solid-phase-extraction followed by RIA. The correlation for methanol extraction is y=1.1x-0.9, R(2)=0.98, P<0.001 and C(18)-extraction y=0.8x-0.03, R(2)=0.99, P<0.001. The distinct advantage of the direct assay of melatonin is that complicated extraction steps can be avoided. The realized advantages of the direct RIA when compared to a commercially available melatonin RIA are its low sample volume and ease of implementation. Exemplary, plasma melatonin was determined at C3H, C57BL, wild-type and melatonin receptor (MT1-/- and MT2-/-) knockout mice kept under L:D=12:12 cycles. The results have indicated that at all mouse strains have plasma melatonin content, with higher levels during the night and lower levels during the day. Because of different melatonin concentrations the direct RIA convince by its low detection limit and wide working range. The determination of serum and plasma melatonin in mice by ELISA or HPLC-technique previously failed due to the required use of a high sample volume and the low sensitivity. In summary, it can be concluded that the developed and validated direct RIA meets the requirements for the determination of melatonin and is especially suitable for the analysis of melatonin in different mouse strains.

Effects of infrared radiation on the diurnal rhythms of melatonin, rectal temperature and heart rate

Infrared radiation presented in the early night was expected to delay the circadian rhythm via suppression of melatonin synthesis. Seven healthy men (16–22 yrs) had completed a constant routine (24-h bedrest, &lt;30 lux, 18°C) and 2 experimental sessions where bright light (BL, 1 500 lux) or infrared radiation (IR, 65°C) was applied from 1700 to 0100 h. Salivary melatonin levels were determined hourly, rectal temperature and heart rate continuously. Melatonin synthesis was suppressed by BL; the onset and the maximum of the melatonin profile and the minima of rectal temperature and heart rate were then delayed thus indicating a circadian effect. IR did not affect melatonin concentration whereas the minima of rectal temperature and heart rate were delayed indicating a dissociation due to the heat-induced elevation of both the latter variables rather than a chronobiologic effect. This study does not indicate that infrared radiation affects the circadian rhythm in night shift workers.

Melatonin secretion in a strictly subterranean mammal, the Damaraland mole‐rat (Cryptomys damarensis)

AbstractSubterranean mammals inhabit an environment that is normally devoid of light and are therefore deprived of photoperiodic information that can be used to time important life‐history events. An assessment was made of whether melatonin secretion in a strictly subterranean rodent, the Damaraland mole‐rat Cryptomys damarensis, can be modified by photoperiod. In experiment 1, a clear diurnal rhythm of melatonin secretion in animals housed under a neutral photoperiod (12L:12D) was observed, with significantly higher melatonin concentrations in the dark compared to the light phase. The same diurnal melatonin rhythm was found 1 day after animals were transferred to either continuous light or continuous dark, suggesting that a circadian rhythm was maintained under acute exposure to light and dark. In experiment 2, melatonin secretion was monitored in a long (14L:10D) and short day (10L:14D) photoperiod and was found to be modified by the photoperiodic change. We therefore suggest that the Damaraland mole‐rat possesses a circadian melatonin rhythm that can be physiologically modulated in response to photoperiod.

Diurnal rhythm of melatonin in young calves and intake of melatonin in milk

AbstractPeripheral concentrations of melatonin in the neonatal mammal are low and the diurnal rhythm is not apparent during the first days or weeks of life. Maternal milk provides a rhythmic although low source of melatonin to the newborn. Diurnal rhythm of serum melatonin was monitored in Friesian calves from birth to 4 weeks of age. Some of the calves under 1 week of age had no clear nocturnal elevation of serum concentrations of melatonin. Thereafter the nocturnal melatonin elevation was apparent in all calves and of the same amplitude as in adult cattle. The effect of oral intake of night-time milk or milk supplemented with low doses of synthetic melatonin was studied during day-time in a group of Ayrshire calves aged 1 to 5 weeks. Two and a half or 3 litres of night-time milk with a mean melatonin concentration of 40 pg/ml, providing 01 to 012 μg melatonin, did not cause any change in the day-time serum concentrations of melatonin, neither did addition of melatonin in doses up to 10 μg per animal. Only the highest dose of melatonin 200 μg in 2 l of milk (01 μg/ml) caused an elevation of day-time serum concentrations of melatonin. These studies show that the pineal gland of a very youg calf is functional and that the concentration of melatonin in blood does not reflect the concentration in milk at the time of feeding. The presence of melatonin in milk suggests, however, its potential significance for the neonate.

Feeding melatonin enhances the phase shifting response to triazolam in both young and old golden hamsters.

Aging alters many aspects of circadian rhythmicity, including responsivity to phase-shifting stimuli and the amplitude of the rhythm of melatonin secretion. As melatonin is both an output from and an input to the circadian clock, we hypothesized that the decreased melatonin levels exhibited by old hamsters may adversely impact the circadian system as a whole. We enhanced the diurnal rhythm of melatonin by feeding melatonin to young and old hamsters. Animals of both age groups on the melatonin diet showed larger phase shifts than control-fed animals in response to an injection with the benzodiazepine triazolam at a circadian time known to induce phase advances in the activity rhythm of young animals. Thus melatonin treatment can increase the sensitivity of the circadian timing system of young animals to a nonphotic stimulus, and the ability to increase this sensitivity persists into old age, indicating exogenous melatonin might be useful in reversing at least some age-related changes in circadian clock function.

Chronic exposure to ELF magnetic fields during night sleep with electric sheet: effects on diurnal melatonin rhythms in men.

The possible effects of repeated night-time exposure to an extremely low frequency magnetic field (ELF-MF) on melatonin were investigated in nine healthy male subjects aged 23-37 yr. The 16-week experiment consisted of 3 weeks of pre-exposure, 11 weeks of night-time exposure to MF generated from a nonheated electric sheet (ES), and 2 weeks of post-exposure recovery observation. The average MF intensity (rms, mainly 50 Hz AC) on the surface of the sheet was 0.7 microT at the head, 8.3 microT at the waist, and 3.5 microT at the feet of the subject. For each of the urine samples collected 5 times a day on scheduled sampling days, the urinary excretion rate (ng/h) of melatonin was determined, and 24 h rhythms were extracted for each subject and each experimental period (pre-exposure, first half and latter half exposure, and post-exposure periods) by the method of complex cosine curve fitting. Although estimates of the peak height, acrophase, and total daily amount of melatonin were characterized by significant variations among individual subjects, they did not reveal any statistically significant difference between exposure periods and nonexposure periods. Thus, the present study indicates that any profound effect of the MF originating from an ES on nocturnal melatonin production and its circadian rhythm is unlikely.

Effects of Lighting Conditions and Melatonin Supplementation on the Cellular and Humoral Immune Responses in Japanese Quail Coturnix coturnix japonica

Two experiments were conducted to determine the effects of lighting conditions and melatonin supplementation on the cellular and humoral immune responses in Japanese quail. The first experiment was designed to evaluate differing light regimes as immune modulators in both adult and juvenile quail. The cellular and humoral immune responses were determined for three lighting conditions; short days (8:16LD), long days (16:8LD), and constant light (LL). In the second experiment, melatonin was administered in varying doses to adult quail placed in LL. The doses used in this experiment were 0.0, 0.5, 5.0, and 50.0 μg/ml melatonin given in the drinking water for 16 h per day for 2 weeks. The cellular and humoral immune responses were evaluated after 1 week of melatonin treatment. In both experiments, a cutaneous basophil hypersensitivity reaction to phytohemagglutinin (PHA-P) was measured to evaluate the cellular immune response. To evaluate the humoral immune response, primary antibody titers were calculated 7 days postintravenous injection with a Chukar red blood cell suspension. In the adult birds of experiment 1, both the 8:16LD and 16:8LD treatments produced similar cellular and humoral immune responses but these responses were significantly greater than those observed in LL. The juvenile birds held under 8:16LD also had significantly greater cellular and humoral immune responses as compared to juvenile birds held in LL. In experiment 2, there was a clear melatonin dose response on immune function in LL. The humoral immune response increased to a peak at the 5.0 μg/ml dose while the cellular immune response increased across all dose levels. From the present study it was clear that quail placed in daily light-dark cycles (LD), possessing a diurnal rhythm of melatonin, had significantly elevated immune responses as compared to those birds in LL. Furthermore, melatonin supplemented to birds exposed to LL was immuno-enhancing. This suggests that melatonin may be a mediator of the differences seen between LD and LL lighting conditions and may have important immune modulating properties.

Diurnal Rhythm of Melatonin in Bovine Milk: Pharmacokinetics of Exogenous Melatonin in Lactating Cows and Goats

We investigated whether the melatonin levels in bovine milk exhibit a similar daily rhythm as serum levels. In 4 Ayrshire cows at the beginning of the lactation period in May the nocturnal rise in milk melatonin was moderate (from 7 +/- 2 pg/ml at noon to 15 +/- 1 pg/ml at night; mean +/- SEM) and did not correlate well with the melatonin level in serum (from 7 +/- 2 pg/ml to 27 +/- 7 pg/ml, respectively). On the other hand, 6 cows in a later phase of lactation, studied in February, showed a clear long-lasting nocturnal melatonin increase both in serum (from 9 +/- 1 pg/ml at noon to 26 +/- 3 pg/ml at night) and in milk (from 12 +/- 5 pg/ml to 26 +/- 7 pg/ml, respectively). Melatonin kinetics during lactation was studied in more detail in 4 Ayrshire cows and 4 dairy goats by giving an intravenous bolus injection of melatonin. A 3-compartment model with melatonin elimination from the central compartment was used to describe the data. The values (mean +/- SD) for the cows and the goats were: elimination half-life 27 +/- 4 min and 27 +/- 1 min, mean residence time 24 +/- 4 min and 18 +/- 4 min, steady state distribution volume 1.0 +/- 0.3 l/kg and 0.6 +/- 0.1 1/kg (p < 0.05), and plasma clearance 0.044 +/- 0.004 l/kg/min and 0.035 +/- 0.011 l/kg/min, respectively. Following injection, the melatonin concentration in milk increased rapidly and exceeded the corresponding serum value 15-30 min later, remaining thereafter above the serum level. Our results suggest that milk melatonin levels reflect blood concentrations of melatonin with a short delay.

Radioimmunoassay of bound and free melatonin in plasma

We describe a nonextraction procedure, and two extraction procedures, for RIA of melatonin in human plasma. All procedures showed a diurnal rhythm of melatonin in human subjects, with interindividual differences greater than interprocedure differences. However, further investigations demonstrated considerable variability of recovery in the nonextraction procedure, suggesting a variability of binding proteins between samples. Combining recovery and dialysis experiments in the extraction procedures, we demonstrated that chloroform was unable to extract albumin-bound melatonin from a human serum albumin solution but, paradoxically, was able to extract bound and free melatonin from a plasma sample. The methanol extraction procedure extracted free and bound melatonin from all sources. These results indicate that albumin binding can substantially affect the RIA procedures. We conclude that assays should be validated against free and bound melatonin and that the two forms should be independently investigated when assessing bioactivity.

Melatonin and deprivation myopia in chickens

Chicken eyes elongate and become myopic if they are covered with translucent diffusors which degrade the retinal image (‘deprivation myopia’). Since it has been shown that dopamine D2 D4 receptors (which mediate inhibition of melatonin synthesis) are also implicated in deprivation myopia, we have studied the role of melatonin in the visual control of eye growth. We have found that (1) diurnal melatonin rhythms and melatonin content in the retina are unchanged during deprivation myopia development despite the breakdown of both diurnal growth rhythms of the eye and diurnal rhythms in retinal dopamine metabolism, (2) diurnal melatonin rhythms and melatonin content in the retina remain unchanged after application of the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) and presumably also after 6-hydroxydopamine (6-OHDA) application which both have a suppressive effect on deprivation myopia and (3) deprivation myopia was slightly reduced in both eyes after unilateral intravitreal injection of melatonin, despite that deprivation myopia is based on a mechanism intrinsic to the eye. We conclude that melatonin is not involved in the retinal signaling pathway translating visual experience to deprivation myopia.

Disruption of the Diurnal Rhythm of Plasma Melatonin in Cirrhosis

To assess 24-hour plasma melatonin profile as a marker of output rhythm from the circadian clock and to study sleep diaries as reflections of subjective sleep quality in patients with liver cirrhosis. Prospective cohort study. Clinical research center in a university hospital. Seven patients with cirrhosis but not alcoholism and seven age-, sex-, and education-matched controls. Neuropsychological testing to confirm subclinical hepatic encephalopathy. Plasma melatonin levels measured every 30 minutes for 24 hours by radioimmunoassay. Sleep diaries kept for 1 week before admission. Patients with cirrhosis had markedly elevated melatonin levels during daytime hours; in addition, the time of onset of melatonin increase and the time at which melatonin levels peaked were consistently and significantly delayed in these patients. Sleep diaries indicated more nocturnal awakenings and more frequent daytime naps in patients with cirrhosis. Disruption of the diurnal rhythm of melatonin may reflect alterations of circadian function that could contribute to the disturbances of the sleep-wake cycle frequently seen in patients with cirrhosis.

Evidence for hypothalamic control of the diurnal rhythms in prolactin and melatonin in the fetal sheep during late gestation.

We have investigated the effect of surgical disconnection of the fetal hypothalamus and pituitary (HPD) on generation of the daily rhythm in fetal plasma melatonin and PRL concentrations under long and short photoperiods. Fetal HPD or a sham operation was carried out at around 110 days gestation. Ewes carrying either HPD fetal sheep (n = 10) or intact fetal sheep (n = 12) were exposed to a long light (LL; 16 h of light and 8 h of darkness) or a short light (SL; 8 h of light and 16 h of darkness) regimen for the remainder of gestation. All ewes were subjected to a 24-h blood-sampling experiment (13 samples collected between 0900-0900 h the following day) between 135-140 days gestation, and fetal and maternal plasma melatonin and PRL concentrations were measured using specific RIAs. The hormonal data were analyzed using multifactorial analysis of variance and cosinor analysis. There was an increase in maternal melatonin concentrations during the dark phase in each lighting regimen in ewes carrying HPD or intact fetal sheep. In the SL regimen, there was also a significant increase in fetal melatonin concentrations during the dark phase in the HPD and intact groups. Under LL conditions, however, fetal melatonin concentrations were only consistently increased during the dark phase in the intact, not the HPD, group. The 24-h mean fetal plasma concentrations of PRL were significantly higher (P < 0.001) in both intact and HPD fetuses in the LL (intact, 111.0 +/- 22.0 pg/ml; HPD, 37.6 +/- 7.3 pg/ml) than in the SL regimen (intact, 37.8 +/- 18.4 pg/ml; HPD, 6.7 +/- 4.3 pg/ml). There was also a significant interaction (P < 0.001) between the effects of fetal surgical treatment and time of day on fetal PRL concentrations. In the intact group, fetal PRL concentrations were significantly higher (P < 0.05) at 1300 and 1700 h than between 0300-0700 h in both lighting conditions. Cosinor analysis also identified a significant rhythm in 8 of the 12 fetal PRL profiles in the intact group. In contrast, in the HPD group, there was no significant effect of time of day on fetal PRL in either the LL or SL regimen, and cosinor analysis only identified a significant rhythm in 2 of the 10 fetal PRL profiles in this group. We have, therefore, demonstrated that in the fetal sheep, HPD resulted in abolition of the diurnal melatonin rhythm under LL conditions and in the loss of the diurnal PRL rhythm under LL and SL conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

A carnivore species (Canis familiaris) expresses circadian melatonin rhythm in the peripheral blood and melatonin receptors in the brain.

Dogs kept under controlled photoperiodic conditions of 12 h light and 12 h dark expressed a clear diurnal melatonin rhythm in the peripheral blood, with a swift peak restricted to the late part of the scotophase. The highest density of high-affinity, G-protein-linked 2-[125I]iodomelatonin binding sites was found in the pars tuberalis of the pituitary gland. Binding sites were found also in the pars distalis, and light microscopy/high-resolution autoradiography showed that binding was located exclusively over the chromophobe and basophilic cells forming the adenopituitary zona tuberalis, well developed in this species, and extending into the gland as a continuation of pars tuberalis. Cords of basophilic cells located in the pars distalis proper also expressed high receptor density. The eosinophils in the adenohypophysis and the neural lobe were devoid of binding. Heavily labeled were the external laminar and the mitral cell layers of the olfactory bulbs, but no binding was detected in the filae nervi olfactorii or tractus olfactorius. The hypothalamic suprachiasmatic nuclei were discernible clearly. Quantitative autoradiography inhibition experiments revealed that the apparent melatonin inhibitory constant (IC50) in all those areas was around 0.1 nmol/l, which is a physiologically appropriate value considering the peripheral blood melatonin levels. Co-incubation with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) led to a consequential decrease in the binding density. The specific binding observed in other areas (hippocampus, frontal, parietal, occipital cortex and cerebellum) was rather weak, diffuse and could not be attributed to a particular layer; the apparent IC50 for melatonin was about 1 mumol/l, and co-incubation with GTP gamma S did not modify the binding density. Collectively, these data show that the dog possess all the prerequisites for an efficient network adapted to photoperiodic time measurements. A circadian melatonin signal in the peripheral blood and an apparently functional readout receptor system located in key positions within the brain are both present in this species.