There are many situations in which it would be useful to know the phase state of the biological clock. It is recognized that measurement of melatonin levels can provide this information, but traditionally blood has been used for the analysis, and there are many problems in extending the measurements into the home or field situations. The aim of this study was to develop and validate a salivary melatonin radioimmunoassay and to compare results obtained against a plasma assay for determining the onset of melatonin secretion. The assay developed was sensitive (4.3 pM) and required only 200 microliters of sample. A rhythm in melatonin was detected in saliva, peaking at approximately 120 pM or 30% of the plasma levels. Using an objective criterion for determining the onset of secretion (mean +/- 2 standard deviations of three daytime samples), the time of onset was shown to exhibit low intraindividual variability (coefficient of variation = 1.5%-4.3%). The time of onset determined using saliva was significantly correlated with the plasma onset (r = .70, p < .05). The onsets determined were 22:30 h +/- 22 min for the saliva and 21:50 h +/- 16 min for plasma for 17 subjects. Similarly, the acrophases of the saliva and plasma melatonin rhythms were significantly correlated. Neither posture alone nor changes in posture affected the calculation of the onset of melatonin secretion using the saliva approach. Very high saliva flow rates induced by citric acid resulted in lower melatonin concentrations compared to the gentle chewing on parafin film. These results firmly establish the use of salivary melatonin measurements for phase typing of the melatonin rhythm in humans.

To evaluate the effects of behavioral interventions on the sleep/wakefulness of infants, parent and infant stress, and later child emotional/behavioral problems, and parent-child attachment. METHODS:A total of 43 infants (6-16 months, 63% girls) were randomized to receive either graduated extinction (n = 14), bedtime fading (n = 15), or sleep education control (n = 14). Sleep measures included parent-reported sleep diaries and infant actigraphy. Infant stress was measured via morning and afternoon salivary cortisol sampling, and mothers' self-reported mood and stress. Twelve months after intervention, mothers completed assessments of children's emotional and behavioral problems, and mother-child dyads underwent the strange situation procedure to evaluate parent-child attachment.RESULTS: Significant interactions were found for sleep latency (P < .05), number of awakenings (P < .0001), and wake after sleep onset (P = .01), with large decreases in sleep latency for graduated extinction and bedtime fading groups, and large decreases in number of awakenings and wake after sleep onset for the graduated extinction group. Salivary cortisol showed small-to-moderate declines in graduated extinction and bedtime fading groups compared with controls. Mothers' stress showed small-to-moderate decreases for the graduated extinction and bedtime fading conditions over the first month, yet no differences in mood were detected. At the 12-month follow-up, no significant differences were found in emotional and behavioral problems, and no significant differences in secureinsecure attachment styles between groups.CONCLUSIONS: Both graduated extinction and bedtime fading provide significant sleep benefits above control, yet convey no adverse stress responses or long-term effects on parent-child attachment or child emotions and behavior. Women's and Children's Health Network, Adelaide, South Australia, Australia Dr Gradisar provided co-conception and design of the study, supervision of most of the analyses and conducted the rest of analyses, signifi cant contribution to the interpretation of analyses, and was main contributor to writing of manuscript; Dr Jackson provided co-conception and design of the study, collected most of the data (pretreatment to 3 months), conducted most of the analyses, provided signifi cant input to the literature reviewed, was a minor contributor to writing of the manuscript, and provided critical evaluation of manuscript drafts; Dr Spurrier provided co-conception and design of the study, critical evaluation of manuscript drafts, and interpretation of fi ndings; Ms Gibson provided input into study design, collected most of the 12-month follow-up data, and provided interpretation of fi ndings and critical evaluation of manuscript drafts; Dr Whitham provided input into the study design, conducted data scoring and analyses of 12-month 2 Large declines in nocturnal wakefulness occur (on average) over the first 6 months of age, and plateau thereafter, 2, 5 occurring after 24-hour circadian rhythm stabilization ...

The light/dark cycle and suprachiasmatic nucleus rhythmicity are known to have important influences on reproductive function of rodents. We studied reproductive function in female heterozygous and homozygous brain and muscle ARNT-like protein 1 (Bmal1, also known as Arntl ) null mice, which lack central and peripheral cellular rhythms. Heterozygous Bmal1 mice developed normally and were fertile, with apparent normal pregnancy progression and litter size, although postnatal mortality up to weaning was high (1.1-1.3/litter). The genotype distribution was skewed with both heterozygous and null genotypes underrepresented (1.0:1.7:0.7; P!0.05), suggesting loss of a single Bmal1 allele may impact on postnatal survival. Homozygous Bmal1 null mice were 30% lighter at weaning, and while they grew at a similar rate to the wild-type mice, they never achieved a comparable body weight. They had delayed vaginal opening (4 days), disrupted estrus cyclicity, and reduced ovarian weight (30%). Bmal1 null mice had a 40% reduction in ductal length and a 43% reduction in ductal branches in the mammary gland. Surprisingly, the Bmal1 mice ovulated, but progesterone synthesis was reduced in conjunction with altered corpora lutea formation. Pregnancy failed prior to implantation presumably due to poor embryo development. While Bmal1 null ovaries responded to pregnant mare serum gonadotropin/human chorionic gonadotropin stimulation, ovulation rate was reduced, and the fertilized oocytes progressed poorly to blastocysts and failed to implant. The loss of Bmal1 gene expression resulted in a loss of rhythmicity of many genes in the ovary and downregulation of Star. In conclusion, it is clear that the profound infertility of Bmal1 null mice is multifactorial.

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Shorter wavelength light has been shown to be more effective than longer wavelengths in suppressing nocturnal melatonin and phase delaying the melatonin rhythm. In the present study, different wavelengths of light were evaluated for their capacity to phase advance the saliva melatonin rhythm. Two long wavelengths, 595 nm (amber) and 660 nm (red) and three shorter wavelengths, 470 nm (blue), 497 nm (blue/green), and 525 nm (green) were compared with a no-light control condition. Light was administered via a portable light source comprising two light-emitting diodes per eye, with the irradiance of each diode set at 65 microW/cm(2). Forty-two volunteers participated in up to six conditions resulting in 15 per condition. For the active light conditions, a 2-hr light pulse was administered from 06:00 hr on two consecutive mornings. Half-hourly saliva samples were collected on the evening prior to the first light pulse and the evening following the second light pulse. The time of melatonin onset was calculated for each night and the difference was calculated as a measure of phase advance. The shorter wavelengths of 470, 495 and 525 nm showed the greatest melatonin onset advances ranging from approximately 40-65 min while the longer wavelengths produced no significant phase advance. These results strengthen earlier findings that the human circadian system is more sensitive to the short wavelengths of light than the longer wavelengths.

The role of peripheral vs. central circadian rhythms and Clock in the maintenance of metabolic homeostasis and with aging was examined by using Clock(Delta19)+MEL mice. These have preserved suprachiasmatic nucleus and pineal gland rhythmicity but arrhythmic Clock gene expression in the liver and skeletal muscle. Clock(Delta19)+MEL mice showed fasting hypoglycemia in young-adult males, fasting hyperglycemia in older females, and substantially impaired glucose tolerance overall. Clock(Delta19)+MEL mice had substantially reduced plasma insulin and plasma insulin/glucose nocturnally in males and during a glucose tolerance test in females, suggesting impaired insulin secretion. Clock(Delta19)+MEL mice had reduced hepatic expression and loss of rhythmicity of gck, pfkfb3, and pepck mRNA, which is likely to impair glycolysis and gluconeogenesis. Clock(Delta19)+MEL mice also had reduced glut4 mRNA in skeletal muscle, and this may contribute to poor glucose tolerance. Whole body insulin tolerance was enhanced in Clock(Delta19)+MEL mice, however, suggesting enhanced insulin sensitivity. These responses occurred although the Clock(Delta19) mutation did not cause obesity and reduced plasma free fatty acids while increasing plasma adiponectin. These studies on clock-gene disruption in peripheral tissues and metabolic homeostasis provide compelling evidence of a relationship between circadian rhythms and the glucose/insulin and adipoinsular axes. It is, however, premature to declare that clock-gene disruption causes the full metabolic syndrome.

Shift work during pregnancy is associated with an increased risk for preterm birth and low birth weight. However, the impact upon the long term health of the children is currently unknown. In this study, we used an animal model to determine the consequences of maternal shift work exposure on the health of the adult offspring. Pregnant rats were exposed to chronic phase shifts (CPS) in their photoperiod every 3–4 days throughout gestation and the first week after birth. Adult offspring were assessed for a range of metabolic, endocrine, circadian and neurobehavioural parameters. At 3 months of age, male pups exposed to the CPS schedule in utero had increased adiposity (+29%) and hyperleptinaemia (+99% at 0700h). By 12 months of age, both male and female rats displayed hyperleptinaemia (+26% and +41% respectively) and hyperinsulinaemia (+110% and +83% respectively). 12 month old female CPS rats displayed poor glucose tolerance (+18%) and increased insulin secretion (+29%) in response to an intraperitoneal glucose tolerance test. In CPS males the glucose response was unaltered, but the insulin response was reduced by 35%. The glucose response to an insulin tolerance test was decreased by 21% in CPS females but unaltered in males. Disruption of circadian rhythmicity during gestation resulted in gender dependent metabolic consequences for the adult offspring. These results highlight the need for a thorough analysis of shift work exposure in utero on the health of the adult offspring in humans.

The development of rhythmic 6-sulfatoxymelatonin excretion in urine was studied in healthy full-term and premature infants during the first 12 months of life. There was little evidence of rhythmic 6-sulfatoxymelatonin excretion before 9 to 12 weeks of age in full-term infants. Over this period, excretion increased five to six times compared to the excretion at 6 weeks (08 +/- 103 vs. 2973 +/- 438 pmol/24 h) with the major proportion of the hormone metabolite being excreted between 0200-1000 h. At 24 weeks of age, total 6-sulfatoxymelatonin excretion was 25% of adult levels. Premature infants (51 +/- 4 days premature) had a delay in the appearance of rhythmic 6-sulfatoxymelatonin of approximately 9 weeks. Even after correcting for gestational age or length of time at home, the premature infants were found to have a 2-3 week delay in the development of 6-sulfatoxymelatonin rhythmicity compared to full-term infants. These results provide evidence that neural centers responsible for rhythm generation and/or the pineal gland fail to accelerate their development after premature delivery. This may be due to the environment the infants are exposed to during their stay in hospital, particularly the pattern and intensity of lighting.