Judie English

The 3111 Clock gene polymorphism is not associated with sleep and circadian rhythmicity in phenotypically characterized human subjects

Mutations in clock genes are associated with abnormal circadian parameters, including sleep. An association has been reported previously between a polymorphism (3111C), situated in the 3′‐untranslated region (3′‐UTR) of the circadian gene Clock and evening preference. In the present study, this polymorphism was assessed in: (1) 105 control subjects with defined diurnal preference, (2) 26 blind subjects with free‐running circadian rhythms and characterized with regard to circadian period (τ) and (3) 16 delayed sleep phase syndrome patients. The control group was chosen from a larger population (n = 484) by Horne‐Östberg questionnaire analysis, from which three subgroups were selected (evening, intermediate and morning preference). Data from sleep diaries completed by 90% of these subjects showed a strong correlation between preferred and estimated timings of sleep and wake. The mean timings of activities for the evening group were at least 2 h later than the morning group. Genetic analysis showed that, in contrast with the previously published finding, there was no association between 3111C and eveningness. Neither was there an association between 3111C and τ, nor a significant difference in 3111C frequency between the normal and delayed sleep phase syndrome groups. To assess the effect of this polymorphism on messenger RNA (mRNA) translatability, luciferase reporter gene constructs containing the two Clock polymorphic variants in their 3′‐UTR were transfected into COS‐1 cells and luciferase activity measured. No significant difference was observed between the two variants. These results do not support Clock 3111C as a marker for diurnal preference, τ, or delayed sleep phase syndrome in humans.

‘Natural’ light treatment of seasonal affective disorder

Patients with seasonal affective disorder (SAD) were treated for 1 week either with a daily 1-h morning walk outdoors (natural light) or low-dose artificial light (0.5 h@2800 lux). The latter treatment (given under double-blind conditions) can be considered mainly placebo and did not improve any of the depression self-ratings, whereas natural light exposure improved all self-ratings. According to the Hamilton depression score, 25% remitted after low-dose artificial light and 50% after the walk. Sleep duration or timing were not crucial for the therapeutic response. The morning walk phase-advanced the onset and/or offset of salivary melatonin secretion, but individual clinical improvement could not be correlated with specific phase-shifts. Morning cortisol was decreased. Low-dose artificial light did not modify melatonin or cortisol patterns. This is the first study to provide evidence for the use of outdoor light exposure as a potential alternative or adjuvant to conventional artificial light therapy in SAD.

24‐HOUR PROFILES OF MELATONIN, CORTISOL, INSULIN, C‐PEPTIDE AND GIP FOLLOWING A MEAL AND SUBSEQUENT FASTING

Melatonin, free and total cortisol, insulin, C‐peptide and glucose‐dependent insulin‐releasing peptide (GIP) were measured in the plasma of twelve normal volunteers (eight women and four men), at hourly intervals for 24 h following a meal and subsequent fasting. One volunteer was excluded from calculations due to a possible effect of stress on melatonin secretion. Melatonin and cortisol showed the normal 24‐h variation with peak values at 0200–0500 h, and 0900 h respectively. Following post‐prandial stimulation, gut hormones remained basal throughout the sampling period. No significant relationship was found between 24‐h melatonin secretion and basal, or stimulated gut hormone secretion. Melatonin secretion did relate significantly to body weight, suggesting that data concerning pineal effects in endocrine physiology and pathology, and effective disease, should be reviewed in the light of these observations.

Melatonin and adjustment to phase shift

SUMMARY  The pineal hormone melatonin has clear circadian phase‐shifting effects in humans which have recently been formalized as a phase response curve. Its potential use in circadian rhythm disorders has been investigated in field studies of jet lag and shift work and in simulated phase shift. A substantial amount of information indicates that in the majority of subjects it hastens adaptation of both subjective and objective measures to forced shifts in time cues with few reported side‐effects. Field studies of its use in adaptation to shift work are sparse and preliminary but the first indications are positive. In some blind subjects with sleep disturbance it can stabilize sleep onset time without necessarily entraining all circadian rhythms and it can advance sleep timing in delayed sleep‐phase insomnia. Acute suppression of core body tempera‐ture may be an integral part of the phase‐shifting mechanism.

A novel prednisolone suppression test for the hypothalamic-pituitary-adrenal axis.

We have developed a suppressive test for the hypothalamic-pituitary-adrenal (HPA) axis using prednisolone, which is similar to endogenous glucocorticoids. We used a single-blind, repeated-measure design in healthy volunteers. In the first phase of the study, we compared placebo or prednisolone 2.5 mg, 5 mg, or 10 mg; in the second phase of the study, we compared placebo or prednisolone 5 mg or dexamethasone.5 mg. On the following day, we collected plasma and salivary cortisol levels from 9 AM to 5 PM. Maximal average prednisolone plasma levels (at 9 AM after the 10-mg dose) were 30 to 35 ng/mL. At all doses, prednisolone caused a larger suppression of salivary cortisol (approximately 20% after 2.5 mg, 30% to 35% after 5 mg, and 70% to 75% after 10 mg) than of plasma cortisol (approximately 5% after 2.5 mg, 10% after 5 mg, and 35% after 10 mg). Dexamethasone.5 mg gave 80% suppression of plasma cortisol and 90% suppression of salivary cortisol. Plasma and salivary cortisol levels were more consistently correlated in each subject after prednisolone than after dexamethasone. We propose that prednisolone at the 5-mg dosage (which gave partial HPA suppression), together with the assessment of salivary cortisol, can be used to investigate both impaired and enhanced glucocorticoid-mediated negative feedback in large samples of patients with psychiatric disorders.

Acute phase-shifting effects of melatonin associated with suppression of core body temperature in humans.

Appropriately administered melatonin is able to phase shift circadian rhythms, to induce transient sleepiness and to suppress core body temperature. The relationships between these phenomena have not been fully explored. In a double-blind, placebo-controlled crossover study, 8 healthy males maintained a regular sleep-wake cycle in a natural environment throughout. From dusk until 2400 h on days (D) 1-4 subjects were in dim artificial lighting (< 100 lux) with darkness (< 1 lux) from 2400-0800 h. Sunglasses were worn during the day when outside. At 1700 h on D3 either melatonin (5 mg) or placebo was administered. Saliva samples were collected at 30 min intervals, 1600-2400 h on D3 and D4, and subjective alertness rated at 30 min intervals from 1600-2400 h on D3 and hourly from 0800-2400 h D4. Sleep quality was rated on nights 2, 3 and 4 and core temperature was recorded throughout. Melatonin induced a significant suppression of temperature and alertness peaking 2.5 h after the dose, together with improved sleep quality on the night of D3 and a phase advance of the endogenous melatonin rhythm on D4. The degree of phase shift was related to the amount of temperature suppression in 6 of 7 subjects with detectable melatonin, suggesting that temperature suppression is an integral part of the phase-shifting mechanism.

Short-Term Variations of Circulating Melatonin in the Ewe

Melantonin levels have been studied in venous blood sampled at different frequencies (0.5‐, 2‐, 60‐min intervals) from intact ewes. All samples were taken during the dark phase of either natural or artificial photoperiods. In one experiment samples were taken simultaneously from both jugular veins to investigate the possible effects of “streaming” on the levels measured. Plasma cortisol was measured to ascertain whether or not the frequent removal of blood activated the ACTH stress axis. Plasma melantonin levels showed considerable variation with peaks of up to 365 pg/ml on a baseline of between 30 60 pg/ml. There was consistent evidence of intermittent peaks, the frequency of which increased with an increase in sampling frequency. Plasma cortisol showed no correlation with either the frequency or the amplitude of the melatonin peaks. When plasma samples were taken from both jugular veins a similar melatonin pattern was seen in the samples from both sides, but samples taken from the left jugular vein invariably showed higher levels than those taken from the right vein. This may be due to differential vascular drainage of the pineal to the two sides.

Hypogonadotrophic hypogonadism and primary amenorrhoea associated with increased melatonin secretion from a cystic pineal lesion

A 17‐year‐old girl presented with primary amenorrhoea and failure to develop secondary sexual characteristics, although her height was above the 90th centile. Endocrine investigations revealed hypogonadotrophic hypogonadism (basal LH and FSH levels <0.5 U/l; FSH rose to 2.0 U/l and LH to 1.0 U/l after GnRH). ACTH, GH, TSH and PRL secretion were normal. A magnetic resonance scan revealed no abnormality in the pituitary, pituitary stalk or hypothalamus but demonstrated a partly cystic enhancing lesion in the pineal region. Melatonin production (assessed as urinary 6‐sulphatoxymelatonin: aMT6s) at baseline was markedly increased: 459–530 ng/kg/24 h compared with aged‐matched controls: 136±69 (P=0.01). However, melatonin production retained a largely normal rhythm with increased production during the night. Treatment with ethinyloestradiol 100 μg daily had no apparent effect on the production of melatonin. Treatment with atenolol, 100 mg daily at 1600 h, was associated with suppression of nocturnal melatonin secretion but a brisk rebound in the morning and a considerably delayed peak excretion time (10.2 h) compared with controls (3.9 h). It is likely the pineal lesion, which may be hyperplasia or possibly even tumour, was responsible for the increased melatonin secretion. These data support the hypothesis that melatonin may have a causal role in hypogonadotrophic hypogonadism.

Diurnal variation in prednisolone kinetics

Plasma concentrations of free and total prednisolone were measured after oral doses at four time points to investigate the possibility of a diurnal variation in the drugs kinetics. There were marked differences in plasma prednisolone concentrations, clearance rates, and bioavailability of both free and total fractions at different times of the day. Changes in the protein binding characteristics of prednisolone with clock time resulted in marked differences between the kinetics of free and total prednisolone. It is recommended that for maximum efficacy and minimum toxicity prednisolone therapy be confined to once‐daily dosing in the morning.

Atenolol facilitates light-induced phase shifts in humans

During time-zone travel, the endogenous melatonin rhythm is often out of phase with the new local time cues. Since endogenous melatonin could act as an endogenous zeitgeber, when its secretory rhythm is out of phase it may hinder adaptation by natural zeitgebers. It is possible that by temporarily suppressing the production of melatonin, by beta-blockers for example, adaptation may be facilitated. In a double-blind, crossover study eight healthy volunteers (aged 23-30 years) took 100 mg atenolol or placebo at 1900 h on Day (D) 1. Volunteers were then exposed to bright light (approx. 1000 lux) from 0000 to 0400 h during the following night and remained in dim light (<50 lux) or darkness until 1200 h on D3. Salivary melatonin (MT) and urinary 6-sulphatoxymelatonin (aMT6s) were measured every 30-60 min and every 2 h (except when asleep), respectively. Subjective alertness and core body temperature (cBT) were also measured. aMT6s and MT were significantly suppressed under atenolol treatment on the night of D1 only. Atenolol significantly phase delayed the salivary melatonin onset by 1.8+/-0.6 h and 1.28+/-0.35 h compared with the onsets on D1 placebo leg and D2 placebo leg (i.e. onset times before and after light treatment), respectively. There were no detrimental effects on cBT or alertness. Temporary suppression of melatonin by beta-blockers may facilitate adaptation to phase shifts.