Associations between nocturnal urinary 6-sulfatoxymelatonin,obstructive sleep apnea severity and glycemic control in type 2 diabetes

Reduced nocturnal secretion of melatonin, a pineal hormone under circadian control, and obstructive sleep apnea have been both identified as risk factors for the development of type 2 diabetes mellitus. Whether they interact to impact glycemic control in patients with existing type 2 diabetes is not known. Therefore, this study explores the relationships between obstructive sleep apnea, melatonin and glycemic control in type 2 diabetes. As diabetic retinopathy may affect melatonin secretion, we also explore the relationship between retinopathy, melatonin and glycemic control. Fifty-six non-shift workers with type 2 diabetes, who were not using beta-blockers, participated. Most recent hemoglobin A1c (HbA1c) levels and the results of ophthalmologic examinations were obtained from medical records. Obstructive sleep apnea was diagnosed using an ambulatory device. Sleep duration and fragmentation were recorded by 7-day wrist actigraphy. The urinary 6-sulfatoxymelatonin/creatinine ratio, an indicator of nocturnal melatonin secretion, was measured in an overnight urine sample. Mediation analyses were applied to explore whether low nocturnal urinary 6-sulfatoxymelatonin/creatinine ratio could be a causal link between increasing obstructive sleep apnea severity [as measured by an Apnea Hypopnea Index (AHI)] and poorer glycemic control, and between the presence of retinopathy and glycemic control. AHI and HbA1c were log-scale (ln) transformed. Obstructive sleep apnea was found in 76.8%, and 25.5% had diabetic retinopathy. The median (interquartile range) of urinary 6-sulfatoxymelatonin/creatinine ratio was 12.3 (6.0, 20.1) ng/mg. Higher lnHbA1c significantly correlated with lower 6-sulfatoxymelatonin/creatinine ratio (p = 0.04) but was not directly associated with OSA severity. More severe obstructive sleep apnea (lnAHI, p = 0.01), longer diabetes duration (p = 0.02), retinopathy (p = 0.01) and insulin use (p = 0.03) correlated with lower urinary 6-sulfatoxymelatonin/creatinine ratio, while habitual sleep duration and fragmentation did not. A mediation analysis revealed that lnAHI negatively correlated with urinary 6-sulfatoxymelatonin/creatinine ratio (coefficient = ?2.413, p = 0.03), and urinary 6-sulfatoxymelatonin/creatinine negatively associated with lnHbA1c (coefficient = ?0.005, p = 0.02), after adjusting for covariates. Mediation analysis indicated that the effect of lnAHI on lnHbA1c was indirectly mediated by urinary 6-sulfatoxymelatonin/creatinine ratio (B = 0.013, 95% CI: 0.0006, 0.0505). In addition, having retinopathy was significantly associated with reduced nocturnal urinary 6-sulfatoxymelatonin/creatinine ratio, and an increase in HbA1c by 1.013% of its original value (B = ?0.013, 95% CI: ?0.038, ?0.005). In conclusion, the presence and severity of obstructive sleep apnea as well as the presence of diabetic retinopathy were associated with lower nocturnal melatonin secretion, with an indirect adverse effect on glycemic control. Intervention studies are needed to determine whether melatonin supplementation may be beneficial in type 2 diabetes patients with obstructive sleep apnea.     

Sex differences in the circadian profiles of melatonin and cortisol in plasma and urine matrices under constant routine conditions

Conflicting evidence exists as to whether there are differences between males and females in circadian timing. The aim of the current study was to assess whether sex differences are present in the circadian regulation of melatonin and cortisol in plasma and urine matrices during a constant routine protocol. Thirty-two healthy individuals (16 females taking the oral contraceptive pill (OCP)), aged 23.8 ± 3.7 (mean ± SD) years, participated. Blood (hourly) and urine (4-hourly) samples were collected for measurement of plasma melatonin and cortisol, and urinary 6-sulfatoxymelatonin (aMT6s) and cortisol, respectively. Data from 28 individuals (14 females) showed no significant differences in the timing of plasma and urinary circadian phase markers between sexes. Females, however, exhibited significantly greater levels of plasma melatonin and cortisol than males (AUC melatonin: 937 ± 104 (mean ± SEM) vs. 642 ± 47 pg/ml.h; AUC cortisol: 13581 ± 1313 vs. 7340 ± 368 mmol/L.h). Females also exhibited a significantly higher amplitude rhythm in both hormones (melatonin: 43.8 ± 5.8 vs. 29.9 ± 2.3 pg/ml; cortisol: 241.7 ± 23.1 vs. 161.8 ± 15.9 mmol/L). Males excreted significantly more urinary cortisol than females during the CR (519.5 ± 63.8 vs. 349.2 ± 39.3 mol) but aMT6s levels did not differ between sexes. It was not possible to distinguish whether the elevated plasma melatonin and cortisol levels observed in females resulted from innate sex differences or the OCP affecting the synthetic and metabolic pathways of these hormones. The fact that the sex differences observed in total plasma concentrations for melatonin and cortisol were not reproduced in the urinary markers challenges their use as a proxy for plasma levels in circadian research, especially in OCP users.     

Magnetic fields and the melatonin hypothesis: a study of workers chronically exposed to 50-Hz magnetic fields

Because epidemiological studies report clinical disorders (mainly neurobehavioral alterations and/or cancer) that may be related to diminished melatonin secretion or to changes in its circadian rhythm in subjects living or working in environments exposed to magnetic fields, research on the effects of these fields in humans is particularly important. In this study, we examine the circadian rhythm of melatonin in 15 men exposed chronically and daily for a period of 1-20 yr, in the workplace and at home, to a 50-Hz magnetic field in search of any cumulative effect from those chronic conditions of exposure. The weekly geometric mean of individual exposures ranged from 0.1 to 2.6 microT. The results are compared with those for 15 unexposed men who served as controls (individual exposures ranged from 0.004 to 0.092 microT). Blood samples were taken hourly from 2000 to 0800. Nighttime urine was also collected and analyzed. This work shows that subjects exposed over a long period (up to 20 yr) and on a daily basis to magnetic fields experienced no changes in their plasma melatonin level, their urinary 6-sulfatoxymelatonin level, or the circadian rhythm of melatonin. Our data strongly suggest that magnetic fields do not have cumulative effects on melatonin secretion in humans and thus clearly rebut the "melatonin hypothesis" that a decrease in plasma melatonin concentration (or a disruption in its secretion) explains the occurrence of clinical disorders or cancers possibly related to magnetic fields.     

Seasonality of Pineal Melatonin Production in the Rat: Possible Synchronization by the Geomagnetic Field

Pineal melatonin production was estimated by means of urinary 6-sulfatoxymelatonin (aMT6s) determination in two groups of female rats for 1 year each. Seasonal changes of nocturnal aMT6s excretion were found with peak levels in summer despite constant photoperiods. We hypothesize that the horizontal component H of the geomagnetic field may act as a seasonal zeitgeber because H shows a similar seasonal rhythm, and changes in the direction and intensity of H can affect pineal activity. The observed seasonal changes of pineal melatonin production stress that despite constant environmental conditions, endocrine experiments require consideration of season, neglect of which may lead to contradictory results.     

Do birth variable data predict melatonin production in 8- to 9-year-old children? Analysis of excreted 6-sulfatoxymelatonin

HYPOTHESES: A cross-sectional study on urinary excretion of 6-sulfatoxymelatonin (aMT.6S) in young adults suggests a relation between melatonin production and body size at birth. As individual melatonin production remains stable during childhood and adolescence, this melatonin-birth size relation should also exist in children. METHODS: Daily urinary output of aMT.6S of 147 healthy white children (78 boys, 69 girls), 8 or 9 years of age, was quantified by ELISA and related to birth variable data. RESULTS: Contrary to expectation, aMT.6S output was not related to the ponderal index at birth but a moderate positive association with body mass index at the age of 8-9 years was seen. CONCLUSION: This study in children contradicts previous findings in adults. As no obvious reason can be identified for this discrepancy, further research (particularly a longitudinal study) is recommended to clarify whether birth variable data may predict melatonin production in certain circumstances during or after puberty.     

Urinary 6-Sulfatoxymelatonin Cycle-To-Cycle Variability

For either clinical or research purposes, the timing of the nocturnal onset in production of the urinary melatonin metabolite 6-sulfatoxymelatonin (UaMT6s-onset), has been proposed as a reliable and robust marker of circa-dian phase. However, given that most circadian rhythms show cycle-to-cycle variability, the statistical reliability of phase estimates obtained from a single study using UaMT6s-onset remains to be determined. Following 2 weeks of sleep diary and wrist actigraphy, 15 young, healthy good sleepers participated in four UaMT6s sampling sessions spaced 1 day apart. During the sampling sessions subjects remained indoors under low light conditions and hourly urine samples were collected from 19:00 to 02:00 h. Samples were subsequently assayed for UaMT6s using standard radioimmunographic techniques. UaMT6s-onset was determined by the time at which melatonin production exceeded the average of three proceeding trials by 100%. Sleep onset times were derived from sleep diary and actigraphic measures taken before the melatonin collection nights. We found that there was no significant variation between nights in group mean UaMT6s-onset times, and intraindividual variability was small. In addition, UaMT6s-onset times were highly and significantly correlated between nights (grand mean r = 0.804). Our results suggest that within 95% confidence interval limits, individual UaMT6s-onset estimates obtained from a single night UaMT6s-onset study can be used to predict subsequent UaMT6s-onset times within ±97 min. A close temporal relationship was also found between the timing of UaMT6s-onset and sleep onset. Overall, our results suggest that under entrained conditions single-session UaMT6s-onset studies can provide reliable individual UaMT6s-onset phase estimates and that the protocol described in this study is a practical and noninvasive methodology. (Chronobiology International, 13(6), 411-421, 1996)     

Urinary 6-Sulfatoxymelatonin Cycle-To-Cycle Variability

For either clinical or research purposes, the timing of the nocturnal onset in production of the urinary melatonin metabolite 6-sulfatoxymelatonin (UaMT6s-onset), has been proposed as a reliable and robust marker of circa-dian phase. However, given that most circadian rhythms show cycle-to-cycle variability, the statistical reliability of phase estimates obtained from a single study using UaMT6s-onset remains to be determined. Following 2 weeks of sleep diary and wrist actigraphy, 15 young, healthy good sleepers participated in four UaMT6s sampling sessions spaced 1 day apart. During the sampling sessions subjects remained indoors under low light conditions and hourly urine samples were collected from 19:00 to 02:00 h. Samples were subsequently assayed for UaMT6s using standard radioimmunographic techniques. UaMT6s-onset was determined by the time at which melatonin production exceeded the average of three proceeding trials by 100%. Sleep onset times were derived from sleep diary and actigraphic measures taken before the melatonin collection nights. We found that there was no significant variation between nights in group mean UaMT6s-onset times, and intraindividual variability was small. In addition, UaMT6s-onset times were highly and significantly correlated between nights (grand mean r = 0.804). Our results suggest that within 95% confidence interval limits, individual UaMT6s-onset estimates obtained from a single night UaMT6s-onset study can be used to predict subsequent UaMT6s-onset times within ±97 min. A close temporal relationship was also found between the timing of UaMT6s-onset and sleep onset. Overall, our results suggest that under entrained conditions single-session UaMT6s-onset studies can provide reliable individual UaMT6s-onset phase estimates and that the protocol described in this study is a practical and noninvasive methodology. (Chronobiology International, 13(6), 411–421, 1996)     

Melatonin production and light exposure of rotating night workers

Decreased melatonin production, due to acute suppression of pineal melatonin secretion by light exposure during night work, has been suggested to underlie higher cancer risks associated with prolonged experience of night work. However, the association between light exposure and melatonin production has never been measured in the field. In this study, 24-h melatonin production and ambulatory light exposure were assessed during both night-shift and day/evening-shift periods in 13 full-time rotating shiftworkers. Melatonin production was estimated with the excretion of urinary 6-sulfatoxymelatonin (aMT6s), and light exposure was measured with an ambulatory photometer. There was no difference in total 24-h aMT6s excretion between the two work periods. The night-shift period was characterized by a desynchrony between melatonin and sleep-wake rhythms, as shown by higher melatonin production during work and lower melatonin production during sleep when working night shifts than when working day/evening shifts. Light exposure during night work showed no correlation with aMT6s excreted during the night of work (p?>?.5), or with the difference in 24-h aMT6s excretion between the two work periods (p >?.1). However, light exposure during night work was negatively correlated with total 24-h aMT6s excretion over the entire night-shift period (p?相似文献   

Emergence and evolution of the circadian rhythm of melatonin in children

OBJECTIVE: To assess the age at which the circadian rhythm of melatonin begins. METHODS: 55 children, divided into groups from the neonatal period to 24 months of life, were studied. Urine samples were taken from 28 newborn babies to measure 6-sulfatoxymelatonin (aMT6s). Salivary samples were collected from infants (27 cases), to measure melatonin (aMT). aMT was measured by RIA and aMT6s by ELISA using commercial kits. Changes in the levels of aMT6s and aMT were evaluated using the Friedman test and Wilcoxon matched pair test. RESULTS: The group aged 27-41 days showed statistically significant differences in daily aMT6s and aMT concentrations. The highest values were always found between 24.00 and 8.00 h. This day/night difference persisted from 2-3 to 13-24 months of age. CONCLUSION: The data indicate that the circadian melatonin rhythm appears at the end of the neonatal period and persists thereafter.     

Epigenetic modification in 4T1 mouse breast cancer model by artificial light at night and melatonin – the role of DNA-methyltransferase

Currently, one of the most disputed hypotheses regarding breast cancer (BC) development is exposure to short wavelength artificial light at night (ALAN) as multiple studies suggest a possible link between them. This link is suggested to be mediated by nocturnal melatonin suppression that plays an integral role in circadian regulations including cell division. The objective of the research was to evaluate effects of 1 × 30 min/midnight ALAN (134 µ Wcm^?2, 460 nm) with or without nocturnal melatonin supplement on tumor development and epigenetic responses in 4T1 tumor-bearing BALB/c mice. Mice were monitored for body mass (W_b) and tumor volume for 3 weeks and thereafter urine samples were collected at regular intervals for determining daily rhythms of 6-sulfatoxymelatonin (6-SMT). Finally, mice were sacrificed and the tumor, lungs, liver, and spleen were excised for analyzing the total activity of DNA methyltransferases (DNMT) and global DNA methylation (GDM) levels. Mice exposed to ALAN significantly reduced 6-SMT levels and increased W_b, tumor volume, and lung metastasis compared with controls. These effects were diminished by melatonin. The DNMT activity and GDM levels showed tissue-specific response. The enzymatic activity and GDM levels were lower in tumor and liver and higher in spleen and lungs under ALAN compared with controls. Our results suggest that ALAN disrupts the melatonin rhythm and potentially leading to increased BC burden by affecting DNMT activity and GDM levels. These data may also be applicable to early detection and management of BC by monitoring melatonin and GDM levels as early biomarker of ALAN circadian disruption.     

Inhibitory action of exogenous melatonin, 5-methoxytryptamine, and 6-hydroxymelatonin on sexual maturation of male rats: activity of 5-methoxytryptamine might be due to its conversion to melatonin

The effect on sexual maturation of 6 different pineal indoles, including melatonin, and of the metabolite 6-hydroxymelatonin was studied in the male rat after daily injections from 20 to 40 days of age. Only 5-methoxytryptamine (5MT) and 6-hydroxymelatonin (6M), in addition to melatonin, inhibited the neuroendocrine-reproductive axis during sexual maturation. Their potencies when injected in the afternoon were in the range of one-twentieth to one-fifth that of melatonin. Like melatonin these two indoles had no effect when injected in the morning. N-acetylserotonin, serotonin, 5-hydroxytryptophol and 5-methoxytryptophol did not influence sexual maturation either when injected in the morning or in the afternoon. Chromatographic separation was performed on plasma extracts from rats injected daily with the biologically active indoles and killed 10-120 min after the last injection. This procedure confirmed that 6M injections did not increase plasma melatonin levels. In contrast, plasma melatonin levels in 5MT-treated rats were increased 1 h after the 5MT injection. These results suggest that 5MT or part of it might be acetylated to melatonin; thus inhibition of sexual maturation might be mainly due to melatonin. These results indirectly support the contention that melatonin is the principal pineal indoleamine playing a role during sexual maturation.     

Evaluation of 5-hydroxytryptophan administration as a test of pineal function in humans

We tested the hypothesis that acute 5-hydroxytryptophan (5-HTP) administration would cause an increase in concentrations of plasma melatonin to levels observed during the spontaneous nocturnal melatonin surge. We administered 5-HTP orally (5-12 mg/kg) to 10 healthy children and 5 healthy adults, and measured melatonin concentrations in plasma samples obtained every 30 min for 3-6 h. There was no appreciable increase in melatonin after 5-HTP stimulation, even though a melatonin increase has been reported in sheep treated with 5-HTP.     

Marker rhythms of circadian system function: a study of patients with metastatic colorectal cancer and good performance status

Cancer patients may exhibit normal or altered circadian rhythms in tumor and healthy tissues. Four rhythms known to reflect circadian clock function were studied in 18 patients with metastatic colorectal cancer and good performance status. Rest–activity was monitored by wrist actigraphy for 72 h before treatment, and its circadian rhythm was estimated by an autocorrelation coefficient at 24h and a dichotomy index that compared the activity level when in and out of bed. Blood samples (9–11 time points, 3–6 h apart) were drawn on day 1 and day 4 of the first course of chronochemotherapy (5-fluorouracil: 800 mg/m²/day; folinic acid: 300 mg/m²/day; oxaliplatin: 25 mg/m²/day). Group 24h rhythms were validated statistically for plasma concentrations of melatonin, 6-α-sulfatoxymelatonin, and cortisol and for lymphocyte counts. Significant individual 24h rhythms were displayed in melatonin by 15 patients, cortisol by seven patients, lymphocytes by five patients, and prominent circadian rhythms in activity were displayed by 10 patients; only one patient exhibited significant rhythms in all the variables. The results suggest the rhythms of melatonin, cortisol, lymphocytes, and rest/activity reflect different components of the circadian system, which may be altered differently during cancer processes. Such 24h rhythm alterations appeared to be independent of conventional clinical factors.     

Physiological effects of shift work on hospital nurses

OBJECTIVE: The purpose of this study was to assess the physiological effects of shift work on the urinary excretion rates of norepinephrine, 6-sulfatoxymelatonin and estriol in hospital nurses. METHOD: Twenty-four hour urine specimens were examined on a daytime/nighttime basis for each work shift of pregnant and non-pregnant subjects. The urinary norepinephrine and 6-sulfatoxymelatonin were measured by enzyme-linked immunosorbent assay and estriol by radio-immunoassay. RESULTS: Urinary norepinephrine level during the night work was higher than the night levels of the days off and the day shift. Urinary 6-sulfatoxymelatonin level during the night work was lower than the night levels of the days off and the day shift. Urinary estriol level of pregnant subjects showed no differences among work shift and also between daytime and nighttime. CONCLUSIONS: Urinary excretion rates of norepinephrine and 6-sulfatoxymelatonin were affected by shift work both for non-pregnant and pregnant subjects. It was unlikely that urinary estriol levels in the pregnant subjects were significantly affected by shift work.     

Sleep duration,nightshift work,and the timing of meals and urinary levels of 8-isoprostane and 6-sulfatoxymelatonin in Japanese women

It has been hypothesized that disruption of circadian rhythms affects human health. Shift work and sleep deprivation are thought to disrupt the normal light–dark cycle, although the disruption due to shiftwork may be dependent on sleep deprivation. Both conditions have been suggested to be associated with an increased risk of cardiometabolic disorders. Non-photic environmental factors, such as the timing of eating, are also thought to regulate circadian rhythm and thus, may have effects on health, but the evidence from human studies is scarce. Oxidative stress is a risk factor of cardiometabolic disorders. Some laboratory studies suggest an involvement of circadian clock genes in the regulation of the redox system. The present study aimed to examine the association of sleeping habits, nightshift work, and the timing of meals with urinary levels of 8-isoprostane, a marker of oxidative stress, and 6-sulfatoxymelatonin, the principal metabolite of melatonin. Study subjects were 542 women who had previously attended a breast cancer mass screening in a community in Japan. Information on bedtimes and wake-up times, history of nightshift work, and the timing of meals was obtained by a self-administered questionnaire. The 8-isoprostane and 6-sulfatoxymelatonin were measured using the first morning void of urine and expressed per mg of creatinine. The geometric mean of 8-isoprostane levels was 12.1% higher in women with ≤6 hours of sleep than that in those with >8 hours of sleep on weekdays, and longer sleep duration on weekdays was significantly associated with lower urinary levels of 8-isoprostane after controlling for covariates (p for trend = 0.04). Women who were currently working the nightshift had a 33.3% higher geometric mean of 8-isoprostane levels than those who were not working nightshift (p = 0.03). Urinary 6-sulfatoxymelatonin levels were unrelated to sleep habits or nightshift work. Women who ate breakfast at irregular times had a 19.8% higher geometric mean of 8-isoprostane levels than those who ate breakfast at a regular time or who did not eat (p = 0.02). Women who ate nighttime snacks at irregular times had a 16.2% higher geometric mean of 8-isoprostane levels than those who did not eat nighttime snacks or who ate nighttime snacks at a regular time (p = 0.003). Among women who ate dinner at a regular time, earlier times for dinner were associated with higher 8-isoprostane and 6-sulfatoxymelatonin levels (p values for trends were 0.01 and 0.02, respectively). However, the times of dinner and nighttime snack are overlapping, and the time of last meal of the day was not associated with 8-isoprostane and 6-sulfatoxymelatonin levels. The time of breakfast or lunch was not associated with these biomarkers among women who ate the meal at regular times. Disturbing the rhythmicity of daily life may be associated with oxidative stress.     

Analysis of trough and peak of plasma melatonin circadian rhythm in ewes

This study evaluates the pattern of plasma melatonin during the trough and the peak of its daily rhythm. Blood samples from 8 ewes were collected every 3 h for a 48-h period. On the third day, blood samples were collected from 10:00 to 13:00 (trough) and from 20:00 to 23:00 (peak) every 20 min. Our results showed a robust daily rhythm of melatonin in both days of monitoring, with nocturnal acrophase. During the trough, a significant decrease was observed starting from the 10:40 with a progressive decrease about every 40 min. During the peak of the plasma melatonin daily rhythm, an increase was observed starting from the 20:40 with a progressive increase about every 40 min. These data could be taken in consideration to monitor the plasma melatonin variations during the 24 h, and for the administration of melatonin for breeding in ewes.     

Association of urinary levels of 6-sulfatoxymelatonin (aMT6s) with prevalent and incident hypertension

Laboratory studies indicate that melatonin has beneficial vascular effects. However, epidemiologic studies on the relationship between endogenous levels of melatonin and hypertension in humans are limited. We examined the association of quartile levels of 6-sulfatoxymelatonin (aMT6s) in first morning urines with prevalent and incident hypertension in 777 postmenopausal women who were originally part of a case–control study of breast cancer nested in the Women’s Health Initiative Observational Study. A total of 321 prevalent and 172 incident cases of hypertension were studied. In cross-sectional analyses, higher quartile level of aMT6s was associated with lower odds of hypertension (Q4 versus Q1; odds ratio = 0.57; 95% confidence interval [CI]: 0.3–0.9), after adjustment for age, body mass index and other risk factors. We also examined the association between baseline aMT6s levels and risk of incident hypertension. Compared to women in the lowest quartile of urinary aMT6s, the multivariable-adjusted hazard ratios and 95% CIs of incident hypertension for women in the second, third and highest quartile were 1.16 (0.8–1.8), 0.96 (0.6–1.5) and 1.02 (0.6–1.6), respectively. The mean change in systolic and diastolic blood pressure over 3 years also did not vary by baseline quartile levels of aMT6s. Although we found no evidence of a prospective association between urinary levels of aMT6s and risk of incident hypertension in postmenopausal women, our cross-sectional results provide some possible evidence of a role for physiologic levels of melatonin in hypertension. Additional larger studies are warranted, preferably with a wider range of ages, both genders and multiple melatonin measurements.     

Rhythmic changes in Fabry disease: Inversion and non-oscillatory pattern in 6-sulfatoxymelatonin daily profile

Fabry disease is a progressive disease characterized by an enzymatic deficiency of acid alpha-galactosidase and glycosphingolipids storage within the lysosomes. The disease has two phenotypes: classic and nonclassic. Excessive daytime sleepiness is a common sign reported by patients and can be caused by a circadian rhythm sleep disorder. Activity and rest cycle, variation of body temperature and melatonin biosynthesis are known rhythmicity markers. In the face of these evidences, our goal was to evaluate the rhythmic profile in Fabry’s disease patients using rhythmicity markers. For this purpose, we recruited 17 patients diagnosed with Fabry disease (11 classic and 6 nonclassic variant) that answered the Epworth Sleepiness Scale and the Morningness–Eveningness questionnaire adapted from Horne and Ostberg; recorded activity and body temperature rhythms by an actigraphy during at least 10 days and collected urine to assess 6-sulfatoxymelatonin excretion load during the day (from the second urine in the morning until 7 p.m.) and night (starting from 7 p.m. until the first urine in the morning of the following day). We observed that control subjects presented higher excretion load of 6-sulfatoxymelatonin during the night (p < 0.05, d = 7.8), as expected. Patients with the nonclassic variant presented an inversion on 6-sulfatoxymelatonin daily profile (p < 0.05, d = 3.8) and there was no difference between the day and night profile of classic variant patients when compared to the other two groups. Patients with the classic variant also presented temperature period greater than 24 hours (p < 0.05, d = 2.0). Therefore, we came to the conclusion that there is an alteration in the circadian rhythms in Fabry disease patients, evidenced by modifications in the 6-sulfatoxymelatonin daily profile and in the body temperature rhythm period.     

Melatonin in rat milk and the likelihood of its role in postnatal maternal entrainment of rhythms

The rhythm of melatonin in rat milk and the capacity of pups to synthesize and metabolize melatonin were studied. Melatonin was undetectable in milk in the light (< 21 pM), but increased rapidly 2-4 h after dark to peak at 357 +/- 66 pM at mid-dark. Oral or subcutaneous administration of melatonin to 5- and 10-day-old pups resulted in peak plasma melatonin levels 30 min after administration and rapid metabolism. Increases in pineal and plasma melatonin levels at night were detected at 5 and 6 days of age, respectively. Isoproterenol administration (2 microg/g body wt) at mid-light to day 10 pups increased plasma melatonin from 312 +/- 40 pM to 1,298 +/- 160 pM, whereas propranolol (2 microg/g body wt) suppressed nocturnal melatonin secretion from 1,270 +/- 128 pM to 395 +/- 66 pM. The rise of pineal and plasma melatonin in day 10 pups occurred 1 and 2 h after dark onset, respectively, preceding the onset in dams by 3 and 4 h, respectively. Propranolol administration to 2- and 5-day lactating dams inhibited plasma and milk melatonin at night but had no effect on their suckling pups. Transfer of melatonin via the milk is unlikely to provide an entraining signal for rat pups.     

Radioimmunoassay of N-acetyl-N-formyl-5-methoxykynuramine (AFMK): a melatonin oxidative metabolite

N-acetyl-N-formyl-5-methoxykynuramine (AFMK) is a melatonin metabolite identified in rat brain by Hirata et al. (The Journal of Biological Chemistry 249 (1974) 1311). Since no assay has been described for its routine measurement, we have developed and validated such a radioimmunoassay. We synthesized AFMK and N-acetyl-5-methoxykynuramine (AMK), in order to produce anti-AFMK antibodies and to standardize the assay. The tracer [3H]-AFMK was obtained from [3H]-melatonin. The assay was preceded by a chromatographic step on Celite microcolumn in order to increase its specificity. The assay was suitable for the measurement of AFMK levels ranging from 59 to 1894 pmol/L. The detection limit of the assay was routinely set at 65 pmol/L. The intra- and inter-assay coefficients of variation were 3.5% and 11% respectively. Investigation of the 24 h plasma pattern in healthy volunteers did not reveal any AFMK levels in plasma samples. In rats, plasma AFMK showed a peak after melatonin injection, which confirmed the in vivo AFMK production as a melatonin metabolite. This AFMK assay is suitable for studies on melatonin metabolism.