Do Plasma Melatonin Concentrations Decline with Age? Jamie M. Zeitzer, PhD, Jessica E. Daniels, AB, Jeanne F. Duffy, MBA, PhD, Elizabeth B. Klerman, MD, PhD, Theresa L. Shanahan, MD, Derk-Jan Dijk, PhD, Charles A. Czeisler, PhD, MD PURPOSE: Numerous reports that secretion of the putative sleep-promoting hormone melatonin declines with age have led to suggestions that melatonin replacement therapy be used to treat sleep problems in older patients. We sought to reassess whether the endogenous circadian rhythm of plasma melatonin concentration changes with age in healthy drug-free adults. METHODS: We analyzed the amplitude of plasma melatonin profiles during a constant routine in 34 healthy drug-free older subjects (20 women and 14 men, aged 65 to 81 years) and compared them with 98 healthy drug-free young men (aged 18 to 30 years). RESULTS: We could detect no significant difference between a healthy and drug-free group of older men and women as compared to one of young men in the endogenous circadian ampli- tude of the plasma melatonin rhythm, as described by mean 24-hour average melatonin concentration (70 pmol/liter vs 73 pmol/liter, P 5 0.97), or the duration (9.3 hours vs 9.1 hours, P 5 0.43), mean (162 pmol/liter vs 161 pmol/liter, P 5 0.63), or integrated area (85,800 pmol 3 min/liter vs 86,700 pmol 3 min/liter, P 5 0.66) of the nocturnal peak of plasma melatonin. CONCLUSION: These results do not support the hypothesis that reduction of plasma melatonin concentration is a general characteristic of healthy aging. Should melatonin replacement therapy or melatonin supplementation prove to be clinically useful, we recommend that an assessment of endogenous melatonin be carried out before such treatment is used in older patients. Am J Med. 1999;107:432– 436. q1999 by Excerpta Medica, Inc. I we controlled for these and other factors that affect melatonin secretion to examine whether changes in the plasma melatonin profile occur with healthy aging. n the past two decades, more than 30 reports have described age-related changes in the amplitude of the melatonin rhythm in adults (1–37). Most of these studies have suggested that, after puberty, the magnitude of the nocturnal melatonin peak declines progressively with age (12–30,32,34 –37). These reports have led to a recent proliferation of strategies for melatonin replacement in the elderly. However, most previous studies failed to verify that older subjects were healthy and nonusers of common drugs that suppress melatonin secretion, such as aspirin, ibuprofen, and b-blockers. Furthermore, prior studies were often performed in ambient illumination that was sufficiently bright to suppress melatonin production and did not control for changes in the posture of subjects, which can affect the plasma concentration of melatonin (38 – 40). In the present study, From the Program in Neuroscience (JMZ, CAC), Harvard Medical School, Boston, Massachusetts; and the Circadian, Neuroendocrine and Sleep Disorders Section (JMZ, JED, JFD, EBK, TLS, D-JD, CAC), Endocrine Division, Department of Medicine, Harvard Medical School, Brigham and Women’s Hospital, Boston, Massachusetts. Supported in part by grants from the National Institute on Aging (P01-AG-09975; R01-AG06072), the National Institute of Mental Health (R01-MH-45130), a General Clinical Research Center grant from the National Center for Research Resources (M01-RR02635), and the National Aeronautics and Space Administration (NAG 5–3952; NAG W-4033). Requests for reprints should be addressed to Charles A. Czeisler, PhD, MD, Circadian, Neuroendocrine and Sleep Disorders Section, Endocrine Division, Department of Medicine, Harvard Medical School, Brigham and Women’s Hospital, 221 Longwood Avenue, Room 438A, Boston, Massachusetts 02115. Manuscript submitted December 7, 1998, and accepted in revised form July 19, 1999. 432 q1999 by Excerpta Medica, Inc. All rights reserved. PATIENTS AND METHODS Study Subjects Between 1990 and 1996, we studied plasma melatonin profiles in 101 young men, 19 older men (65 years of age and older), and 24 older women, all of whom were in good physical and mental health, as confirmed by history, physical examination, electrocardiography, psychological questionnaires, chest radiographs (older subjects only), and routine laboratory tests. All participants were recruited from the greater Boston area by radio and print advertising. They were free of any sleep complaints and did not use prescription or nonprescription medication. Of the original 144 subjects, 12 were excluded from the present analysis as follows: 4 older men, 4 older women, and 2 young men because of an incomplete series of hourly blood samples, 1 young man because of technical difficulties with the melatonin assay, and 1 older man because of an absence of rhythmic melatonin levels (although this subject had an average plasma melatonin concentration within 1 SD of the average of the older male subjects). Plasma melatonin data from the remaining young men [n 5 98, ages 18 to 30 years, mean (6SD) age of 23.2 6 3.8 years], older women (n 5 20, ages 65 to 81 years, 68.9 6 4.2 years), and older men (n 5 14, ages 64 to 75 years, 67.7 6 3.3 years) were included in this analysis. 0002-9343/99/$–see front matter PII S0002-9343(99)00266-1 Do Plasma Melatonin Concentrations Decline with Age?/Zeitzer et al Prestudy Conditions All subjects maintained a regular sleep-wake schedule for at least 1 week before entering our facility, as confirmed by wrist actigraphy and call-in phone logs. During that week, as well as throughout the protocol, subjects refrained from the use of nicotine, alcohol, caffeine, and all other prescription and nonprescription medications, as confirmed by comprehensive urinary toxicologic analysis during the prestudy screening and upon admission to the laboratory. To determine a subject’s sleep-wake schedule, the bed and wake times during the week before entry were averaged and the midpoint was scheduled as the midpoint of an 8-hour scheduled sleep episode while in the laboratory. Study Conditions The laboratory portion of the protocol included 3 baseline days and nights in the Intensive Physiological Monitoring Unit of the Brigham and Women’s Hospital General Clinical Research Center. Subjects were exposed to a 16-hour:8-hour light:dark regimen (an ambient illumination measured in angle of gaze of approximately 150 lux: ,0.03 lux), and were scheduled to sleep and wake at their typical bed and wake times. On laboratory day 4, subjects awoke to a constant routine that lasted at least 30 hours (41– 43). Throughout the constant routine, subjects were required to remain awake and in bed in a semirecumbent position under dim ambient illumination (,15 lux). Equicaloric snacks and fluids were provided hourly. Subjects were constantly monitored to ensure compliance with the protocol. These conditions minimize or hold constant factors such as postural changes (38 – 40) that affect plasma melatonin levels. The protocol was approved by the Brigham and Women’s Hospital Human Research Committee, and all subjects gave informed, written consent. Biochemical Assays Blood samples were drawn every 20 to 60 minutes with an indwelling intravenous catheter inserted on laboratory day 2, approximately 40 hours before the initiation of sampling. Samples were immediately centrifuged and a portion of the plasma was frozen and later assayed for melatonin concentration by radioimmunoassay (assay sensitivity of 22 pmol/liter, intra-assay coefficient of variation of 8%, interassay coefficient of variation of 13%; DiagnosTech, Osceola, Wisconsin). Amplitude Analysis Several measures of amplitude were calculated: mean 24hour plasma melatonin concentration, duration of the nocturnal peak of melatonin secretion, mean melatonin concentration during the nocturnal peak, and the integrated area of the nocturnal peak. The 24-hour mean concentration was calculated as the mean of the values between hours 5 and 29 of the constant routine (the first 5 hours of the constant routine were excluded because of the potential effect of the change in posture at the start of the routine). The duration of the nocturnal peak was calculated as the number of hours that plasma melatonin concentrations were in excess of the 24-hour mean. The mean of the melatonin values and the integrated area under the curve during this time were determined. This method of determining the timing of the peak was used to account for the intersubject variability in nocturnal melatonin concentrations, which was greater than one log unit in these subjects. Statistical Analysis Comparisons between amplitude parameters were made using two-tailed Student’s t test except as noted. Comparison of the variance of the individual amplitude parameters were made using the F test. Data are shown as means with 95% confidence intervals. RESULTS Amplitude in Young Men and in Older Men and Women The young male subjects had a mean 24-hour melatonin concentration of 70 pmol/liter [95% confidence interval (CI): 31 to 116 pmol/liter], similar to that in the older male and female subjects of 73 pmol/liter (95% CI: 23 to 124 pmol/liter, P 5 0.97 for comparison with younger subjects). Nocturnal melatonin peaks in the young men averaged 9.3 hours (95% CI: 8.5 to 9.9 hours) in length, with a mean plasma melatonin concentration of 162 pmol/liter (95% CI: 72 to 269 pmol/liter) and integrated area under the curve of 85,800 pmol 3 min/liter (95% CI: 38, 700 to 144,000 pmol 3 min/liter). These values were similar in the older male and female subjects, who had nocturnal peaks that averaged 9.1 hours (95% CI: 8.5 to 9.7 hours) in length (P 5 0.43), with a mean melatonin concentration of 161 pmol/liter (95% CI: 54 to 267 pmol/ liter, P 5 0.63) and integrated area under the curve of 86,700 pmol 3 min/liter (95% CI: 26, 400 to 147,000 pmol 3 min/liter, P 5 0.66). The lack of a statistical difference between young and older subjects in any of the four described amplitude parameters is also evident from the average curves of the young and older subjects (Figure 1) and scatter plots of the individual data (Figure 2). There was also no significant difference in the variance of any of the amplitude parameters in the young as compared with the older subjects (all P . 0.19). Visual inspection of the scatter plots suggested that there may have been some distributional difference between young and older subjects. Thus, we compared the upper and lower fifteenth percentile of each amplitude category using onetailed t tests (Figure 3). This analysis revealed that there was no significant difference in the upper fifteenth percentiles between the young and older subjects in 24-hour November 1999 THE AMERICAN JOURNAL OF MEDICINEt Volume 107 433 Do Plasma Melatonin Concentrations Decline with Age?/Zeitzer et al between the lower fifteenth percentiles of the young and older subjects in 24-hour average melatonin concentration (P 5 0.04), average nocturnal peak melatonin concentration (P 5 0.004), and integrated nocturnal peak (P 5 0.006). There was no difference in the average duration of the nocturnal melatonin peak (P 5 0.46). DISCUSSION Figure 1. Plasma melatonin concentrations during a constant routine in young men and older men and women. Plasma melatonin concentrations were binned hourly based on each subject’s habitual wake time (beginning of constant routine 5 0). Binned values were averaged within and across subjects. Bars indicate the standard error of the mean. The dashed line indicates the approximate regular bedtime and the solid line indicates the approximate regular waketime. average melatonin concentration (P 5 0.25), average nocturnal peak concentration (P 5 0.39), integrated nocturnal peak (P 5 0.30), or duration of the nocturnal peak (P 5 0.11). However, there was a significant difference Our results do not support the contention that nocturnal plasma melatonin concentrations decrease with healthy aging. Rather, the endogenous circadian melatonin rhythm in most healthy, drug-free older people has an amplitude comparable to that of young adults. There may be a small group of older adults who have significantly lower melatonin amplitude, as has been observed in one previous study (44). Our findings challenge the conventional belief that plasma melatonin concentrations decline systematically with advancing age because of an increase in pineal calcification (45– 47) or other biochemical change (48). This decline has been thought to act as an “aging clock” (47,49), a hypothesis that our data do not support. It has been hypothesized that low melatonin amplitude reflects a diminished amplitude of the light-sensitive endogenous circadian pacemaker (38), located in the hypo- Figure 2. Comparison of plasma melatonin amplitudes in young men and older men and women. Melatonin values from the 98 young men, 14 older men, and 20 older women are represented by a single point in each plot. Means with 95% confidence intervals for older and younger subjects are also shown. 434 November 1999 THE AMERICAN JOURNAL OF MEDICINEt Volume 107 Do Plasma Melatonin Concentrations Decline with Age?/Zeitzer et al Figure 3. Plasma melatonin concentrations during a constant routine in young men and older men and women, in upper and lower fifteenth percentiles. The upper plot shows the 15 young subjects and 5 older subjects with the greatest plasma melatonin amplitudes for their age group (upper fifteenth percentile), and the lower plot shows the 15 young subjects and 5 older subjects with the lowest plasma melatonin amplitudes for their age group (lower fifteenth percentile). Mean values with standard error bars are shown. The dashed line indicates the approximate regular bedtime and the solid line indicates the approximate regular waketime. thalamic suprachiasmatic nucleus. If the duration of the melatonin peak indicates the amplitude of this pacemaker (38), then our results suggest that there is not a generalized age-related reduction in its amplitude. It should be emphasized, however, that our study sample underwent an extensive medical screening exam, and only healthy subjects free of medical conditions and medication use, including alcohol, caffeine, and nicotine, were included. One possible explanation for the difference between our results and those of other investigators may be that use of melatonin-suppressing medications commonly used by older people (eg, aspirin, nonsteroidal anti-inflammatory agents, b-blockers) (50), poorly controlled or uncontrolled environmental lighting (51), or other medical conditions (2) may have accounted for the reported decline in melatonin secretion with age. Furthermore, because the older subjects in this study were 65 to 81 years old, we cannot assess whether melatonin levels decline after the eighth decade. Because of the inherently large intersubject variability in plasma melatonin amplitude, however, the minimum differences we had 90% power to detect (at two-sided a 5 0.05) were 32 pmol/liter in the 24-hour average melatonin concentration, 0.40 hours in the average duration of the nocturnal peak, 69 pmol/liter in the average melatonin concentration during the nocturnal peak, and 37,500 pmol 3 min/liter in the average integrated area under the curve of the nocturnal peak. It thus remains possible that a much larger sample might have revealed small differ- ences in these parameters. However, the data presented in Figure 1 suggest that the magnitude of any such difference is likely to be small. There has been a spate of recent books, print and television advertisements, and newspaper and magazine articles asserting as fact that melatonin secretion decreases with age, beginning as early as age 40 years, and that this decline may be causally linked to sleep disruption in older people (52,53). Melatonin is being heavily marketed as a hypnotic agent in the United States in widely variable dosage forms of unknown purity (54). Advertisement of these health claims to the general public have been permitted to continue even though large-scale clinical trials have never evaluated the efficacy and safety (54 –56) of melatonin treatment for insomnia. Our results demonstrate, however, that most healthy older people have plasma melatonin concentrations comparable with those of young adults. 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