Melatonin secretion occurs at a constant rate in both young and older men and women

J. B. Fourtillan, A. M. Brisson, M. Fourtillan, I. Ingrand, J. P. Decourt, and J. Girault

CEMAF s.a., 86000 Poitiers, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The magnitude and duration of melatonin (MLT) secretion were measured over a period of 25 h with pharmacokinetic studies employing administration of D7 MLT at midday and at midnight in two separate studies and two groups of subjects, 12 young and 11 older men and women. Plasma levels of endogenous MLT and D7 MLT were quantified separately by use of a specific and sensitive method (gas chromatography-mass spectrometry) previously developed in our laboratory, enabling us to measure endogenous and exogenous MLT levels down to 0.5 pg/ml in plasma. In the two groups of subjects, MLT secretion occurred only at night: onset time of secretion was from 1915 to 2205 (Greenwich mean time), and offset was from 0305 to 0545. No MLT peak was observed in individual nocturnal MLT profiles that were similar to curves obtained for a rate-constant infusion. Modelization demonstrated the superimposition of observed data and simulated curves. MLT concentrations decreasing from the offset of secretion might correspond to the elimination of MLT present in the body at the end of nocturnal secretion. By use of the MLT clearance given by pharmacokinetics, the amount of secreted MLT was found to be 35.7 and 21.6 µg for men and women, respectively, and the rate of secretion was 4.6 and 2.8 µg/h, respectively. No significant gender difference was observed for these two parameters when normalized to body weight. No significant gender difference was observed for onset times of secretion or duration of secretion (7.6-8.6 h) within the two groups, or between young and older subjects.

endogenous melatonin; nocturnal secretion; young and elderly subjects; amount of melatonin; rate of secretion


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ALTHOUGH MELATONIN (MLT, N-acetyl-5-methoxytryptamine) may be synthesized in several tissues (2), the circulating hormone in humans is principally of pineal origin. In the pineal gland, serotonin (5-hydroxytryptamine) is converted into MLT through a two-step enzymatic process involving N-acetylation followed by methylation. Circulating MLT is inactivated in the liver, where it is first converted to 6-hydroxy-MLT and then conjugated principally to 6-sulfatoxy-MLT, excreted by the kidneys. The pineal gland is regulated by a circadian (i.e., ~24-h) rhythm-generating system located in the suprachiasmatic nuclei of the hypothalamus (26, 36). The neurohormone is produced during the dark phase of the day-night cycle.

Measurement of the timing (from onset to offset times of secretion) of endogenous MLT production is a common method for determination of the state of the biological clock. This timing has been determined by evaluation of the MLT profile in plasma (32, 43) as well as in saliva (9, 21, 37) samples.

In a review of the literature, Brown and colleagues (5, 6) indicate that reported concentrations of MLT depend on the reliability of the assay procedure. Many different analytical approaches, including RIA (7, 10, 11), HPLC with electrochemical detection (34), and gas chromatography-mass spectrometry (GC-MS) (22), have been previously used to analyze MLT in plasma. Hitherto, RIA has been the fastest and simplest technique but the least specific due to cross-reactivity with related structural compounds. Although the resolution of MLT RIA has improved considerably, among the factors that govern primarily plasma MLT levels, "error in the MLT immunoassay" is still cited (5). For example, although some reports conclude that diurnal plasma levels are "undetectable" if the assay method is specific (14), others find that there are "very low" concentrations (i.e., 10 pg/ml) during most of the day (11), variably characterized as <10 pg/ml (30), or averaging from 3.3 to 7.8 pg/ml (35).

At present, capillary GC-MS remains the gold standard for quantitative determination of MLT down to 0.5 pg/ml of MLT in a complex biological matrix, but the few methods that have been described (22) are particularly delicate to set up and not appropriate for routine use. In our laboratory, we have developed a highly specific and sensitive GC-MS assay in negative ion chemical ionization mode (12). MLT has been routinely and accurately measured down to 0.2 pg/ml; the limit of quantification of 0.5 pg/ml has been validated for plasma samples.

Our specific method has permitted us to distinguish, in the same biological sample, endogenous MLT from exogenously administered D7 MLT, a molecule in which seven deuterium atoms replace seven hydrogen atoms (3). Despite widespread use of exogenous MLT, its absolute bioavailability in humans has not been well characterized, as noted by Yeleswaram et al. (40). Moreover, all previous results do not separate endogenous MLT from exogenously administered MLT.

Although an almost flat pattern of nocturnal MLT levels has occasionally been observed and reported in recent papers (5, 38), most results indicate that nocturnal secretion reaches a peak (or several peaks) at night. Comparing the 24-h profiles of endogenous MLT in two groups of subjects of different ages, Zhdanova and colleagues (42, 43) have found significantly lower peak serum levels in the older group, but both groups displayed high interindividual variability in this parameter.

Therefore, with the aim of studying both the pharmacokinetics of D7 MLT and the endogenous MLT pattern, particularly to specify onset and offset times of MLT secretion, we quantified D7 MLT and endogenous MLT simultaneously in a total of 44 plasma samples obtained over a period of 25 h in subjects who received two doses of D7 MLT.

The present paper describes the results obtained in our study of endogenous MLT in 23 healthy subjects, 12 young and 11 older adults, of both genders.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Design of the Study

The first part of this study (study A, May) was undertaken in 12 healthy young subjects (6 men, 6 women) whereas study B (in October) included 11 healthy elderly subjects (6 men, 5 women). All subjects participated in two experiments: one with 250 µg of D7 MLT at midday and, after a washout period of 1 wk, one with 250 µg of D7 MLT at midnight. Blood was therefore collected twice for each subject; the results after the two administrations (midday vs. midnight dosing) were combined to produce the 25-h profile of endogenous MLT secretion described in the present paper. In addition, in study A, the young subjects participated in a third study, involving a 23-µg D7 MLT infusion. The protocol of these studies is detailed in the following sections.

Study A. Twelve healthy young subjects successively received, with washout periods: a) 250 µg of D7 MLT orally at 1200 noon; endogenous and exogenous MLT levels were quantified in 22 plasma samples collected between 0800 and 2100 at the following times: -4, -3, -2, and -1 h, immediately before administration (0 h), and 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, and 9.0 h after administration; b) 250 µg of D7 MLT orally at midnight; endogenous and exogenous MLT levels were quantified in 22 plasma samples collected between 2000 and 0900 at the following times: -4, -3, -2, and -1 h, immediately before administration (0 h), and 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, and 9 h after administration; and c) 23 µg of D7 MLT in 500 ml of isotonic solution in a 2-h late-morning (1000-1200) infusion. Endogenous and exogenous MLT levels were quantified in 26 plasma samples collected between 1000 and 1900 at the following times: before administration, then 10, 20, 30, 45, 60, 80, 100, and 120 min after the beginning of infusion, and 5, 10, 15, 20, 30, 40, 60, 80, 100, 120, 150, 180, 210, 240, 300, 360, and 420 min after the end of infusion.

The data provided by the three parts of the study enabled us to obtain: 1) the profile of endogenous MLT secretion over a 25-h period, combining data from parts a and b, 2) MLT total body clearance from part c of study A used to calculate the rate of secretion and the amount of nocturnal endogenous MLT (part b), 3) MLT bioavailability (parts a-c), and 4) comparison of D7 MLT pharmacokinetic parameters (parts a-b).

Study B. Eleven healthy elderly subjects completed parts a and b as described for study A but did not undergo intravenous infusion of D7 MLT. Thus we were unable to calculate total clearance and the amount of endogenous MLT secreted by night in older adults.

Subjects

Plasma MLT profiles were determined in 12 healthy young self-reported noninsomniac subjects (6 men, 6 women). Values for mean ± SD age, height, body weight, and body mass index (BMI) were 26.7 ± 4.4 yr, 171.1 ± 11.1 cm, 64.5 ± 11.3 kg, and 21.9 ± 1.5 kg/m2, respectively (detailed in Tables 1 and 2). An identical study was conducted in 11 healthy elderly self-reported noninsomniac subjects (6 men and 5 women). Values for mean ± SD age, height, body weight, and BMI were 70.0 ± 3.3 yr, 165.8 ± 8.6 cm, 67.0 ± 14.3 kg, and 24.4 ± 4.3 kg/m2, respectively (see Tables 3 and 4). All but one subject (elderly woman 01) were within 10% of the ideal body weight range for height. No subject had any clinically significant allergic disorders or any history of drug hypersensitivity, and none was taking any systemic or topical medicine. A full clinical evaluation, including measurement of vital signs, a 12-lead EKG, and routine laboratory tests (hematology, enzymology, blood chemistry, immunology, urinalysis, and urine toxic screening) was performed ~2 wk before the start of the study. The same tests (except immunology) were repeated at the end of the study. No medical treatment was allowed for 14 days (young subjects) or 7 days (older adults) before the study. The ethics committee approved the study, and written informed consent was obtained from all subjects.

                              
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Table 1.   Kinetics of nocturnal melatonin secretion in six healthy young men


                              
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Table 2.   Kinetics of nocturnal melatonin secretion in six healthy young women


                              
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Table 3.   Kinetics of nocturnal melatonin secretion in six healthy elderly men


                              
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Table 4.   Kinetics of nocturnal melatonin secretion in six healthy elderly women

Study Protocol

There were two parts to each study. Each subject was dosed orally, in fasting conditions at midday, with a solution containing 250 µg of D7 MLT (Besins-Iscovesco, Montrouge, France) added to 200 ml of water and again, after a washout period of 1 wk, with 250 µg of D7 MLT at midnight.

Subjects were confined to our Biomedical Research Clinical Center for 14 h (midday administration) or 5 h (midnight administration) before dosing. Caffeine and nicotine were not allowed.

Subjects fasted for a minimum of 3 h before D7 MLT administration and for the first 2 h posttreatment. For the midnight treatment they were in a supine position, because it has been reported that posture may influence MLT concentrations in humans (9, 29), and they were in total darkness from 2200 until 0900.

For study A (young subjects), D7 MLT was administered to every subject at midnight on May 29. At that time of year, the duration of night was ~8.3 h. It was not possible to dose elderly subjects at the same time of year. For this study (study B), D7 MLT midnight dosing was on October 19-24, when it was dark for ~13.3 h.

Sampling

In each study, blood samples (7 ml) were collected in heparinized Vacutainer tubes. The subjects remained in total darkness, and blood samples were taken with no supplementary lighting in the room but with use of the light in the corridor (not measured) with minimal disturbance (1, 4, 18, 28). Blood samples were taken with an indwelling cannula inserted into a vein in the forearm. After centrifugation, plasma samples were immediately frozen at -20°C until analysis.

Analytical Methodology

The quantitative measurement of D7 MLT and endogenous MLT in biological samples was performed by combined GC-MS with an HP 5988A mass spectrometer (Hewlett-Packard) focused to monitor the negative ions provided by chemical ionization with methane as reactant gas. A simple liquid-liquid extraction procedure with methylene chloride was used to isolate endogenous MLT and D7 MLT from the complex biological matrix. This assay was conducted with 1 ml of plasma, and the limit of quantification was statistically calculated as being 0.5 pg/ml [n = 10, relative standard deviation (RSD) = 7.95%]. The method was linear over plasma concentrations ranging from 1 to 200 pg/ml. The between-day precision (RSD) and accuracy (mean percentage of error) were determined over the entire analytical period at levels of 5 and 100 pg/ml in quality control samples: n = 45 and 54 (study A) and n = 38 and 42 (study B). The between-day precision did not exceed 6.8%, and accuracy was <3.9% for MLT.

Pharmacokinetic Analysis

Pharmacokinetic parameters characteristic of D7 and endogenous MLT were calculated by standard noncompartmental linear techniques (15) with the Siphar program (16) (Simed, Creteil, France). Total areas under plasma concentration vs. time curves (AUC) were determined by the linear trapezoidal rule from 0 to t (t = 9 h posttreatment) and from t to infinity, dividing the t concentration by the elimination constant k. Elimination half-life (t1/2) was calculated from the slope of the terminal portion of the log plasma concentration time curve by linear least squares regression. D7 MLT clearance value (Cl) was calculated as Cl = dose/AUC after intravenous infusion of 23 µg D7 MLT to the same subjects.

The Cl value obtained for D7 MLT is also the value for endogenous MLT clearance. The amount of endogenous MLT was obtained by Cl divided by AUC, where AUC is area under the curve of endogenous MLT, and the rate of secretion was obtained by amount divided by duration of endogenous secretion. Observed nocturnal endogenous (D0) MLT data were fitted by a monoexponential equation corresponding to an open one-compartment model (zero-order absorption, first-order elimination). Superimposition of experimental data (endogenous MLT concentration vs. time) and representative curve of monoexponential function was visually observed, and results were verified by the value of the coefficient of correlation.

Statistical Analysis

All results are expressed as means ± SD with maximum and minimum values. Comparisons of pharmacokinetic parameters of exogenous D7 MLT and endogenous MLT secretion between men and women and between young and elderly subjects were performed by Student's t-test or by a Mann-Whitney nonparametric test when no homogeneous variance between groups was observed (31).

The comparison of onset MLT secretion in young and older and in male and female subjects, as well as offset secretion, was performed with the Kruskall-Wallis test. Two-factor parametric ANOVA (gender and age, with interaction) was used, and the results of this ANOVA were confirmed by a nonparametric ANOVA on the ranks of the values. Statistical significance was accepted at P < 0.05 for all the tests.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Study A: Young Adults

Clearance. Total body clearance was calculated from data obtained after a D7 MLT intravenous infusion (13). At the end of infusion, D7 MLT plasma concentrations ranged from 88.8 to 164.4 pg/ml in men and from 102.7 to 195.3 pg/ml in women. There was no significant gender difference in volume of distribution at steady state (0.99 ± 0.06 vs. 0.97 ± 0.13 l/kg) and total body clearance (1.27 ± 0.20 vs. 1.18 ± 0.22 l · h-1 · kg-1) normalized to body weight. The present total clearance (Tables 1 and 2) is higher than data reported by Mallo et al. [0.90 ± 0.12 l · h-1 · kg-1 (24)], but lower than the value of 1.62 ± 0.66 l · h-1 · kg-1 found by Cavallo and Ritschel (8).

Oral D7 MLT pharmacokinetics. Only the results obtained for midnight administration of D7 MLT on plasma samples concerning nocturnal MLT secretion are described in the present report.

Peak exogenous MLT maximal concentration (Cmax) and time to peak maximal time (tmax) were determined by visual inspection of results. Although D7 MLT profiles were fairly similar for men and women, considerable intersubject variability was observed particularly in women: Cmax = 134.4 ± 72.8 pg/ml at tmax (= 0.58 ± 0.70 h) for men and 475.8 ± 640.7 pg/ml at tmax (= 0.42 ± 0.20 h) for women.

There was no significant gender difference in half-life: 1.14 ± 0.15 h (range, 0.90-1.34) and 0.88 ± 0.11 h (range, 0.76-1.06) for men and women, respectively.

MLT endogenous secretion. So far in the present report, times have been expressed in local clock time. In the following tables and in RESULTS and DISCUSSION, all times will be expressed in universal time (Greenwich mean time): in France, in May (study A), clock time was universal time + 2 h, whereas in October (Study B), it was universal time + 1 h. Use of universal time enabled us to compare MLT onset and offset times with sunset and sunrise times, respectively.

For all subjects, endogenous MLT concentrations were lower than 0.5 pg/ml (limit of quantification) between 1000 and 1800.

Individual profiles given in Fig. 1, A and B (square symbols) and in the data (Tables 1 and 2) show large interindividual variations in concentrations measured in plasma samples collected at night. The observed individual time courses of MLT levels in these plasma samples resembled a rate-constant infusion (33). Thus, using a one-compartment open-body model, we were able to fit experimental endogenous MLT plasma concentrations according to a single zero order intravenous infusion. Qualities of data fittings are shown for all subjects by superimposition of one-compartment-model representative curves on the experimental data (Fig. 1, A and B).


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Fig. 1.   Experimental plasma concentration time curves () and representative curves (---) corresponding to one-compartment open-body model with zero order iv infusion for endogenous melatonin (MLT) in young men (A) and young women (B). Vertical lines, onset and offset times of secretion; black horizontal bars, darkness period from sunset to sunrise; universal time, Greenwich mean time.

Reviewing the literature, we found it difficult to compare our onset and offset times of secretion with those previously given, and it was especially difficult to use the dim-light melatonin onset time (DLMO), defined by Lewy and Sack (23) as a fixed concentration of 10 pg/ml. Nagtegaal et al. (27) have calculated "start time" as the time after which the next two consecutive measurements exceed a threshold, defined as twice the mean of the 10 lowest MLT values over 24 h and "stop time" as the time after the two consecutive measurements were <10 pg/ml on the declining slope. Voultsios et al. (37) and others have defined onset and offset as the time points at which evening or morning MLT reached a concentration 2 standard deviations (SD) above the mean daytime level as measured between 1100 and 1800. The nocturnal rise time was taken by Sekula et al. (35) as that time after the nadir (mean of the three lowest sequential diurnal values) at which the MLT value first exceeded the nadir + 2 SDs of the nadir.

Given all of these different definitions, we decided to determine other parameters, which were expected to provide a better description of the profile of endogenous MLT. Estimation of offset time of secretion was determined from a regression line extrapolated back in time through the decay phase to intersect the steady-state level (Fig. 1, A and B). It is possible to estimate onset time in two ways: either as the first point for which there is a quantifiable rise (>= 0.5 pg/ml) in MLT in the evening [given in tables as "onset time of secretion" (tsec) or as the model-based "onset time of infusion" (tinf)]. To verify whether tsec correlated with tinf, multiple combinations of duration of secretion were chosen. For all subjects, visual inspection indicated a good fit of model predictions when zero was set at the first quantifiable MLT data. Different criteria, such as analysis of residuals and coefficient of correlation between observed and theoretical values, were used to assess the validity of fitted data. For all subjects (except one), the concentration of the first quantifiable level (higher than the limit of quantification) was <10 pg/ml (the DLMO used by many authors for the onset) and for eight subjects this concentration was <6 pg/ml. Mean concentration was 5.2 ± 3.9 pg/ml for the first quantifiable level.

Using pharmacokinetics, we can estimate the exact time of onset of infusion. In a rate-constant infusion, the plasma concentration Cinf at a given time (t) toward the steady state (Cmean = Css) is given by the formula: Cinf = Css (1 - e-kt). For each subject, we can replace Cinf, Css, and t1/2 by their values: Cinf is the concentration of the first quantifiable level, Css is the mean concentration at steady state, and k is given by 0.693 divided by t1/2. The estimates for onset ranged from tsec plus 2 min to tsec plus 24 min (mean = 10.9 ± 7.1 min). The present results suggest that, for these twelve young subjects, tsec is equivalent to tinf, and the duration of secretion is equivalent to the duration of infusion.

A rapid rise was observed for all subjects, and steady state was reached in <3-4 h (3 or 4 half-lives). Secretion started at 2010 ±50 min (1915-2125) and 2040 ±40 min (1950-2145) in young men and women, respectively. Offset of secretion was at 0405 ±50 min (range, 0305-0525) and at 0420 ±35 min (range, 0335-0510) for men and women, respectively. No significant gender difference in duration of secretion (7.9 ± 0.8 h and 7.6 ± 0.8 h), Cmax (54.7 ± 23.1 and 54.2 ± 26.4 pg/ml) and AUC (375.5 ± 178.6 and 349.7 ± 174.8 pg · h · ml-1) was observed.

The values of terminal half-life, t1/2 = 1.2 ± 0.3 and 1.1 ± 0.5 h, determined by regression of the terminal portion of the log-plasma concentration-time profile, showed no significant gender difference and also no significant difference with t1/2 of nocturnal D7 MLT: 1.1 ± 0.1 and 0.9 ± 0.1 h, determined from the same plasma samples.

By use of pharmacokinetics for a rate-constant infusion, the duration of infusion can also be estimated in another way. It takes one half-life to reach one-half of Css (mean plasma concentration at steady state); it also takes one half-life to decrease from steady state to Css/2. MLT secretion duration can therefore be estimated as the duration for which plasma concentrations are higher than Css/2. As an example, for subject 1 (Css = 41.8 pg/ml) we can observe (Fig. 1A) that the duration for which plasma concentrations are >20.9 pg/ml (Css/2) is equal at offset minus onset time, i.e., 7.25 h. For all subjects, Css/2 was obtained at ~1 h, i.e., one half-life (at the 2nd data point). Duration of secretion obtained this way correlates with the results obtained by offset time minus onset time.

When we take into account the clearance values observed for intravenously administered D7 MLT (Table 1), the daily production of MLT may be estimated by means of the relationship: amount of nocturnal secretion (in µg) = Cl × AUC. Our results show no significant gender difference in the amount of nocturnal secretion normalized to body weight of subjects: 0.48 ± 0.23 and 0.40 ± 0.19 µg/kg in men and women, respectively.

Furthermore, we estimate the rate of nocturnal secretion by means of the relationship: rate of nocturnal secretion (µg/h) = amount of secretion/duration of secretion obtained from tsec to offset time. The values of rate of secretion normalized to body weight were, respectively, 0.062 ± 0.028 and 0.050 ± 0.023 µg · h-1 · kg-1.

Css calculated by dividing the rate of nocturnal secretion by clearance was equal to 47.2 ± 20.4 pg/ml (range, 17-85.7) and 46.3 ± 25.5 pg/ml (range, 18.7-91.5) in men and women, respectively, with no significant gender difference. As shown in Fig. 1, A and B, there is a good correlation between graphically observed Css and calculated Css.

Study B: Elderly Subjects

The elderly subjects were not administered intravenous infusion, so we were unable to calculate total clearance and the amount of endogenous MLT secreted by night.

D7 MLT pharmacokinetic study. Pharmacokinetic parameters for the midnight administration of D7 MLT are reported in the following section for men and women, respectively. Cmax = 526.8 ± 141.2 pg/ml was observed at tmax = 0.54 ± 0.29 h, and Cmax = 685.9 ± 418.2 pg/ml was observed at tmax = 0.40 ± 0.22 h. No significant gender difference was observed for Cmax, tmax, or for elimination half-life (t1/2= 1.09 ± 0.14 and 0.91 ± 0.09 h).

A significant age difference was noted in men for pharmacokinetic parameters characterizing D7 MLT bioavailability (Cmax, AUC). These parameters were significantly higher in young than in older men (P < 0.01), but there was no significant age difference in men for tmax and half-life. No significant age difference was observed in women for any parameter: Cmax, tmax, AUC, and half-life.

Nocturnal endogenous MLT secretion. Levels of endogenous MLT were measured in samples collected after D7 MLT administration at midday and at midnight, 1 wk apart. As in young subjects, MLT secretion started at night (Fig. 2, A and B and Tables 3 and 4): at 2010 ±70 min (range, 1910-2205) for men and 2005 ±35 min (range, 1920-2100) for women. In three subjects, levels of MLT were <0.5 pg/ml at 0800. For all subjects, there was no detectable MLT from 0900 and throughout the afternoon. The mean duration of secretion was 8.6 ± 0.9 h and 8.0 ± 0.3 h in men and women, respectively.


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Fig. 2.   Experimental plasma concentration time curves () and representative curves (---) corresponding to one-compartment open-body model with zero-order iv infusion for endogenous MLT in elderly men (A) and elderly women (B).

Endogenous MLT Cmax was not significantly different in young (54.4 ± 23.6 pg/ml) and old (45.8 ± 31.9 pg/ml) subjects; AUC showed no significant age difference (362.6 ± 169.1 and 316.7 ± 235.9 pg · h · ml-1; Table 5).

                              
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Table 5.   Secretion of endogenous melatonin in young and elderly subjects

As in young subjects, endogenous MLT secretion could be approximated by a constant-rate infusion, and there were considerable interindividual variations in plasma concentrations and AUCs. The most significant intersubject variability was observed in men: Cmax varied from 9.6 pg/ml (subject 04) to 124 pg/ml (subject 05). This elderly subject (73 yr old) exhibited the highest levels (AUC, Cmax) of all volunteers, irrespective of gender and age. For nine subjects, plasma concentration of the first level above the limit of quantification was <3 pg/ml, and the highest value for that first quantifiable level was observed at 12.2 pg/ml.

Good data fittings were observed for all subjects; nevertheless, in several older subjects and particularly in women, infusion started after a delay. Subjects 03, 04, 05, and 11 had secretory profiles similar to those observed in young subjects. For the other older subjects, the curves showed evidence of a delay in the start of the infusion. The data were thus modeled as a zero-order infusion after deletion of the first one or two points: one point for subjects 07*, 08*, 12*, 06*, and 09* and two points for subjects 01* and 02*, as shown in Fig. 3.


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Fig. 3.   Experimental data for subjects 01 and 02 (elderly women) analyzed in a zero input order: 0 at onset of secretion time (tsec; 01*-02*) or 0 at onset of infusion time (tinf), i.e., tsec + 2 h (01-02).

For five subjects, therefore, tinf was tsec + 1 h, and for two subjects it was tsec + 2 h. Despite this observation, MLT secretion can be approximated by a constant rate infusion for all subjects, and the duration of secretion is given by offset time minus tsec. No gender difference was observed for Css graphically obtained: Css = 40.9 ± 31.5 and 39.7 ± 17.1 pg/ml for older men and women, respectively.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The validity of investigations of the circadian rhythm of MLT is critically dependent on the accuracy and precision of the raw data. Based on our assay method, and contrary to most common reports, our results clearly demonstrate that, irrespective of gender and age, human MLT secretion occurs only at night. A sampling interval of 15 min between 2200 and 2330 and of 30 min between 2330 to 0300 led us to observe a steady-state pattern of MLT secretion rather than a distinct peak. We were therefore able to visually equate MLT secretion to an infusion at a constant rate. MLT clearance derived from pharmacokinetics allowed us to calculate the amount of secreted MLT and the rate of secretion.

Our data are consistent with the most recent observations that diurnal levels are undetectable if the assay method is specific (5, 14) and also confirm the observations of a few recent papers that reported rather flat curves (5, 38) instead of the MLT peaks described by most previous studies. Although the MLT pattern has been studied by use of mathematical models (5, 35), as far as we know, our study is the first that indicates that endogenous MLT secretion may be approximated as a constant-rate infusion. In a constant-rate infusion, the amount of drug in the body rises until the rate of elimination matches the rate of infusion (33); at that moment, a steady state is obtained (after about three or four half-lives). The only factor controlling the approach to the steady state is the half-life. At the end of infusion, the plasma concentrations decrease as for an intravenous bolus. For example, in our data for young women in May (Table 2), MLT secretion begins at around 2040 and proceeds at a constant rate until cessation at 0420. However, a steady-state concentration is not reached in the blood until 3-4 h after onset. This delay is equivalent to three or four MLT half-lives (half-life = 1 h), and that is the time required for the rate of secretion of MLT to become equal to the rate of elimination of MLT from the plasma. At 0420, MLT secretion ceases, and MLT levels decrease as for an intravenous bolus, i.e., by one-half each half-life. MLT levels observed in the morning might correspond to the elimination of the MLT present in the body at the end of nocturnal secretion and not to a low-level secretion, as described by several authors. Our observations are confirmed by the values of Css (mean concentration at steady state) obtained by rate of secretion/clearance that correlate for each young subject with those observed visually and those given for Cmax, the maximum observed concentration in the data. There is a large interindividual variation in concentrations, as reported in Tables 1 and 2, and these findings are consistent with the findings in other reports (43). Because it was previously demonstrated that the amplitude of MLT production is not affected by MLT administration (19, 25) and that MLT only has an influence on circadian timing when administered several hours before onset time (30), it can be considered that endogenous MLT was not modified by administration of D7 MLT.

Various parameters have been used for the definition of onset and offset times of secretion and the duration of MLT elevation above some (fairly low) threshold level. Contrasting opinions were expressed regarding the validity of the DLMO, the dim-light melatonin onset time defined by Lewy and Sack (23), as the time at which MLT concentration in serum reached a level of 10 pg/ml (43 pmol/l) and duration of secretion defined as the time during which MLT concentrations are higher than 10 pg/ml. For Nagtegaal et al. (27), the DLMO presents the best estimate of the timing of the nocturnal MLT curve, but some recent reports (6) argue that the DLMO, based on an absolute concentration, is not the best physiologically interpretable phase marker. However, we calculated the duration of MLT secretion for all our subjects, with the use of the definition of the DLMO (set at 10 pg/ml). For one subject (elderly subject 04), all MLT concentrations were lower than 10 pg/ml, but examination of the secretory profile (Fig. 2) does not permit us to conclude that there was no secretion. Seven subjects (06, 07, 08 in young and 01, 02, 03, 08 in elderly) had a duration of secretion correlating with the data given by the DLMO. For these subjects, the mean concentration at steady state was a value of ~20 pg/ml. With use of the definition of the duration of infusion that permitted us to obtain the duration of secretion (i.e., the time during which the plasma concentrations are higher than Css/2, i.e., 10 pg/ml), the two methods clearly provided highly correlated results. On the other hand, no correlation was observed for the 15 other subjects due either to the higher plasma concentration values measured at steady state or to a longer half-life. Nevertheless, we compared the mean duration of secretion obtained by the two methods (DLMO and our method): 10.3 ± 1.5 vs. 7.9 ± 0.8 h (young men), 9.6 ± 2.4 vs. 7.6 ± 0.8 h (young women), 10.3 ± 2.2 vs. 8.9 ± 0.5 h (5 elderly men), and 9.2 ± 1.0 vs. 8.0 ± 0.3 h (elderly women). We conclude that DLMO is useful only when steady state is close to 20 pg/ml. In all subjects, the duration of secretion was significantly lower with our method, but the difference was particularly due to the estimation of offset time. We defined that time visually and graphically on the representative curves of data points, and our results correlate with those of Brown et al. (5), obtained with a mathematical model. Expressed in clock time, onset time of secretion was 2156 (range, 1940-0029) in the Brown et al. study vs. 2225 (2115-2345) in our study, whereas offset time of secretion was equal to 0554 (0326-0817) in Brown et al. vs. 0612 (0505-0725) in our study. Values for offset observed in our two studies (around 0400) correspond to those given for the "peak" in most of the previous reports in which the peak is the maximum value before the decrease in concentrations. We defined the start of secretion either by tinf, or by tsec, but the latter seems to be the better indicator for the onset of secretion in the elderly, because rather high MLT concentrations are observed before the start of infusion. Advance in circadian phase in elderly subjects has been reported by several authors (42) but was not observed in the present study. On the contrary, we noted some delay in the start of infusion in the elderly, particularly in two women.

We found no significant age difference for the amount of endogenous melatonin defined by Cmax and AUC (Table 5). Most previous investigators have reported that MLT production decreases with age; nevertheless Zeitzer et al. (41) have very recently demonstrated that the values describing the amplitude of MLT secretion were not significantly different between young and old subjects. For these authors, a possible explanation for the difference between their results and others' may be melatonin-suppressing medications commonly used by old people, uncontrolled environmental lighting, etc. In our study, young and elderly people were included only when they had no medication before and throughout the study and when environmental lighting was controlled. Nevertheless, by considering the individual results of our study (the highest MLT level was found in one older subject, and low values were found in several young), the insufficient number of young and elderly subjects, along with the fact that our oldest subject was only 73 yr old, does not enable us to conclude that MLT secretion decreases or otherwise with age.

Our studies were performed at two different times of the year; the young subjects were studied (study A) in May and the older subjects in October (study B; Table 6), when the duration of night was 8.3 and 13.3 h, respectively. A mean value for onset time of secretion was observed at 2010 in the two studies, whereas sunset occurred at 1940 and 1655 in May and October, respectively. The nonparametric test showed no significant difference either for onset (P = 0.61) or for offset (P = 0.33) among the four groups. Parametric analysis of variance demonstrated no significant interaction and no gender or age effect. For onset, the confidence intervals (95%) were (-1.07 h; +0.47 h) and (-0.55 h; +0.99 h) for age and gender, respectively; for offset, the confidence intervals were (-0.27 h; +0.90 h) and (-0.84 h; +0.33 h) for age and gender, respectively. These intervals seem to be narrow enough to conclude that gender and age do not influence the time of onset and offset MLT secretion.

                              
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Table 6.   Onset time and offset time of melatonin secretion in young and elderly men and women in two times of the year (May and October)

Onset time of secretion correlates exactly with sunset in young subjects but not in older adults. This difference could be explained by differences in light exposure. In May, from late afternoon to 2000 (bedtime at 2200) subjects lived in natural light; in October, from 1700 to 2100 (bedtime at 2200), elderly subjects were in artificial light. Whereas sunrise occurred at 0400 and 0620, respectively, in the two seasons, MLT offset was observed around 0400 with a delay at 0450 for elderly men. A good correlation is also observed between offset time and sunrise in young subjects but not in older adults. From our determinations for onset and offset time and duration of secretion, identical values were therefore observed in young and elderly subjects, in men and women, and in two different seasons for these parameters. There is a wealth of information in animal literature to indicate the importance of MLT duration in regulating the timing of seasonal or annual rhythms in reproduction, body weight, etc. (17), but no significant seasonal effect was noted in humans by Sekula et al. (35). Wehr's studies (38, 39) indicate that the human MLT rhythm is capable of adapting its wave form to the photoperiod and, therefore, of transmitting day length information. Nevertheless, urban men and some women show no winter-summer difference in the intrinsic duration of nocturnal MLT due to their exposure to artificial light at night. Our data agree with these recent observations, in that, for the two first hours of the present studies, the subjects were in natural (in May) or artificial (in October) light. A much larger sample size would probably be required to test for physiologically meaningful and/or genetically based differences in the phase, amplitude, or mean level of MLT secretion that might be related, for example, to age, mental illness, or sleep pathology.

In conclusion, pharmacokinetic study of D7 MLT after intravascular infusion enabled us to obtain total MLT body clearance. Using this parameter, we calculated the amount of endogenous MLT secreted by night, the rate of secretion, and the mean concentration at steady state. Clearance values were in the range of previously described values; nevertheless, to our knowledge, it is the first time that the amount and rate of MLT secretion have been quantitatively estimated.

An important finding of the present study is that melatonin secretion seems to occur essentially at a constant rate in normal individuals from its onset in the evening until pineal synthesis ceases in the early morning (offset). We also observed that, despite the small number of subjects in different age groups and in two different seasons of the year, the mean timings of onset and offset of MLT secretion were similar. Moreover, unlike results found by several authors, ours indicate that duration of nocturnal MLT release does not correlate with the duration of the dark phase.


    FOOTNOTES

Address for reprint requests and other correspondence: J. B. Fourtillan, CEMAF s.a., 6, Avenue Mozart, 86000 Poitiers, France (http://www.cemaf.com).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7 January 2000; accepted in final form 5 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 280(1):E11-E22
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