Prediction of nocturnal plasma melatonin from morning urinary measures

Copyright 0 Munksgaard, 1998 J Pineal Res 1998; 24:230-238 Printed in the United States of America-all rights reserved

Journal of Pineal Research ISSN 0742-3098

Prediction of nocturnal plasma melatonin from morning urinary measures

Graham C, Cook MR, Kavet R, Sastre A, Smith DK. Prediction of nocturnal plasma melatonin from morning urinary measures. J. Pineal Res. 1998; 24:230-238.0 Munksgaard, Copenhagen

Abstract: A growing literature indicates that blood levels of the hormone melatonin may have important implications for human health and well- being. Melatonin is synthesized and released into the general circulation at night, however, and it is seldom feasible to draw blood samples at night in epidemiological studies. There is some evidence that levels of urinary melatonin and of 6-sulfatoxymelatonin (aMT6s), the major metabolite of melatonin, accurately reflect nocturnal plasma melatonin. If this is the case, urinary assays could be powerful tools for epidemiological studies. A laboratory-based study was performed to examine the relationships between nocturnal plasma melatonin, morning urinary melatonin, and morning urinary aMT6s levels in 78 men. The relationship between total nocturnal plasma melatonin and both urinary aMT6s corrected for creatinine and urinary melatonin is significant. Combining the two urinary measures accounts for 72% of the variance in total plasma melatonin. Peak nocturnal plasma melatonin also was significantly related to urinary melatonin and to aMT6s. The urinary measures show good sensitivity and specificity in identifying individual differences in nocturnal plasma melatonin levels. These results support the inclusion of morning urine samples to assess the contribution of the hormone melatonin in occupational or residential studies involving healthy, young men.

Charles Graham,' Mary R. Cook,' Robert Kavet,' Antonio Sastre,' and Deborah K. Smith' 'Midwest Research Institute, Kansas City, Missouri 64110; 'EPRI, Palo Alto, CA 94303

Key words: cancer - human - aMT6s - 6- sulfatoxymelatonin - EMF

Address reprint requests to Dr. Charles Graham, Midwest Research Institute, 425 Volker Boulevard, Kansas City, MO 641 10. E-mail: [email protected]

Received October 17, 1997; accepted December 10,1997.

Introduction [Brzezinski, 1997; Liu et al., 1997; Reiter, 1997;

Melatonin is a highly lipophilic and hydrophilic molecule that diffuses readily to all cells and to all subcellular and extracellular spaces [Menendez- Pelaez and Reiter, 19931. There is now considerable evidence that melatonin is a free radical scavenger [Tan et al., 1993; Reiter et al., 19941, immune system stimulant [Maestroni, 19931, oncostatic agent [Blask et al., 1993; Panzer and Viljoen, 19971, and a promoter of physiologichehavioral changes associated with reproduction in seasonal breeders [Arendt et al., 19851. This hormone also appears to be involved in mood disorders associated with seasonal changes [Checkley et al., 1993; Rice et al., 19951. Perhaps its best established role is as a fundamental mediator of photoperiodism and seasonality [Reiter, 1991abl. Thus, a growing literature indicates that blood levels of this multi- functional hormone may have important implications for human health and well-being

Stevens et al., 19971. Recently, melatonin also has become a focal point

of research on the biological effects of exposure to the electric and magnetic fields (EMF) associated with the generation and distribution of electric power. Various epidemiological investigations have evaluated the possibility that occupational or residential EMF exposure may be associated with increased cancer risk [for reviews, see references Coleman and Beral, 1988; Savitz et al., 1989; Theriault, 1991; National Academy of Science/ Na- tional Research Council, 19961. Beginning in the early 1980s, a number of laboratory studies, prima- rily with rodents, suggested that night-time levels of melatonin are reduced by EMF exposure [Wil- son et al., 1986; Reiter, 1988; Lerchl et al., 1990; Kato et al., 19931. Results are, however, inconsis- tent [Kavet, 19961. Stevens [1987] proposed in his original hypothesis and its subsequent elaboration

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Prediction of nocturnal melatonin

[e.g., Wilson et al., 1990; Stevens, 1992; Stevens, 19951 that exposure-related suppression of nocturnal melatonin might provide a plausible biological mechanism to account for some of the epidemiological findings. This so-called “melatonin hypothesis” posits that EMF decreases pineal secretion of melatonin and that decreased melatonin leads to a cas- cade of hormonal and immune system changes relevant to the body’s cancer surveillance capacity. Laboratory studies with human volunteers have been in progress to examine several aspects of this hypothesis [Graham et al., 1996; Graham et al., 1997; Graham and Gibertini, 1997; Selmaoui et al., 19961, and evaluation of melatonin in epidemiological studies has now become a matter of some interest.

Melatonin is synthesized and released into the general circulation at night, however, and it is seldom feasible to draw blood samples at night in occupational or residential studies. One promising technique is to collect morning urine samples and analyze them for concentrations of melatonin itself, or of 6-sulfatoxymelatonin (aMT6s), the major urinary metabolite of melatonin [Arendt, 19951. If these urinary measures accurately reflect integrated or peak blood values of melatonin over the night, they could be of value for studies in the field.

Like plasma and serum melatonin levels, urinary melatonin excretion shows a clear circadian rhythm [e.g., Akerstedt et al., 1979; Lynch et al., 1995; Kabuto, 19971, and there are some data about the relationship between melatonin in plasma and urine. Wetterberg [ 19781 reported a correlation of 0.89 between plasma melatonin level at 02:OO hr and melatonin in morning urine and suggested that morning urinary melatonin levels might have clinical signifi- cance. Lang et al. [ 198 11 reported a correlation of 0.74 between plasma melatonin at midnight and urine collected between 21.00 and 07.00 hr. The correlations were lower when urine samples from 21 .OO to 24.00 hr (r = 0.61) and between 24.00 and 08.00 hr (r = 0.51) were examined. Nowak and co-workers [ 19871 found significant correlations between serum melatonin and aMT6s excretion (r = 0.72) in five subjects from whom blood and urine samples were obtained repeatedly every 2 4 hr over a 26 hr period. Fernandez et al. [1990] studied premenopausal, perimenopausal, and postmenopausal women. They drew blood for melatonin assays between 08:OO and 09:OO hr and correlated the melatonin level with the level obtained from 12 hr nocturnal urine samples. In premenopausal women, the correlation was higher during the follicular (r = 0.89) than during the luteal (r = 0.73) phase of the cycle. In perimenopausal women, the correlation was 0.92, and in postmenopausal women it was 0.89. Several investigators have provided data rel-

evant to the relationship between serum or plasma melatonin levels and aMT6s in 24 hr urine samples obtained from male volunteers [e.g., Arendt et al., 1985; Markey et al., 1985; Bojkowski et al., 1987; Bartsch et al., 19921. Significant relationships, rang- ing from r = 0.62 to r = 0.84, were typically found between aMT6s and either total or peak melatonin.

This report describes a laboratory-based study of healthy, young men in which sufficient data were collected to make a detailed examination of the relationships between nocturnal plasma melatonin, and urinary melatonin and aMT6s levels obtained from a single morning urine sample. The data were sufficient to address the following questions. How well do morning levels of urinary aMT6s and urinary melatonin reflect total plasma melatonin secretion over the night? Do morning levels of urinary aMT6s or urinary melatonin also predict peak nocturnal blood levels of melatonin for an individual? By using urinary data alone, how accurately can morning urinary aMT6s or melatonin values iden- tify individuals with naturally high and low levels of plasma melatonin? Is the relationship between blood and urinary measures improved by taking into account the pre-sleep (23:OO hr) values? Finally, will correcting for individual differences in urine volume using creatinine, a well-known indicator of lean body mass, improve the relationship between nocturnal plasma melatonin and aMT6s?

Materials and methods

Data were collected from 78 healthy, young men according to a protocol reviewed and approved by the Institute’s Human Subjects Committee. All volunteers met the following criteria: 18 to 35 years of age, not on medication, no chronic disease or dis- ability, no recent serious acute illness, regular sleep habits and diet, no night work, and no shift work. In order to determine how well morning urinary measures reflected the nocturnal plasma melatonin curve, we concentrated on the hours between 23:OO and 07:OO hr. Each volunteer slept overnight (23:OO-07:OO hr) in the laboratory. Blood was drawn via an indwelling catheter inserted in an arm vein into EDTA tubes each hour through the night. The samples were immediately centrifuged, and the plasma extracted and frozen in 0.6 mL aliquots at -20°C.

Melatonin was assayed with the Buhlmann RIA kit (ALPCO, Ltd., Windham, NH), which uses the Kennaway G-280 anti-melatonin antibody. Under this method, melatonin is extracted from the plasma samples by a reverse-phase column extraction. The extracted samples and reconstituted standards and controls are incubated with the anti-melatonin anti-

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Graham et al.

body and ‘251-melatonin. I-Melatonin competes with melatonin present in samples, standards, and controls for binding sites on the anti-melatonin antibody. After incubation, a solid-phase second antibody is added to the mixture in order to precipitate the antibody-bound fraction. The unbound fraction is discarded, and the radioactive bound fraction is counted in a gamma counter. Quantitation of unknowns is determined by comparing their activity with a response curve prepared from the known standards. The melatonin present in the samples is inversely related to the radioactivity in the bound fraction.

Validation of this assay for the quantitation of melatonin in human serum was undertaken in our laboratory under USFDA Good Laboratory Practice Standards (21 CFR 58) to determine precision, accuracy, selectivity, sensitivity, freeze-thaw stability, and parallelism. Intra-assay precision at six concentration levels ranged from 1.9 to 5.2% (n = 5 at each level). Inter-assay precision over 3 days at three concentration levels ranged from 3.6 to 9.7% (n = 2 on each day). Accuracy was determined by calculating the mean percent recovery (MPR) at six concentration levels. MPR ranged from 100 to 117% (n = 5 at each level). At a level of 5 pg/mL, MPR was 106%, and at a level of 30 pg/mL level (a 300 pg/ mL spike diluted 1 :lo), it was 11 1 % (n = 5). Three samples were assayed for freeze-thaw stability. Each sample was taken through three freeze-thaw cycles. After each cycle, melatonin was assayed for and compared to the melatonin value obtained before the samples were frozen (FTO). After the third freeze- thaw cycle, the mean percent compared to F T O at each level (n = 2) was 101% at 400 pg/mL, 98% at 200 pg/mL, and 108% at 15 pg/mL. There was no evidence of decreased melatonin levels after the three freeze-thaw cycles. The 300 pg/mL spike was diluted seven times in buffer to achieve parallelism of dilution. The percent observed compared to expected ranged from 96 to 116%. This assay has proven highly reliable in our laboratory, with a de- tection limit of 0.3 pg/mL.

Additional studies were performed with plasma samples to compare the results obtained using the ALPCO assay with those found using the “Rollag” assay [Rollag and Niswender, 19761 and the Elias USA assay [Zimmermann, et al., 19901. These comparative studies were performed by us and by Pro- fessor Rollag in his laboratory under blind test conditions using identical aliquots drawn from each of 80 frozen plasma samples. The samples, origi- nally collected in EDTA tubes, were selected to span a wide range of human melatonin concentrations (6 pg/mL to 200 pg/mL). The correlation (Pearson’s r) between ALPCO and Rollag was 0.98, between ALPCO and Elias, it was 0.99, and between Elias

232

125 and Rollag, it was 0.99. The data suggest that the three assays produce similar results over a wide range of concentrations. Intra-assay coefficient of variation for the ALPCO assay for plasma ranged from 1.9 to 8.6%. Inter-assay variability was 5.4% for high standards and 11.3% for low standards. As- say dilution recovery (1:2 to 1:16) results ranged from 94 to 114%.

All urine voided at the start and end of each ses- sion was collected, and the volume recorded. The 07:OO hr sample included all urine voided after the 23:OO hr sample. Aliquots (5 mL) were frozen at -20°C. Assays for urinary melatonin used the method described above for plasma melatonin. As- says for aMT6s used the RIA kit distributed by Stockgrand (Guildford, Surrey, UK). The validation of this assay is well described in Arendt et al. [1985]. A specific sheep anti-aMT6s anti-serum is added to the samples, and after a 30 min incubation, IZ5I-aMT6s is added. The aMT6s in the urine samples and the I-aMT6s compete for a limited number of high-affinity binding sites on the anti- aMT6s. The amount of radioactivity bound to the anti-serum is inversely related to the amount of un- labeled aMT6s present in the urine. The antibody- free aMT6s is absorbed with a dextran-coated charcoal suspension, centrifuged, and the radioactivity counted in a gamma counter. Quantitation of unknowns is achieved by comparing their activity with a response curve prepared using known standards.

In our laboratory, the lower limit of sensitivity determined for the Stockgrand assay was 3.13 pg/mL. Intra-assay coefficients of variation were determined at three levels over the range of the curve and were below 10%. Inter-assay coefficients of variation ranged from 5.8 to 9.5%. Since individuals differ in the volume of urine they produce, urinary creatinine also was assayed in the present study using a COBAS MIRATM (kinetic Jaffe reaction). The aMT6s results were normalized to creatinine concentration and expressed as nanograms aMT6s per milligram creatinine. All plasma and urine assays were performed blind to collection time. To estimate the total amount of plasma melatonin secreted over the night, the hourly values were summed to provide an approximation of area-under-the-curve (AUC).

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Results

Figure 1 shows hourly arithmetic means and standard errors for plasma melatonin over the night for the 78 volunteers. The distribution of melatonin over the night was as expected; values rose from 31 f 3 pg/mL (mean, standard error) at midnight to about 60 f 3 pg/mL (mean, standard error) between 03:OO and 05:OO hr, and then gradually declined. As ex-


701 I

0 1 2 3 4 5 6 7 - (AM)

Fig. 1. Mean (Standard Error) hourly plasma melatonin curve obtained overnight on 78 healthy, young men.

pected from previous literature, individuals differed in their levels of plasma melatonin and in the time at which peak melatonin was observed. Figure 2 il- lustrates this variability in individual melatonin pro- files. Descriptive statistics for the four measures included in this report are shown in Table 1 .

Urinary aMT6s

While both corrected and uncorrected aMT6s Val- ues were log-normally distributed by the Shapiro- Wilk Test, AUC was not. A nonparametric measure of correlation, Spearman’s r, was therefore used to evaluate the strength of the relationships among the measures. Figure 3 shows scatter plots of the relationship between log-transformed AUC and log- transformed morning level of aMT6s, uncorrected and corrected for creatinine. For reference, each panel displays the line of least-squares fit. The Spearman’s r between uncorrected urinary aMT6s and AUC was 0.69 ( P < 0.0001). Correcting urinary aMT6s for creatinine increased Spearman’s r to 0.76 ( P < 0.0001). Corrected urinary aMT6s also was a

1 8

2 I 6

4

2

0

0 1 2 3 4 5 6 7

TimeOfpenlrmumUM&hlNU * (AM)

Fig. 2. Observed variability in time to reach peak plasma melatonin concentration.

good predictor of peak melatonin (r = 0.73). The relationship was not improved by taking into account the change in corrected aMT6s values from 23:OO to 07:OO hr.

Urinary melatonin

Figure 4 shows the scatter plot of log-transformed urinary melatonin and AUC. Urinary melatonin was significantly related to both plasma AUC (Spear- man’s r = 0.74, P < 0.0001) and peak melatonin value (Spearman’s r = 0.68, P < 0.0001). Correct- ing urinary melatonin for creatinine did not improve its relationship to plasma melatonin. This was not surprising since plasma melatonin and kidney tubule melatonin readily equilibrate with one another due to melatonin’s rapid diffusion across lipid membrane structures. Measures of urinary melatonin and urinary aMT6s also were highly correlated (Spear- man’s r = 0.63, P < 0.0001). To further examine this relationship, urinary melatonin and aMT6s concentrations were converted to absolute molar values excreted. Both were log-normally distributed. Me- latonin was clearly the minor quantity of the two, with a geometric mean (GM) of 0.24 pmole (geometric standard deviation [GSD] of 1.8). aMT6s was present with a GM of 53 pmole (GSD of 1.7), more than two orders of magnitude greater than melatonin. The median of the melatonin-to-aMT6s ratio was 0.0045, and only two of 78 subjects had ratios greater than 0.01. The close relationship between urinary melatonin and aMT6s is shown graphically in Figure 5.

Classification of subjects

We next addressed the question of whether urinary values could be used to accurately divide subjects into groups with high and low levels of melatonin. The distributions of plasma AUC and the three urinary parameters were split into tertiles to determine categorical concordance between plasma and urinary measures. As shown in Table 2, all three urinary measures displayed comparable concordance, with the highest (62%) associated with aMT6s corrected for creatinine, and the lowest with the uncorrected aMT6s value (55%). Urinary melatonin had a 59% concordance with AUC. The chi-square value for all three urinary values was highly significant ( P < 0.0001).

Regression model

Because creatinine-corrected urinary aMT6s and urinary melatonin each display a reasonably strong relationship with AUC, we introduced a linear re-

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Graham et al. Table 1. Descriptive statistics for all plasma and urinary measures obtained

Geometric Geometric Measure N Mean SE Range mean SD

Plasma melatonin AUC (pg/ml) 78 418.9 20.89 62.1-854.3 366 1.7 Urinary melatonin (pg/ml) 78 64.4 4.18 7.0-185.0 55 1.8 Urinary aMT6s 78 28.8 3.26 4.1-145.5 31 2.1

(Uncorrected: ng/mL)

(Corrected: ndmg creatinine) Urinary aMT6s 78 9.7 1.1 3.9-52.3 18 1.7

3.00

2.75

1.75

3.00 I I I 1 b i 1

250

225

200

1.75 I I I 1

0.5 1.0 1.5 2.0

Log [vriwy aMTb (ng(mg creatinine)]

Fig. 3. The relationship between log-transformed nocturnal plasma area-under-the-curve (AUC) and log-transformed morning urinary levels of 6-sulfatoxymelatonin (aMT6s), uncorrected (panel A) and corrected (panel B) for creatinine in 78 healthy, young men.

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Table 2. Concordance between nocturnal plasma melatonin area- under-the-curve (AUC) and morning urinary measures of melatonin and 6-sulfatoxymelatonin (aMT6s)

3.00

275

250

225

200 * *

4

n-- I 90

80

I 1'

I I I {

Fig. 4. The relationship between log-transformed nocturnal plasma AUC and log-transformed morning levels of urinary melatonin in 78 healthy, young men.

gression model to test if the use of both terms (log- transformed) provided improved correlation with log (AUC). The Pearson correlation coefficient between log-transformed, creatinine-corrected urinary aMT6s (UaMT6s) and log-transformed, urinary melatonin (UMLT) is 0.63, suggesting they are not completely redundant. The model is as follows:

The analysis result appears below in Table 3. For the entire sample of 78 men, the model accounts for 72% of the variability in log (AUC), marginally increased over the 67% explained by log (UaMT6s) alone. None- theless, log (UMLT) provides a statistically significant increment (P < 0.001) to the variability already ac- counted for by the metabolite term. The model's re- siduals were normally distributed overall, with no statistically significant differences among their means

0.1 1 10 100 1000 Amount (pmoles) in morning urine

Fig 5. The relationship between urinary melatonin (MLT) and 6-sulfatoxymelatonin (aMT6s).

AUC

Low Medium High Total

Urinary Low 15 11 0 26 aMT6 Med 8 10 8 26 Uncorrected High 3 5 18 26

Total 26 26 26 78 Urinary Low 16 9 1 26 aMT6s Med 7 13 6 26 Correcteda High 3 4 19 26

Total 26 26 26 78 Urinary Low 17 8 1 26 Melatonin Med 9 10 6 25

High 0 8 19 27 Total 26 26 26 78

aUrinary values normalized to creatinine to adjust for individual differences in urine volume, Chi Square for all urine levels = P < o.ooai.

(one-way ANOVA) or among the absolute deviation from their means (Levene test) across tertile catego- ries of each of the two covariates. To determine whether urinary aMT6s or urinary melatonin clearly dominate the prediction of plasma melatonin, log (UaMT6s) and log (UMLT) were both standardized with a mean of 0.00 and a standard deviation of 1.0. The ensuing comparison of estimates showed the coefficient for aMT6s significantly greater than the coefficient for urinary melatonin (P < 0.01). After removing the two highest leverage points, the ensuing model explained 70% of log (AUC) variance, but the difference between the coefficients was no longer statistically significant (P > 0.2).

Table 3. Results of regression model

Panel A

Analysis of Variance Source DF C Squares Mean sq. F Ratio

Model 2 2.808 1.40 95.28a Error 75 1.105 0.01 Total 77 3.91 3 > 0.0000 Panel B

~

Parameter Estimates Term Estimate Std Error t Ratio Prob> It I

P O 1.339 0.097 13.88 0.0000 P1 0.654 0.079 8.25 0.0000 I32 0.235 0.068 3.45 0.0009 Panel C

Effect Test Source DF zSquares F ratio Prob>F

Log (UaMT6) 1 1.003 68.089 0.0000 Log (UMLT) 1 0.175 11.902 0.0009

aF > 0.000

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Graham et al.

Relationship between urinary aMT6s and hourly plasma values

Lastly, we examined the relation of urinary aMT6s (corrected for creatinine) and urinary melatonin with the hourly plasma melatonin concentrations. As noted previously, plasma values peaked between 03:OO and 0590 hr (Fig. 1). The correlation between urinary aMT6s and hourly plasma melatonin peaked between 01:OO and 03:OO hr, and the correlation between urinary melatonin and hourly plasma melatonin peaked between 01:OO and 02:OO hr (Fig. 6a). Since morning urinary measures reflect integration of overnight levels, we also plotted (Fig. 6b) the relation between urinary measures and the cumulative AUC from midnight to each subsequent hour. As shown, the correlation of cumulative plasma melatonin with urinary metabolite peaked at 03:OO hr and with urinary melatonin at 04:OO hr. Thus, for both hourly and cumulative plasma levels, the relationships of urinary melatonin to plasma melatonin were stronger for the times preceding the observed plasma peak.

Spearman Correlation to hour

0 1 2 3 4 5 6 7

Time (9

0.6

0.5 -1 I I 1 I I I

0 1 2 3 4 5 6 7

T I (AM)

Fig. 6. The relationship between morning measures (urinary aMT6s and melatonin) and hourly (A) and cumulative (B) values of nocturnal plasma melatonin (MLT) in 78 healthy, young men.

Discussion

The results reported here both replicate and extend previous research [e.g., Lynch et al., 1975; Wetterberg, 1978; Bojkowski et al., 1987; Selmaoui et al., 19961. Using a sample of volunteers almost four times larger than any previously reported in the literature, morning urinary melatonin and urinary melatonin metabolite levels correlated significantly with the integrated value of melatonin in blood samples collected over the entire night. The study also demonstrated that peak plasma melatonin is correlated with these urinary measures.

The morning urinary aMT6s values were not op- timally related to plasma melatonin unless corrected for creatinine, not surprising in light of expected subject-to-subject variability with respect to urinary volume. Such a correction was not necessary for urinary melatonin since plasma melatonin and kidney tubule melatonin readily equilibrate with one another due to melatonin’s rapid diffusion across lipid membrane structures. On a molar basis, the major urinary product of melatonin excretion was aMTbs, present (in all but two subjects) in quantities at least two orders of magnitude greater than the parent molecule. Using non-parametric correlation and categorical concordance analyses, urinary melatonin was related to plasma melatonin about as well as creatinine-corrected metabolite.

A linear model incorporating both measures explained 72% of AUC variability, marginally better than metabolite alone but substantially better than melatonin alone. However, with two high leverage points removed, the difference between the predic- tive strength of the two urinary parameters narrowed and was not statistically significant. Thus, when ana- lyzing such data, one must be aware of the poten- tially influential nature of even a small number of observations. Nonetheless, even though present in small quantities relative to its metabolite, urinary melatonin correlates remarkably well and highly significantly with plasma melatonin.

Although urinary measures were significantly correlated with AUC, the higher correlations occurred for time points prior to the peak of the sample’s average plasma melatonin, which occurred between 03:OO and 05:OO hr. When considering plasma melatonin integrated from midnight to each hour, the peak correlation remained in advance of the pooled overnight peak. These relations were similar for both urinary melatonin and urinary aMT6s, although the peak correlation for the former occurred about 1 hr in advance of the latter. This small discrepancy might reflect clearance differences between aMT6s and melatonin, since the time for melatonin-to-aMT6s urinary excretion depends

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on hepatic processing, as well as renal clearance, whereas urinary melatonin accumulation depends on renal clearance and the perfusion of the kidneys, ureters, and bladder. Metabolic processing of melatonin in the liver and its ultimate clearance occurs over a 1 to 2 hr period [Selmaoui et al., 19961, which is consistent with our observations. The early occurrence of the correlation peaks may reflect dif- ferent patterns of secretion and metabolism as the overnight period progresses. For example, increased secretion and hepatic metabolism in the earlier por- tion of the observation period could conceivably result in the observed correlation patterns. Further pharmacokinetic research would help unravel the basis for the observed relationships.

In conclusion, the present results provide a solid basis for the inclusion of morning urine samples as a tool in epidemiological studies involving healthy, young men. The data suggest that creatinine-corrected aMT6s or melatonin are both acceptable urinary measures of overnight plasma melatonin and that the comparative relationship of these two measures to plasma melatonin might be sensitive to a small number of observations. For cases in which subjects have suspected or known metabolic or liver disease [Iguchi et al., 19821, urinary melatonin may be considered as a measure of choice because of its independence of hepatic degradation pathways, Al- though Bojkowski and Arendt [ 19901 have reported that there are no gender or adult age differences in aMT6s, further studies are needed to determine whether the relationships between plasma melatonin, urinary aMT6s, and urinary melatonin are the same in women and in men, and whether the relationships vary as a function of age.

Acknowledgments

The work reported here was supported by National Institute of Environmental Health Sciences grant ES07053 and by Elec- tric Power Research Institute contract W04307. The authors thank Donald W. Riffle and Nancy E. Phelps for data collection and Mary M. Gerkovich and Kirsten E. Kakolewski for data management and initial statistical analysis.

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Prediction of nocturnal plasma melatonin from morning urinary measures