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

Pippa J Gunn, Benita Middleton, Sarah K Davies, Victoria L Revell, Debra J Skene, Pippa J Gunn, Benita Middleton, Sarah K Davies, Victoria L Revell, Debra J Skene

Abstract

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.

Keywords: 6-sulfatoxymelatonin; Sex differences; circadian rhythms; constant routine; cortisol; human; melatonin.

Figures

Figure 1.
Figure 1.
Melatonin production and excretion of 6-sulfatoxymelatonin (aMT6s) in males and females measured over 32 hours during a constant routine (CR) protocol. (A) Plasma melatonin concentrations measured at hourly intervals in males (black circles) and females (open triangles) over 32 hours. Data are plotted relative to each person’s DLMOn25%, designated to occur at 0 h. *p < 0.05 between sexes. Two-way ANOVA with Sidak’s multiple comparisons test, time effect p < 0.0001; sex effect p < 0.05; time x sex interaction p < 0.0001. (B) Urinary aMT6s excretion expressed per hour from eight timed collections in males (dark bars) and females (light bars). Two-way ANOVA with Sidak’s multiple comparisons test, time effect p < 0.0001; sex effect and time x sex interaction not significant (p > 0.05). Data are mean ± SEM, n = 14 for each group.
Figure 2.
Figure 2.
Cortisol production and excretion in males and females measured over 32 hours during a constant routine (CR) protocol. (A) Plasma cortisol concentration measured at hourly intervals in males (black circles) and females (open triangles) over 32 hours. Data are plotted relative to each person’s DLMOn25%, designated to occur at 0 h. *p < 0.05 between sexes at indicated time points. Two-way ANOVA with Sidak’s multiple comparisons test, time effect p < 0.0001; sex effect p < 0.0001; time x sex interaction p < 0.0001. (B) Urinary cortisol excretion expressed per hour from eight timed collections in males (dark bars) and females (light bars). *p < 0.05 between sexes at indicated time points. Two-way ANOVA with Sidak’s multiple comparisons test, time effect p < 0.0001; sex effect p < 0.05; time x sex interaction not significant (p > 0.05). Data are mean ± SEM, n = 14 for each group.
Figure 3.
Figure 3.
Individual plots of plasma melatonin (A) and cortisol (B) concentrations measured at hourly intervals during a constant routine (CR) protocol in female participants according to hormone dose: 0 µg ethinylestradiol and 75 µg progestin (long-dashed line, n = 1), 30 µg ethinylestradiol and 150 µg progestin (solid grey line, n = 10), 30 µg ethinylestradiol and 3000 µg progestin (short-dashed line, n = 2), 35 µg ethinylestradiol and 250 µg progestin (solid black line, n = 1). Data are plotted relative to each person’s DLMO25%, designated to occur at 0 h.

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