Daytime melatonin and light independently affect human alertness and body temperature

Light significantly improves alertness during the night (Cajochen, Sleep Med Rev, 11, 2007 and 453; Ruger et al., AJP Regul Integr Comp Physiol, 290, 2005 and R1413), but results are less conclusive at daytime (Lok et al., J Biol Rhythms, 33, 2018 and 589). Melatonin and core body temperature levels at those times of day may contribute to differences in alerting effects of light. In this experiment, the combined effect of daytime exogenous melatonin administration and light intensity on alertness, body temperature, and skin temperature was studied. The goal was to assess whether (a) alerting effects of light are melatonin dependent, (b) soporific effects of melatonin are mediated via the thermoregulatory system, and (c) light can improve alertness after melatonin‐induced sleepiness during daytime. 10 subjects (5 females, 5 males) received melatonin (5 mg) in dim (10 lux) and, on a separate occasion, in bright polychromatic white light (2000 lux). In addition, they received placebo both under dim and bright light conditions. Subjects participated in all four conditions in a balanced order, yielding a balanced within‐subject design, lasting from noon to 04:00 pm . Alertness and performance were assessed half hourly, while body temperature and skin temperature were measured continuously. Saliva samples to detect melatonin concentrations were collected half hourly. Melatonin administration increased melatonin concentrations in all subjects. Subjective sleepiness and distal skin temperature increased after melatonin ingestion. Bright light exposure after melatonin administration did not change subjective alertness scores, but body temperature and proximal skin temperature increased, while distal skin temperature decreased. Light exposure did not significantly affect these parameters in the placebo condition. These results indicate that (a) exogenous melatonin administration during daytime increases subjective sleepiness, confirming a role for melatonin in sleepiness regulation, (b) bright light exposure after melatonin ingestion significantly affected thermoregulatory parameters without altering subjective sleepiness, therefore temperature changes seem nonessential for melatonin‐induced sleepiness, (c) subjective sleepiness was increased by melatonin ingestion, but bright light administration was not able to improve melatonin‐induced sleepiness feelings nor performance. Other (physiological) factors may therefore contribute to differences in alerting effects of light during daytime and nighttime.     

Peripheral vision suppression of melatonin.

The suppression of melatonin by bright light is probably mediated by the suprachiasmatic nucleus (SCN) in humans. In animals, SCN cells have broad visual receptive fields, suggesting that peripheral bright light could be effective for melatonin suppression. Twelve healthy subjects were subjected to 1000 lux illumination for 2 hr from 0100 to 0300 on two occasions: once lighting the central visual field 5 degrees from the center of gaze and once lighting the peripheral visual field 60 degrees lateral to the direction of gaze. Six subjects were observed on a third occasion in dim light. The three conditions differed significantly, with less melatonin secreted in 1000 lux, but melatonin levels with central and peripheral illumination did not differ. This suggests that phototherapy using bright light in the visual periphery may be effective.     

Peripheral vision suppression of melatonin

Abstract: The suppression of melatonin by bright light is probably mediated by the suprachiasmatic nucleus (SCN) in humans. In animals, SCN cells have broad visual receptive fields, suggesting that peripheral bright light could be effective for melatonin suppression. Twelve healthy subjects were subjected to 1000 lux illumination for 2 hr from 0100 to 0300 on two occasions: once lighting the central visual field 5° from the center of gaze and once lighting the peripheral visual field 60° lateral to the direction of gaze. Six subjects were observed on a third occasion in dim light. The three conditions differed significantly, with less melatonin secreted in 1000 lux, but melatonin levels with central and peripheral illumination did not differ. This suggests that phototherapy using bright light in the visual periphery may be effective.     

One-hour exposure to moderate illuminance (500 lux) shifts the human melatonin rhythm

Abstract: Salivary melatonin levels were measured in 12 healthy volunteers in order to determine whether a moderate light intensity, which suppresses the nocturnal rise of melatonin, was able to shift the melatonin rhythm. The samples were collected at 1-hr intervals under lighting of < 100 lux (experiment 1) or < 10 lux (experiment 2). The control melatonin profiles were determined during the first night. In the second night the subjects were exposed to light of 500 lux for 60 min during the rising phase of melatonin synthesis. The third series of samples was collected during the third night. The mean decrease of melatonin levels by the exposure to light was 56% of the prelight concentrations. The melatonin onset times were delayed significantly (about 30 min) the night after the exposure to light. The melatonin offset times tended to be delayed in experiment 2. The shifts of the melatonin offset correlated positively with the amount of the melatonin suppression. The results suggest that a relatively small and short lasting light-induced interruption of melatonin synthesis may affect the melatonin rhythm in humans.     

The effects of prior light history on the suppression of melatonin by light in humans

We investigated the impact of light exposure history on light sensitivity in humans, as assessed by the magnitude of the suppression of melatonin secretion by nocturnal light. The hypothesis was that following a week of increased daytime bright-light exposure, subjects would become less sensitive to light, and that after a week of restriction to dimmer light they would become more sensitive. During the bright week, subjects (n = 12) obtained 4.3 +/- 0.4 hr of bright light per day (by going outside and using light boxes indoors). During the dim week, they wore dark goggles (about 2% light transmission) when outside during daylight and spent 1.4 +/- 0.9 hr per day outside. Saliva samples were obtained every 30 min for 7 hr in dim light (<15 lux) on two consecutive nights (baseline and test night) at the end of each week. On the test night, 500 lux was presented for 3 hr in the middle of the collection period to suppress melatonin. There was significantly more suppression after the dim week compared with after the bright week (to 53 versus 41% of the baseline night values, P < 0.05). However, there were large individual differences, and the difference between the bright and dim weeks was most pronounced in seven of the 12 subjects. Possible reasons for these individual differences are discussed, including the possibility that 1 wk was not long enough to change light sensitivity in some subjects. In conclusion, this study suggests that the circadian system's sensitivity to light can be affected by a recent change in light history.     

Evening melatonin and bright light administration induce additive phase shifts in dim light melatonin onset

In healthy young men, administration of a single light pulse (5000 lux for 3 hr) or a single melatonin pill (5 mg) at 20:40 hr under controlled constant routine conditions of <10 lux, yielded a phase delay and a phase advance, respectively, in the circadian marker of dim light melatonin onset 24 hr later. Phase shifts after combining the two interventions were additive. Melatonin suppression is not necessary for a phase shift by light, and melatonin is not a 'weak' Zeitgeber relative to bright light when ambient lighting is strictly controlled.     

Prolonged suppression of serum concentrations of melatonin in prepubertal heifers

Our objective was to suppress the daily surge of melatonin in serum of prepubertal dairy heifers by manipulating intensity of light (Experiment 1) and duration of exposure to light (Experiment 2). Heifers in Experiment 1 were exposed to either 12 hr of darkness (000 lux, control), or 400, 800, or 1,200 lux of light during the last 6 hr of their usual 12-hr nocturnal period. During this 6-hr exposure to various intensities of light, melatonin concentrations were similar to their respective daytime baseline values measured under 400 lux of light, but were 62% to 82% lower than melatonin concentrations during their nocturnal surge period. Suppression of melatonin concentrations was similar between 400 and 1,200 lux of light. In Experiment 2, heifers were exposed to LD 8:16, LD 16:8, LD 20:4, or LD 24:0 photoperiods (1,200 lux) for 4 months. Throughout treatment, concentrations and durations of the melatonin surge were suppressed in the LD 24:0 group and were greatest (during the nocturnal period) in the LD 8:16 group. Concentrations of prolactin in serum were elevated in animals under long days relative to LD 8:16 treatment and respective pretreatment periods. In conclusion, continuous light at an intensity of 1,200 lux suppressed the nocturnal surge of melatonin, but increased secretion of prolactin for at least 4 months in prepubertal heifers.     

Advancing human circadian rhythms with afternoon melatonin and morning intermittent bright light

CONTEXT: Both light and melatonin can be used to phase shift the human circadian clock, but the phase-advancing effect of the combination has not been extensively investigated. OBJECTIVE: The objective of the study was to determine whether phase advances induced by morning intermittent bright light and a gradually advancing sleep schedule could be increased with afternoon melatonin. PARTICIPANTS: Healthy adults (25 males, 19 females, between the ages of 19 and 45 yr) participated in the study. DESIGN: There were 3 d of a gradually advancing sleep/dark period (wake time 1 h earlier each morning), bright light on awakening [four 30-min bright-light pulses (approximately 5000 lux) alternating with 30 min room light < 60 lux] and afternoon melatonin, either 0.5 or 3.0 mg melatonin timed to induce maximal phase advances, or matching placebo. The dim light melatonin onset was measured before and after the treatment to determine the phase advance. RESULTS: There were significantly larger phase advances with 0.5 mg (2.5 h, n = 16) and 3.0 mg melatonin (2.6 h, n = 13), compared with placebo (1.7 h, n = 15), but there was no difference between the two melatonin doses. Subjects did not experience jet lag-type symptoms during the 3-d treatment CONCLUSIONS: Afternoon melatonin, morning intermittent bright light, and a gradually advancing sleep schedule advanced circadian rhythms almost 1 h/d and thus produced very little circadian misalignment. This treatment could be used in any situation in which people need to phase advance their circadian clock, such as before eastward jet travel or for delayed sleep phase syndrome.     

A model for the study of the acute effects of melatonin in man

The role of the pineal hormone melatonin in human physiology is uncertain. Previous studies correlated plasma melatonin levels with several physiological parameters or determined the responses to pharmacological doses of melatonin during daylight hours. We established an acute model that is more rigorously physiological. Constant nocturnal bright light in sleep-deprived normal men resulted in low plasma melatonin levels throughout the night, in contrast to sleep in the dark and dim light sleep deprivation nights. Subsequently, melatonin was infused during bright light exposure to approximate physiological levels. Plasma GH and PRL measurements in these four conditions revealed an effect of sleep deprivation independent of the presence or absence of melatonin. A subsample of these men had an intermediate level of melatonin suppression with 500 lux light intensity, relative to those during sleep and bright light. The results suggest that melatonin has no acute modulatory effect on the secretion of these two sleep-related hormones.     

Plasma melatonin levels in Japanese quail exposed to dim light are determined by subjective interpretation of day and night, not light intensity

Plasma melatonin levels were measured in male Japanese quail exposed to lighting schedules consisting of combinations of bright light (2000 or 1500 lx), darkness, or dim light (2 lx) or to constant dim light. Melatonin levels in dim light were dependent upon the relative intensity of accompanying phases, being significantly higher when dim light was subjective night than when it was subjective day. There was no significant melatonin rhythm in constant dim light, even on the first day of constant dim light exposure. Melatonin levels were intermediate when dim light was accompanied by both bright light and darkness. These results indicate that melatonin secretion in birds does not depend solely on light intensity. Furthermore, these results suggest that the avian circadian system may be more sensitive to environmental cues than its mammalian counterpart.     

Transition from dim to bright light in the morning induces an immediate elevation of cortisol levels

The only well documented effect of light exposure on endocrine function is the suppression of nocturnal melatonin. Bright light exposure has behavioral effects, including the alleviation of sleepiness during nocturnal sleep deprivation. The present study examines the effects of bright light on the profiles of hormones known to be affected by sleep deprivation (TSH) or involved in behavioral activation (cortisol). Eight healthy men participated each in three studies involving 36 h of continuous wakefulness. In one study, the subjects were exposed to constant dim light (baseline). In the two other studies, dim light exposure was interrupted by a 3-h period of bright light exposure either from 0500-0800 h (early morning study) or from 1300-1600 h (afternoon study). Blood samples were obtained every 15 min for 24 h to determine melatonin, cortisol, and TSH concentrations. Alertness was estimated by the number of lapses on two computerized vigilance-sensitive performance tasks. The early morning transition from dim to bright light suppressed melatonin secretion, induced an immediate, greater than 50% elevation of cortisol levels, and limited the deterioration of alertness normally associated with overnight sleep deprivation. No effect was detected on TSH profiles. Afternoon exposure to bright light did not have any effect on either hormonal or behavioral parameters. The data unambiguously demonstrate an effect of light on the corticotropic axis that is dependent on time of day.     

Daily illuminance levels affect pituitary prolactin in male rats

The 24-h patterns of melatonin, PRL, and gonadotropins in male rats maintained under natural lighting conditions have been found to differ from the patterns in rats kept under artificial lighting. In the present experiments we studied the role of different daily illuminances as a possible causative factor for the variation of the hormonal patterns. Three groups of male rats were kept under artificial lighting conditions (12 h on/12 h off), where the daily illuminance was 550, 110 or 25 lux. After a 7-day adaptation period the pineal content of melatonin, the serum levels of LH, FSH and PRL, and the pituitary content of these hormones were measured by RIAs in samples taken at 10.00, 13.00, 22.00 and 01.00 h. The patterns of pineal melatonin were equal in all three groups. The variation of daily illuminance did not change the serum levels of LH, FSH and PRL or the pituitary content of the gonadotropins. However, the pituitary content of PRL during the light phase was inversely related to the illuminance. The results suggest that the intensity of daily lighting in the studied range does not affect the patterns of melatonin or gonadotropins, but the synthesis of prolactin may be significantly regulated by the daily illuminance level.     

Diurnal Changes in Serum Melatonin Concentrations Under Indoor and Outdoor Environments and Light Suppression of Nighttime Melatonin Secretion in the Female Japanese Monkey

To examine whether artificial light with the intensity commonly used for animal experimentation can mimic natural sunlight with respect to diurnal changes in serum melatonin, and to determine the minimum light intensity required to suppress nocturnal melatonin, serum melatonin profiles were examined in groups of female Japanese monkeys (Macaca fuscata fuscata). Under outdoor environment, light intensities at the level of the monkey's eyes varied during daytime (0900-1500 h) depending on weather conditions (minimum and maximum on particular experimental days: 170 lux at 0900 h on a rainy day and 9500 lux at 0900 h on a slightly cloudy day); under indoor environment, light was provided by ordinary fluorescent bulbs that resulted in intensities of 400-500 lux at the level of monkey's eyes. No difference was found in diurnal changes in serum melatonin concentrations regardless of weather or housing conditions: Serum melatonin remained low during daytime and increased during nighttime. Following exposure to light, irradiances of 10,000, 400-500, 100-140, 50-100, and 10-30 lux at midnight resulted in a rapid decrease in serum melatonin to daytime levels within 1 to 2 h. After the onset of dark, serum melatonin reverted to previous nighttime levels within 2 h. Exposure to a light irradiance of 2-5 lux, however, did not suppress nocturnal melatonin secretion. It is concluded that artificial light can mimic natural sunlight with respect to melatonin secretion in the female Japanese monkey, and that light of 10-30 lux irradiance was sufficient to suppress serum melatonin to near daytime levels.     

Normal pupillary size in fluorescent and bright light

STUDY OBJECTIVE: Despite its common clinical use, the range of normal pupillary size has been described only crudely. The objective of this report is to describe the distribution of normal pupillary sizes in 2 light conditions that are available in clinical settings. METHODS: Pupillary size measurements were taken from healthy patients by the principal investigator using a modified Haab scale. Measurements were obtained in areas with fluorescent lighting with an intensity of between 2,700 and 5,400 lux and by using bright handheld light sources producing a light intensity of greater than 54,000 lux. The effect of varying the type of handheld device (otoscope, ophthalmoscope, or penlight) on mean pupillary size was analyzed on the basis of intervals calculated from the t distribution. RESULTS: One hundred twenty-eight patients were enrolled, with a mean age of 35+/-9 years. The mean pupillary size in fluorescent light was 3.6+/-0.7 mm, and the mean size in bright light was 2.6+/-0.5 mm. Extreme values in fluorescent light were 2.6 mm (5th percentile) and 5.0 mm (96th percentile). Extreme values in bright light were 1.9 mm (3rd percentile) and 3.6 mm (96th percentile). The type of bright light source had no effect on pupillary size measurement. CONCLUSION: Pupillary sizes of greater than 5.0 mm or less than 2.6 mm are rare (<10%) in normal individuals in fluorescent lighting (2,700 to 5,400 lux), and sizes of greater than 3.6 mm or less than 1.9 mm are rare (<10%) in bright light.     

Twenty-four-hour patterns of pineal melatonin and pituitary and plasma prolactin in male rats under 'natural' and artificial lighting conditions

Natural lighting differs from usual artificial lighting mainly as follows: it has larger spectral composition, fluctuations of intensity during the day, higher intensity levels during the night (moonlight, starlight), and gradual changes of illuminance at dawn and dusk. The present experiment was performed in order to study whether these features of lighting affect the 24-hour patterns of melatonin and prolactin in male rats. The rats were kept 7 days in 'natural' lighting (sunlight through windows) or in artificial lighting (cool white fluorescent lamps) of similar periodicities (13/11 h light/dark). The samples were collected at 3-hour intervals during a 24-hour period. Pineal melatonin contents, pituitary prolactin contents, and plasma prolactin concentrations were measured radioimmunologically. The nocturnal pineal melatonin contents were higher and the daytime contents lower in natural than in artificial lighting conditions. A corresponding 'strengthening of rhythm' of prolactin was found in natural lighting. A reason for the higher amplitude variation of melatonin in the natural lighting conditions may be the gradual changes of illuminance at dawn and dusk. The different pituitary and plasma prolactin patterns of the rats kept in the two lighting conditions might partly be explained by a stimulatory effect of melatonin on the production and secretion of prolactin, but other regulatory factors had to be involved, too.     

Scheduled Bright Light for Treatment of Insomnia in Older Adults

OBJECTIVES: To determine whether bright light can improve sleep in older individuals with insomnia.
DESIGN: Single-blind, placebo-controlled, 12-week, parallel-group randomized design comparing four treatment groups representing a factorial combination of two lighting conditions and two times of light administration.
SETTING: At-home light treatment; eight office therapy sessions.
PARTICIPANTS: Thirty-six women and fifteen men (aged 63.6±7.1) meeting primary insomnia criteria recruited from the community.
INTERVENTION: A 12-week program of sleep hygiene and exposure to bright (∼4,000 lux) or dim light (∼65 lux) scheduled daily in the morning or evening for 45 minutes.
MEASUREMENTS: Within-group changes were observed for subjective (sleep logs, questionnaires) and objective (actigraphy, polysomnography) sleep measures after morning or evening bright light.
RESULTS: Within-group changes for subjective sleep measures after morning or evening bright light were not significantly different from those observed after exposure to scheduled dim light. Objective sleep changes (actigraphy, polysomnography) after treatment were not significantly different between the bright and dim light groups. Scheduled light exposure was able to shift the circadian phase predictably but was unrelated to changes in objective or subjective sleep measures. A polymorphism in CLOCK predicted morningness but did not moderate the effects of light on sleep. The phase angle between the circadian system (melatonin midpoint) and sleep (darkness) predicted the magnitude of phase delays, but not phase advances, engendered by bright light.
CONCLUSION: Except for one subjective measure, scheduled morning or evening bright light effects were not different from those of scheduled dim light. Thus, support was not found for bright light treatment of older individuals with primary insomnia.     

Effects of a two-hour early awakening and of bright light exposure on plasma patterns of cortisol, melatonin, prolactin and testosterone in man.

Bright light is a synchronizing agent that entrains human circadian rhythms and modifies various endocrine and neuroendocrine functions. The aim of the present study was to determine whether and how the exposure to a bright light stimulus during the 2 h following a 2 h earlier awakening could modify the disturbance induced by the the sleep deprivation on the plasma patterns of hormones whose secretion is sensitive to light and/or sleep, namely melatonin, prolactin, cortisol and testosterone. Six healthy and synchronized (lights on: 07.00-23.00) male students (22.5 +/- 1.1 years) with normal psychological profiles volunteered for the study in winter. The protocol consisted of a baseline control night (customary sleep schedule) followed by three shortened nights with a rising at 05.00 and a 2 h exposure to either dim light (50 lux; one week) or bright light (2000 lux; other week). Our study showed a phase advance of the circadian rhythm of plasma cortisol without significant modifications of the hormone mean or peak concentration. Plasma melatonin concentration decreased following bright light exposure, whereas no obvious modifications of plasma testosterone or prolactin patterns could be observed in this protocol.     

Sensitivity of Goats to a Light Pulse During the Night as Assessed by Suppression of Melatonin Concentrations in the Plasma

This study investigates the ability of a 1 h light pulse of different intensities at night to suppress plasma melatonin in goats. Six female Saanen dairy goats, about 2 yr old, were housed in a light-tight shed. The goats were habituated for 1 wk to an 8L:16D photoperiod (40.70 +/- 4.16 microW/cm2; 137 +/- 14 lux), lights on 0800 h. A 1 h light pulse, of different intensity on each occasion, was given from 1900 to 2000 h. Light intensity was measured by using a lux meter (mean of 36 measurements at goat's eye level). Five different light intensities were given during December in the order 4.22 +/- 0.62 microW/cm2 (14.2 +/- 2.1 lux), 0.68 +/- 0.09 microW/cm2 (2.3 +/- 0.3 lux), 0.26 +/- 0.004 microW/cm2 (0.87 +/- 0.14 lux), darkness, 40.70 +/- 4.16 microW/cm2 (137 +/- 14 lux), with 1-3 d between treatments. The goats were bled hourly from 1500 to 1900 h and every 15 min from 1900 to 2100 h, and a last bleed occurred at 2200 h. Dark-phase samples were taken in dim red light (less than 0.03 microW/cm2; 0.1 lux). Plasma was assayed for melatonin by radioimmunoassay. Suppression of melatonin concentrations increased as light intensity increased as follows: Darkness, 0%; 0.26 +/- 0.004 microW/cm2; 0%; 0.68 +/- 0.09 microW/cm2; 43.1%; 4.22 +/- 0.62 microW/cm2, 71.1%; 40.70 +/- 4.16 microW/cm2, 81.2%. Suppression was significant (P less than 0.05) at light intensities greater than 0.68 microW/cm2, 2.3 lux. A hyperbolic relationship existed between percent suppression and light intensities.     

Human circadian melatonin rhythm phase delay during a fixed sleep-wake schedule interspersed with nights of sleep deprivation

The human circadian pacemaker, with an intrinsic period between 23.9 and 24.5 hr, can be reset by low levels of light. Biomathematical models of the human clock predict that light-dark cycles consisting of only approximately 3.5 lux during 16 hr of wakefulness and 0 lux during 8 hr of sleep should entrain approximately 45% of the population. However, under real-life conditions, sleep-wake schedules and the associated light-dark exposures are often irregular. It remains unclear whether the phase of the pacemaker would remain stable under such conditions. We investigated the stability of the circadian phase in dim light by assessing the plasma melatonin rhythm during nine consecutive circadian cycles. Ten subjects were scheduled to sleep for 8 hr (0.03 lux) and to be awake for 16 hr (5-13 lux) during all days except on days 4 and 8, during which the subjects were sleep deprived for 40 hr (5-13 lux), either in a sitting/standing or supine body posture. In all subjects, the phase of the melatonin rhythm occurred at a later clock time on day 9 than on day 2 (average delay: 1.4 hr). Largest delays in the melatonin onset were observed in subjects with low amplitude melatonin rhythms. The area under the curve during active melatonin secretion was significantly reduced when subjects were sleep deprived in the 40-hr supine body posture condition compared with either the 40-hr sitting/standing sleep deprivation (SD) or the ambulatory condition under non-SD conditions. Posture differences did not significantly affect the relative phase position of the melatonin profiles. The data indicate that under conditions of reduced zeitgeber strength, the phase of the human circadian pacemaker, using plasma melatonin as a marker, can be phase delayed by one night of SD and the associated dim light exposure.     

Crcadian patterns of melatonin, corticosterone, and progesterone in male rats subjected to chronic stress: Effect of constant illumination

Plasma and pineal melatonin and plasma corticosterone and progesterone concentrations have been shown to be altered by several types of stressors. This study was designed to define the circadian patterns of the hormones mentioned above in rats subjected to chronic stress and to investigate the influence of constant illumination. The results revealed that melatonin and corticosterone circadian patterns deteriorated and their plasma concentrations were significantly elevated. The constant illumination (2,500 lux) during the dark period (from 2000 to 0600) was not able to suppress melatonin production in stressed animals, while the plasma content of corticosterone was decreased at the end of experimental period compared to control rats. Plasma levels of progesterone were increased in stressed animals as well. Constant illumination, however, provoked also an increase of progesterone secretion in controls. Statistical comparisons between hormonal secretory patterns showed that melatonin and corticosterone correlated negatively in controls (r = -0.58, P less than 0.05) during the nighttime. However, in stressed animals correlation was observed only between melatonin and progesterone secretion during the light and dark period (r = -0.43, P less than 0.05). Surprisingly, the correlation during the nighttime in rats subjected to constant illumination was negative (r = -0.60, P less than 0.02) compared to positive correlation (r = 0.60, P less than 0.02) in rats kept under normal lighting regimen. These results suggest that melatonin release is affected by stress and, possibly, under these circumstances, interacts with adrenal steroid secretion.