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Melatonin, pituitary function and stress in humans
Psychoneuroendocrtnology,Vol.4, pp. 351 to 362. © Pergamon Pr--~ Ltd. 1979. Printed in GreatBritain.
0306--4530/79/1001--0351$02,00/0
MELATONIN, PITUITARY FUNCTION AND STRESS IN HUMANS GEORGE M. VAUGHAN,* STEPHEN D. MCDONALD,'~ RICHARDM. JORDAN, JOHN P. ALLEN,~ RODNEY BELL and EDWIN A. STEVENS Audie Murphy Memorial Veterans Hospital, Department of Medicine, The University of Texas Health Science Center at San Antonio and Wilford Hall Medical Center, San Antonio, Texas, U.S.A.
(Received 23 April 1979) SUMMARY (1) The nocturnal rise in plasma melatonin concentration continued through two cycles with continuous light occlusion by blindfolds in normal subjects. A daytime nap did not disturb the rhythm. (2) The plasma melatonin rhythm was present in patients with pituitaryadrenal and pituitary-gonadal failure, but appeared diminished or absent in patients bearing lesions at different points in the pineal afferent pathway. (3) In other patients and subjects who showed normal response of cortisol, growth hormone or prolactin after stimuli including hypoglycemia, pneumoencephalography, exercise or administration of L-dopa during the daytime, there was no stimulation of plasma melatonin concentration. (4) Melatonin was not correlated with prolactin in blood or cerebrospinai fluid of patients with a wide range of plasma prolactin levels. (5) The adult human melatonin rhythm is relatively independent from pituitary, gonadal, and adrenal function, but may rely on a neural pathway similar to that controlling the rhythm in lower animals. The human melatonin rhythm may represent the output of a stable oscillator with a signal relatively free from acute perturbation by sleep, darkness, or stress sufficient to cause changes in other hormones.
Key Words--melatonin; human; stress; pituitary; L-dopa; cerebrospinal fluid; exercise; growth hormone; prolactin; cortisol. INTRODUCTION MELATONIN is considered a psychoactive hormone because its administration can produce oniric episodes and sleep (Ant6n-Tay, 1974; Cramer, Rudolph, Consbruch & Kendel, 1974), elevation of serotonin metabolite in urine (Papavasiliou, Cotzias, Diiby, Steck, Bell & Lawrence, 1972) and cerebrospinal fluid (Carman, Post, Buswell & Goodwin, 1976) in humans, and elevated brain serotonin levels in rats (Ant6n-Tay, 1974) and mice (Cotzias, Tang, Miller & Ginos, 1971). Although the melatonin rhythm is probably normal in depressed patients (Jimerson, Lynch, Post, Wurtman & Bunney, 1977), there is a controversey over whether nmlatonin treatment may be of benefit for neurologic (Ant6n-Tay, 1974; Papavasiliou et al., 1972) and psychiatric (Carman et ai., 1976; Baron, 1977) patients. Although very little has been published on the endocrine effects of melatonin in humans, a rich literature is available suggesting effects of melatonin on pituitary-dependent hormones * Address reprint requests to Dr. George M. Vaughan at the U.S. Army Institute of Surgical Research, Fort Sam Houston, Texas 78234, U.S.A. t Present address: Wright-Patterson Air Force Base, Ohio, U.S.A. ~/Present address: Department of Neurosciences, Peoria School of Medicine, Peoria, Illinois 61605, U.S.A. 351
352
GEORGEM. VAUGnANet al.
in animals (Reiter, Vaughan, Vaughan, Sorrentino & Donofrio, 1975; Reiter, Rollag, Panke & Banks, 1978). Furthermore, since several hormones manifest nyctohemeral rhythmicity, which is also a characteristic feature o f melatonin (Vaughan, Pelham, Pang, Loughlin, Wilson, Sandock, Vaughan, Koslow & Reiter, 1976; Arendt, Wetterberg, Heyden, Sizonenko & Paunier, 1977; Lynch, Jimerson, Ozaki, Post, Bunney & Wurtman, 1978), the discussion o f melatonin in h u m a n s has been couched in its possible relationship to hormones controlled by the pituitary. With few exceptions (Wetterberg, Arendt, Paunier, Sizonenko, van Donselaar & Heyden, 1976; Fevre, Segel, Marks & Boyar, 1978), this possible relationship depends experimentally on a small n u m b e r o f observations o f the in vivo (Nordlund & Lerner, 1977; Smythe, C o m p t o n & Lazarus, 1976) and in vitro (MacPhee, Cole & Rice, 1975) pharmacologic effects o f melatonin. One o f our goals has been to seek a possible link between naturally occurring levels o f h u m a n melatonin and pituitary hormones. Two approaches have been undertaken: (1) to look for correlation between levels o f melatonin and other hormones in normal subjects during periods o f spontaneous fluctuation in pituitary h o r m o n e concentration; and (2) to observe the relationship between melatonin and pituitary hormones when the latter have been altered by disease states or physiological stimuli. The first approach has uncovered no special relationship between melatonin and anterior pituitary hormones in adult men (Vaughan, Allen, Tullis, Siler-Khodr, de la Pefia & Sackman, 1978b). The aim o f the present report is to describe our effort at the second approach, and t o make initial observations on the possible neurological basis for the nocturnal melatonin surge. SUBJECTS AND METHODS The hormonal status of subjects and patients in this study was determined utilizing radio-immunoassays (RIA) for cortisol, New England Nuclear kits, Boston; corticotrophin (ACTH), Allen, Cook, Kendall & McGilvra (1973); growth hormone (GH), luteotrophin (LH), thyrotrophin (TSH), and follicuiotrophin (FSH), Rakoff, VandenBerg, Siler & Yen (1974); prolactin (PRL), Sinha, Selby, Lewis & Vanderlaan (1973); testosterone, Furuyama, Mayes & Nugent (1970); thyroxine, and tri-iodothyronine uptake (radioassay), Nuclear Medical Laboratories kits, Dallas; and melatonin, Vaughan et al., (1978b). Cyclic and prolonged darkness (Fig. l)
Two young healthy men were housed for 6 consecutive days (24-hr cycles) on a ward in a light cycle with darkness and sleep between 2300 and 0700 hr. Beginning at 0800 hr on day 3 and continuing for 48 hr without interruption, they wore blindfolds that totally occluded all light perception. For the final 2 days, they were again exposed to the normal light cycle. On days 2, 4 and 6, they took a 2 hr nap, during which they invariably reported normal sleep and this was also observed by experienced nursing personnel. Venous blood was sampled through a heparin lock for cortisol and melatonin assay at representative times of the day and night, including beginning, middle and end of the nap. Endocrine disease (Fig. 2)
Plasma was sampled from male patients at various times throughout the 24-hr cycle to determine melatonin levels. Ages and diagnoses are indicated in Fig. 2. The endocrine status of these patients is described below. ADX was bilaterally adrenalectomized and had pituitary irradiation for Cushing's disease 3 yr prior to the study. He was off all medications for 10 days, and received 50 mg cortisone acetate orally at midnight of the second of 2 consecutive 24-hr melatonin sampling cycles. Measurements on these samples (not shown in the figure) revealed undetectable cortisol (except after cortisone administration) and quite elevated ACTH levels clearly lacking the normal nyctohemeral rhythm and without suppression after the cortisone dose. Visual fields and polytomographic radiograms of the sella were normal. IAD (isolated ACTH deficiency) had low plasma cortisol which was stimulated in normal fashion by ACTH infusion. GH, but not the low levels of ACTH or cortisol, responded normally after administration of propranolol and glucagon and after induction of hypoglycemia by insulin. Plasma testosterone and free
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FIG. 1. Six consecutive days for each of 2 normal male subjects (NS). The numbers before and after the NS are age and month of sampling, respectively. The hatched area represents darkness. For the middle two days, they wore light-occluding blindfolds. Sleep occurred between 2300 and 0700 hr each night and during the times (nap) indicated by the boxes. thyroxine index were normal. He was off all medication for 7 previous days and during the 2 consecutive days of sampling for melatonin levels. A C R e had a pituitary tumor without suprasellar extension and active acromegaly with elevated (9-47 ng/ml) G H levels throughout the preoperative 24-hr period of sampling at 20 min intervals. Plasma cortisol rose in a normal progressive fashion from 2 ~tg/dl at 0100 hr to 20 ~tg/dl at 0900 hr. Because of tumor characteristics encountered during surgery, he underwent complete hypophysectomy. By 6 weeks following transsphenoidal surgery, he developed evidence of pan-hypopituitarism including new impotence; low testosterone, LH, and FSH levels; failure of cortisol and G H response to insulin hypoglycemia and of G H response to L-dopa; and complete diabetes insipidus verified by standard water deprivation testing. At this time, he underwent another 24 hr, 20 min interval sampling cycle during which G H remained undetectable and cortisol remained less than 2 Fg/dl throughout. His only medication was thyroxine replacement. The melatonin values for the postoperative 24 hr sampling cycle are presented in Fig. 2 for comparison with the preoperative cycle values. H H had ideopathic hypogonadotrophie hypogonadism (low testosterone, LH and FSH levels with small testes). He was off testosterone replacement therapy for a year prior to the time of study. LCT had a leydig cell tumor with normal plasma testosterone level, gynecomastia and elevated urinary excretion of estrogens. DEP had an endogenous depression without demonstrable endocrine abnormality. PT had a pineal tumor with large hypothalamic ectopic sites demonstrated by pneumoencephalogram that had almost completely regressed following irradiation one year prior to testing. There had been no biopsy taken. At the time of testing, he had residual hypothalamic damage expressed as hypogonadotrophic hypogonadism, low plasma cortisol responding to ACTH but not hypoglycemia and partial diabetes
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FIG. 2. Twenty-four hr profiles in male patients whose age and month of sampling are given before and after the symbol, respectively. ADX, bilateral adrenalectomy and high ACTH levels; IAD, isolated ACTH deficiency with secondary adrenal insufficiency; ACRO, active acromegaly preoperatively, and pan-hypopituitarism 6 weeks postoperatively; HH, ideopathic hypogonadotrophic hypogonadism; LCT, leydig cell tumor with gynecomastia; DEP, endogenous depression; PT, pineal tumor with hypothalamie ectopic sites; SDS, Shy-Drager syndrome; PSP, progressive supranuclear palsy; HT, hypothalamic tumor; HD, hypothalamic destruction. Unless otherwise indicated, when data for more than one cycle length from one patient are shown, they represent consecutive 24-hr cycles.
indipidus demonstrated on a formal water deprivation test. Sellar films remained normal. He was on no replacement therapy. SDS had the Shy-Drager syndrome with severe orthostatic hypotension and normal basal norepinephrine levels unresponsive to upright posture, characteristic of a preganglionie sympathetic lesion (Kopin, Lake & Ziegler, 1978). PSP had progressive supranuclear palsy, without evident endocrine abnormality. HT had a hypothalamic tumor (metastatic carcinoma from an undetermined primary site) replacing most of the hypothalamus, with a normal sella. He had pituitary-adrenal and pituitary-thyroid failure, although he was on thyroxine replacement at the time of testing. HD had hypothalamic damage from a pituitary tumor with suprasellar extension that had been partially resected and irradiated 4 yr prior to testing. At the time of testing, he was on replacement therapy for pituitary-adrenal and pituitary-thyroid failure, and his hypothalamic area was mostly replaced by recurrent tumor or cerebrospinal fluid visualized on contrast-enhanced computerized tomographie radiograms. Insulin tolerance test (1TT, Fig. 3) Insulin hypoglycemia was used as part of the regular evaluation of 3 women and 8 men referred for suspected pituitary abnormality. Plasma was sampled at 15 min intervals after i.v. bolus injection of insulin, 0.1 U/kg, for determination of cortisol, G H and PRL. Three patients were ultimately assessed to have no endocrine disease, 2 had empty sella syndrome and 6 had pituitary tumors. Pneumoencephalography (PEG) stress (Fig. 3) PEG was used when clinically indicated as part of the evaluaton of 16 women and 7 men referred for suspected pituitary disease. Plasma and lumbar cerebrospinal fluid (CSF) were sampled at the beginning of the PEG, near the middle, and at the end of the procedure for determination of PRL and melatonin. In
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Fie. 3. Insulin tolerance test (ITT) and pneumocncephalographic (PEG) test values for plasma glucose (GLU), cortisol (CORT), growth hormone (GH), melatonin (MEL) and prolactin (PRL). Average time zero was 0814 hr for the IT'I" and 0929 hr for the PEG. These tests were performed at various times throughout the year. The bottom portions represent data only from those patients with normal prolactin ( < 15 ng/ml) at time zero. One-way analysis of variance followed by a t-test for several means gave p values si~-,nitled by a, <0.05; b, <0.01; c, <0.001 compared with the mean marked by an asterisk. addition, cortisol and G H were determined on plasma. Four patients had the empty sella syndrome and 19 had pituitary adenomata. Eight patients were also in the group whose ITT's are reported. Melatonin levels on 6 of these 23 PEG patients have been reported in a previous rapid communication (Vaughan, McDonald, Jordan, Allen, Bohmfalk, Abou-Samra & Story, 1978c).
Plasma and cerebrospinal fluid (CSF) PRL and melatonin (Fig. 4, 5) Plasma and CSF from the PEG's mentioned above were analyzed for PRL (Fig. 4). Melatonin analysis was also performed on these samples and on paired plasma and lumbar CSF samples taken from 12 additional patients being evaluated for possible pituitary or pineal tumor (Fig. 5). All CSF samples were assayed in the same run that included the comparable plasma samples. Suprasellar extension (SSE) of a pituitary tumor was diagnosed on PEG by observing tumor mass above a line extending between the superior aspect of the posterior clinoids and the posterior ridge of the sphenoid. CSF/plasma ratios were calculated for PRL and melatonin and compared among the groups of patients as described in Figs. 4 and 5.
Exercise (Fig. 6) Two untrained, normal men, aged 21 and 24 yr, each ran 3 sprints of 100 yards at maximal speed in August. Plasma was sampled through a heparin lock before, between and after the sprints, and subjected to RIA of GH, cortisol, and melatonin.
L-dopa administration (Fig. 7) Five healthy subjects, 1 woman and 4 men, aged 21-35 yr, received 500 mg L-dopa orally in July or August. At the same time of day L-dopa was given, 4 of the same 5 subjects also received a placebo in single-blind fashion on the day preceding (2 subjects) or following (2 subjects) L-dopa administration. Plasma was sampled before and at various times after the tablet was administered.
356
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FIG. 4. Prolactin (PRL) concentrations in the initial sample of cerebrospinal fluid (CSF) and paired plasma from the 23 patients receiving PEG in Fig. 3. Open circles indicate patients with normal time-zero PRL in plasma ( < 15 ng/ml). Closed circles represent patients with basal plasma PRL ranging from 16 to 2740 ng/ml. Small arrows pointing down from a symbol indicate CSF PRL below the detectability limit. The separating line in the upper panel at 26 ng/ml was placed to include below the line all patients in the present group having pituitary (Pit) tumor without suprasellar extension (SSE). The line in the bottom panel at ratio 0.2 was placed at the level previously found to include below the line almost all patients with pituitary tumor without SSE (Jordan et al., 1979). Five patients were excluded from the bottom panel because their undetectable CSF PRL (using the detectability level of 2 ng/ml) would produce a CSF/plasma ratio >0.2. Arrows in the upper panel indicate whether the melatonin (M) concentration was above or below 11 pg/ml. The p value indicates the level of association between the group having pituitary tumor with SSE and PRL >26 ng/ml (Chi-square test). RESULTS
Cyclic and prolonged darkness (Fig. 1) B o t h subjects showed the n o r m a l n o c t u r n a l rise o f cortisol a n d m e l a t o n i n levels w h e t h e r o r n o t their eyes were e x p o s e d to light the previous day. A n a f t e r n o o n n a p h a d no consistent effect on levels o f these h o r m o n e s . W h e n sampling was m o r e frequent n e a r the m i d d l e o f the light period, occasional high levels o f m e l a t o n i n a n d cortisol indicated an episodic p a t t e r n o f b l o o d levels o f these h o r m o n e s .
Endocrine disease (Fig. 2) The n y c t o h e m e r a l r h y t h m a n d episodic p a t t e r n o f p l a s m a m e l a t o n i n levels was evident in the p a t i e n t with bilateral a d r e n a l e c t o m y with or w i t h o u t cortisone replacement. T h e r h y t h m was also present in A C T H deficiency a n d in acromegaly. Postoperatively, the acromegalic h a d p a n - h y p o p i t u i t a r i s m b u t retained his m e l a t o n i n r h y t h m . The m e l a t o n i n r h y t h m was also evident in the patients with h y p o g o n a d o t r o p h i c h y p o g o n a d i s m , leydig cell
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FIG. 5. Melatonin (MEL) levels in the initial sample of CSF of the patients receiving PEG, and in 12 additional patients receiving lumbar puncture for whom PRL levels are not available. The small arrows downward indicate CSF levels below the detectability limit. The separating line in both panels is placed at a level that includes below the line the group having pituitary tumor without SSE. Arrows in the upper panel indicate whether prolaetin (P) concentration was above or below 26 ng/ml. The p values indicate the level of association between the group having pituitary tumor with SSE and MEL > 11 pg/ml (Chi-square test).
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Fzo. 6. Two healthy young men ran a 100-yd dash at the times marked by the vertical lines. MEL, melatonin; CORT, cortisol; GH, growth hormone. t u m o r a n d e n d o g e n o u s depression. H o w e v e r , only a questionable r h y t h m with a n a t t e n u a t e d n o c t u r n a l rise (to < 6 0 pg/ml) was evident in patients with pineal t u m o r , S h y - D r a g e r syndrome, supranuclear palsy or hypothalamic tumor.
Insulin tolerance test (Fig. 3) S y m p t o m s o f h y p o g l y c e m i a a n d m e a s u r e d suppression o f glucose c o n c e n t r a t i o n s o c c u r r e d in each subject. Despite a significant s t i m u l a t i o n o f cortisol, G H a n d P R L c o n c e n t r a t i o n s , m e l a t o n i n levels d i d n o t rise.
GEORGEM. VAUGHANet al.
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Pneumoencephalography stress (Fig. 3) Despite a rise in cortisol and P R L levels, and elevated levels ( > 7 ng/ml) of G H throughout, melatonin showed no consistent change. Baseline plasma levels of melatonin and P R L at the beginning of the I T T ' s or P E G ' s showed no correlation on regression analysis.
CSF and plasma PRL and melatonin (Fig. 4, 5) Baseline P E G plasma P R L ranged from normal ( < 15 ng/ml) to over 2000 ng/ml. P R L in the CSF above 26 ng/ml was associated with suprasellar extention (SSE) of a pituitary tumor. However, this association lost its significance when CSF]plasma P R L concentration ratio was considered in this small group of patients (Fig. 4). Like PRL, melatonin concentration was always lower in CSF than in plasma (Fig. 5). However, the higher levels of CSF melatonin were associated with SSE of a pituitary tumor, whether melatonin concentrations were expressed directly for CSF or as the CSF/plasma ratio. In this small series, an elevated CSF/plasma melatonin ratio (>0.3) was the most specific CSF hormonal indicator for SSE, although it was only 50 ~o sensitive. During PEG, no change occurred in CSF levels of P R L or melatonin. A typical example was one patient in which plasma P R L rose from 100 to 418 ng/ml while P R L and melatonin in the CSF remained undetectable and plasma melatonin remained stable (41, 40, 38 pg/ml).
Exercise (Fig. 6) Although a dramatic rise in G H levels occurred after sprinting in both subjects, no change in melatonin or cortisol levels could be seen related to the exercise.
L-dopa (Fig. 7) Although an impressive rise of G H concentration occurred in 3 subjects after admini-
MELATONIN,PITUITARYANDSTRESS
359
stration of L-dopa, none of the subjects showed a melatonin rise following L-dopa beyond the melatonin levels observed following placebo. DISCUSSION Lack of an acute effect of daytime sleep (naps) on melatonin levels was consistent with previous reports (Vaughan et al., 1976; Jimerson et aL, 1977). The findings suggesting an episodic profile and occasional high levels of melatonin during the light phase also have precedent (Vaughan et al., 1976; Weinberg, D'Elleto, Weitzman, Erlich & Hollander, 1979). It is not yet known what factors exert moment to moment control over melatonin secretion. The presence of a nocturnal rise in melatonin in the patient without adrenal glands, with or without cortisol administration, conformed to the finding in rats that the nocturnal rise in pineal melatonin synthesis does not depend on the adrenals (Lynch, Ho & Wurtman, 1977). Independence of the melatonin rhythm from the ACTH-cortisol axis was confirmed by the melatonin rhythm in the patient with ACTH deficiency, and in the patient with acromegaly following removal of his pituitary. This latter patient also showed that the melatonin rhythm does not depend upon GH. The hypogonadal patient and the post-surgical acromegalic (HH and ACRO, Fig. 2) demonstrated that the melatonin rhythm can occur with a deficient pituitary-gonadal axis. Since all our measurements were taken in patients who were euthyroid, we cannot comment on any possible role of thyroid function in controlling the melatonin rhythm. It appears that an absent or attenuated melatonin rhythm was exhibited by the patient with Shy-Drager syndrome (SDS, Fig. 2). This disease is associated with failure of the preganglionic component of the sympathetic nervous system (Kopin et al., 1978). The patient with progressive supranuclear palsy (PSP) demonstrating brainstem involvement with severe cranial nerve deficits, also appeared to have an attenuated rhythm. The patient with an ectopic pinealoma (PT) could have had compromise of his melatonin rhythm as a result of hypothalamic damage or pineal irradiation. The patients with a hypothalamic lesion (HT and HD) also lacked a melatonin rhythm. It is possible that the above 5 patients had, in effect, lesions at various points in a pathway which normally would extend from the suprachiasmatic nucleus caudally through the hypothalamus and brainstem to the thoracic spinal cord, then through the sympathetic nervous system to the pineal. It is known that this pathway is responsible for the nocturnal rise of pineal melatonin synthesis in experimental animals (Moore, 1978). Kneisley, Moskowitz & Lynch (1978) have observed the absence of a nocturnal urinary melatonin surge in patients with cervical spinal cord transection. Our results with plasma melatonin tend to support their suggestion that the same afferent neural pathway to the pineal may also be operative in control of the human melatonin rhythm. Previous reports have indicated that stress applied to rats by inducing hypoglycemia (Lynch, Eng & Wurtman, 1973a), immobilization (Lynch et al., 1977), or ether anesthesia (Vaughan, Allen, Tullis, Sackman & Vaughan, 1978a) can result in increased synthesis of melatonin in the pineal. Administration of melatonin has reportedly resulted in stimulation of GH levels in humans (Smythe et al., 1976) and PRL levels in rats (Kamberi, Mical & Porter, 1971 ; Lu & Meites, 1973). Thus, it is reasonable to consider whether the rise in GH and PRL which follow stress are mediated by a rise in melatonin, We have shown that in
360
GEORGEM. VAUOHANet aL
humans this mechanism does not explain the responses of GH, PRL, or cortisol which variously occurred in the stresses of ITT, PEG and exercise, because we observed no increase in plasma melatonin concentration. Wetterberg (1978) has seen a similar dissociation between prolactin and melatonin levels after electrogenic convulsions. It is of interest that oral melatonin administration did not affect plasma prolactin in humans (Smythe et al., 1976). During the ITT, the melatonin levels tended to fall. Although this could have resulted from the test stimulus, we think it is more likely due to the normal tendency of melatonin to fall at that early time of the day. Although our studies indicate no stimulatory role for melatonin in GH responses, Smythe & Lazarus (1974) found some inhibition of the GH response to insulin after pre-treatment with melatonin. Whether the latter results indicate a pharmacologic effect or a physiologic role for melatonin in modulating brain serotonin is not known. The previously observed stimulation of plasma PRL in rats given melatonin into the blood (Lu & Meites, 1973) or into the cerebrospinal fluid (Kamberi et al., 1971; Iwazaki Kato, Ohgo, Abe, Imura, Hirata, Senoh, Tokuyama & Hayaishi, 1978) could raise the question of whether high PRL levels in patients with pituitary tumors might be related to high plasma or CSF melatonin levels. This seems not to be the case because baseline plasma prolactin levels over a wide range were not correlated with plasma or CSF melatonin in the PEG patients. Although high PRL and melatonin in the CSF seemed to be related to SSE of a pituitary tumor, the two hormones tended not to be high in the CSF of the same patients (top panels, Figs. 4, 5). Elevated PRL level in the CSF in SSE has already been reported. In a larger group of patients than reported herein, the CSF/plasma PRL ratio was greater in the presence than in the absence of SSE (Jordan, McDonald, Stevens & Kendall, 1979). We are not able to explain the association of SSE with elevated CSF melatonin levels. L-Dopa was observed to result in a striking elevation of pineal melatonin in rats (Lynch, Wang & Wurtman, 1973b), and was, therefore, suggested as the basis for a possible pineal function test in humans (Wurtman & Moskowitz, 1977). Despite GH stimulation by L-dopa in our subjects, there was no stimulation of melatonin levels. Arendt (1978) and Wetterberg (1978) also found no change in human melatonin after L-dopa. Our results suggest that it is unlikely that either stress or L-dopa would provide the basis for a human pineal function test, at least with respect to melatonin. In the rat, some authors (Lynch et aL, 1973a; Vaughan et aL, 1978a) have been able to produce a rise in pineal melatonin synthesis, using various forms of stress. Furthermore, pineal denervation potentiated the melatonin response to stress (of immobilization), whereas adrenalectomy prevented it (Lynch et al., 1977). Utilizing the same species, other workers found that stress (of swiming) failed to stimulate pineal melatonin synthesis. They provided evidence that this failure was due to protection of pinealocytes against stimulation by circulating catecholamines (presumably partly from the adrenal medulla), as a result of uptake of these amines in the intact pineal sympathetic nerve endings (Parfitt & Klein, 1976). Perhaps, such a mechanism is operative in humans and prevents stress-activated plasma melatonin increments. Whether a response in pineal melatonin could have occurred without a corresponding change in plasma melatonin is not yet known; nor is it known whether stresses other than those presently employed would have affected human pineal or plasma melatonin.
MELATON~q,PITUITARYAND STRESS
361
Wetterberg (1978); Wietzman, Weinberg, D'Eletto, Lynch, Wurtman, Czeisler & Erlich (1978); and L y n c h et al., (1978), have shown that after a prolonged (week or more) exposure to a shifted cycle o f light-dark and activity-sleep, the h u m a n melatonin r h y t h m shifts accordingly. Nevertheless, acute light exposure and wakefulness at night do not reduce melatonin levels in h u m a n s (Wetterberg, 1978; Arendt, 1978; Vaughan, Bell & de la Pefia, 1979). Similarly, the present results show that sleep, darkness or stress during the day do not elevate melatonin levels, and that the melatonin r h y t h m remains intact in altered h o r m o n a l states. This m a y allow the h u m a n melatonin r h y t h m to continuously reflect a stable oscillator which signifies at a given time the b o d y ' s exposure to a retrospective light-dark or sleep-activity schedule for the previous several days. The stresses o f day to day living probably would not produce acute perturbations in the melatonin signal. We c a n n o t exclude the possibility that a chronically shifted or modified melatonin r h y t h m resulting f r o m long-term exposure to an altered light-dark schedule might alter pituitary function. We thank the nurses of the Special Diagnostic and Treatment Unit of the V. A. Hospital; Dr. A. de la Pefia for use of the sleep laboratory; C. Ney and G. Ortiz for technical assistance; Dr. T. Siler-Khodr for GH assays; Dr. M. Ziegler for norepinephrine assays and informative consultation; Drs. G. Bohmfalk, W. Brown and M. Abou-Samra for surgical care of some of the patients; and Bess Mitchell for typing. This work was partially supported by NIH Grant No. P30 HI) 10202. REFERENCES
ALLEN,J. P., COOK,D. M., KENOALL,J. W. & McGILvRA, K. (1973) Maternal-fetal ACTH relationship in man. J. Clin. Endocrinol. Metab. 37, 230-234. AWr6N-TAY,F. (1974) Melatonin: effects on brain function. Adv. Biochem. Psychopharmacol. 11, 315-324. ARENDT,J. (1978) Melatonin in body fluids. J. Neural Transmission, Suppl. 13, 265-278. ARENDT,J., WETTERBERG,L., HEYDEN,T., SIZONENKO,P. C. ~ PAUNIER,L. (1977) Radioimmunoassay of melatonin: human serum and cerebrospinal fluid. Hormone Res. 8, 65-75. BARON,M. (1977) Melatonin, MSH and psychosis. Amer. J. Psychiat. 134, 583. CARMAN,J. S., POST, R. M., BUSW~LL,R. & GOODWlN, K. F. (1976) Negative effects of melatonin on depression. Amer. J. Psychiat. 133, 1181-1186. COTZaAS,G. C., TANG, L. C., MILER, S. T. & GINOS, J. Z. (1971) Melatonin and abnormal movements induced by L-dopa in mice. Science 173, 450-452. ~R, H., RLrOOU,H, J., CONSBRUCH,U. & KENDEL,K. (1974) On the effects of melatonin on sleep and behavior in man. Adv. Biochem. Psychopharmacol. 11, 187-191. FEw~L M., SEGEL,T., MARKS,J. F. & BOYAR,R. M. (1978) LH and melatonin secretion in pubertal boys. J. Clin. Endocrinol. Metab. 47, 1383-1386. FLrgL~tA, S., MAX,.S, D. M. & NuG~rcr, C. A. (1970) A radioimmunoassay for plasma testosterone. Steroids 16, 415-428. IWASAKI,Y., KATO,Y., OHC,O, S., AaE, H., IMURA,H., HIRATA,F., SENOH,S., TOKt~AMA,T. & HAYAISI-H,O. (1978) Effects of indoleamines and their newly identified metabolites on prolactin release in rats. Endocrinol. 103, 254-258. JIMERSON,D. C., LYNCH,H. J., POST,R. M., WURTMAN,R. J. & BUNNEY,W. E. (1977) Urinary melatonin rhythms during sleep deprivation in depressed patients and normais. Life Sciences 20, 1501-1508. JORDAN,R. M., McDoNALD, S. D., STEVENS,E. A. & KENDALL,J. W. (1979) Cerebrospinai fluid prolactin. A re-evaluation. Arch. Int. Med. 139, 208-211. KAMBERI,L A., MICAL,R. S. & PORTER,J. C. (1971) Effects of melatonin and serotonin on the release of FSH and prolactin. Endocrinol. 88, 1288--1293. KNEISLEY,L. W., MOSKOWITZ,M. A. & LYNCH,H. J. (1978) Cervical spinal cord lesions disrupt the rhythm in human melatonin excretion. J. Neural Transmission, Suppl. 13, 311-323. KOPIN, I. J., LAKE,R. C. & ZIEOLER,M. (1978) Plasma levels of norepinephrine. Ann. Int. Med. 88, 671-680. Lu, K. & MEITES,J. (1973) Effects of serotonin precursors and melatonin on serum prolactin release in rats. Endocrinol. 93, 152-155.
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LYNCH, H. J., ENG, J. P. & WURTMAN, R. J. (1973a) Control of pineal indole biosynthesis by changes in sympathetic tone caused by factors other than environmental lighting. Proc. Nat. Acad. Sciences (U.S.A.) 70, 1704-1707. LYNCH, a . J., WANG, P. (~. WURTMAN, R. J. (1973b) Increase in rat pineal melatonin content following L-dopa administration. Life Sciences 12, 145-151. LYNCH, H. J., HO, M. & WURTMAN,R. J. (1977) The adrenal medulla may mediate the increase in pineal melatonin synthesis induced by stress but not that caused by exposure to darkness. J. Neural Transmission 40, 87-97. LYNCH, H. J., JIMERSON,D. C., OZAKI, Y., POST, R. M., BUNNEY, W. E., JR. ~. WURTMAN, R. J. (1978) Entrainment of rhythmic melatonin secretion in man to a 12-hr phase shift in the light/dark cycle. Life Sciences 23, 1557-1564. MACPHEE,A. A., COLE, F. E. & RICE, B. F. (1975) The effect of melatonin on steriodogenesis by the human ovary in vitro. J. Clin. Endocrinol. Metab. 40, 688-696. MOORE, R. Y. (1978) Neural control of pineal function in mammals and birds. J. Neural Transmission, SuppL 13, 47-58. NORDLUND, J. J. & LERNER, A. B. (1977) The effects of oral melatonin on skin color and on the release of pituitary hormones. J. Clin. Endocrinol. Metab. 45, 768-774. PAPAVASILIOU,P. S., COTZIAS, G. C., DUBY, S. E., STECK, A. J., BELL, M. & LAWRENCE,W. H. (1972) Melatonin and Parkinsonism. J. Amer. Med. Assoc. 221, 88. PARrH r, A. G. & KLEIn, D. C. (1976) Sympathetic nerve endings in the pineal gland protect against acute stress-induced increase in N-acetyltransferase (E.C. 2.3.1.5.) activity. EndocrinoL 99, 840-851. RAKOEr, J., VANDENBERG,G., SLIER, T. M. & YEN, S. S. C. (1974) An integrated direct functional test of the adenohypophysis. Amer. J. Obstet. GynecoL 119, 358-368. REnmR, R. J., ROLLAG, M. D., PANKE, E. S. & BANKS, A. F. (1978) Melatonin: reproductive effects. J. Neural Transmission, Suppl. 13, 209-223. REITER, R. J., VAUGHAN,M. K., VAUGHAN,G. M., SORRENTINO,S., JR. • DONOFRIO, R. J. (1975) The pineal as an organ of internal secretion. In Frontiers o f Pineal Physiology, M. D. Altschule (Ed.), pp. 54-174. MIT Press, Cambridge, Mass. SINHA, Y. A., SELBY,F. W., LEWIS, V. J. & VANDERLAAN,W. P. (1973) A homologous radioimmunoassay for human prolactin. J. Clin. Endocrinol. Metab. 36, 509-516. SMYTHE, G. A. & LAZARUS,L. (1974) Suppression of human growth hormone secretion by melatonin and cyproheptadine. J. Clin. Invest. 54, 116--121. SMYTHE, G. A., COMPTON, P. J. & LAZARUS,L. (1976) Serotoninergic control of human growth hormone secretion: the actions of L-dopa and 2-bromo-ct-ergocryptine. In Growth Hormone and Related Peptides, A. Pecile and E. E. MfiUer (Eds.), pp. 222-235. Excerpta Medica, Amsterdam. VAUGHAN, G. M., BELL, R. & DE LA PEI~A, A. (1979) Nocturnal plasma melatonin in humans: episodic pattern and influence of light. Neuroscience Letters (In press). VAUGHAN, G. M., ALLEN, J. P., TULLIS, W., SACKMAN,J. W. ~; VAUGHAN,M. K. (1978a) Stress-induced increase of pineal N-acetyltransferase activity in intact rats. Neuroscience Letters 9, 83-87. VAUGHAN,G. M., ALLEN,J. P., TULLIS,W., SILER-KHODR,T. M., DE LA PEIZlA,A. & SACKMAN,J. W. (1978b) Overnight plasma profiles of melatonin and certain adenohypophyseal hormones in men. J. Clin. EndocrinoL Metab. 47, 566-571. VAUGHAN,G. M., MCDONALD,S. D., JORDAN,R. i . , ALLEN, J. P., BOHMFALK,G. L., ABou-SAMRA, M. & STORY,J. L. (1978c) Melatonin concentration in human blood and cerebrospinal fluid: relationship to stress. J. Clin. Endocrinol. Metab. 47, 220-223. VAUGHAN,G. M., PELHAM,R. W., PANG, S. F., LOUGHLIN,L. L., WILSON,K. M., SANDOCK,K. L., VAUGHAN, M. K., KOSLOW, S. H. & RErrER, R. J. (1976) Nocturnal elevation of plasma melatonin and urinary 5-hydroxyindoleacetic acid in young men: attempts at modification by brief changes in environmental lighting and sleep and by autonomic drugs. J. Clin. EndocrinoL Metab. 42, 752-764. WEINBERG,U., D'EIETTO, R. D., WErrZMAN,E. D., ERLICH,S. & HOLLANDER,C. S. (1979) Circulating melatonin in man: episodic secretion throughout the light-dark cycle. J. Clin. Endocrinol. Metab. 48, 114-118. WEITZMAN,E. D., WEINBERG,U., D'ELETTO,R., LYNCH, H., WURTMAN,R. J., CZEISLER,CH. t~ ERLICH, S. (1978) Studies of the 24-hr rhythm of melatonin in man. J. Neural Transmission, Suppl. 13, 325-337. WETrERBERG, L. (1978) Melatonin in humans: physiological and clinical studies. J. Neural Transmission, SuppL 13, 289-310. WETrERBERG, L., ARENDT, J., PAUNIER, L., SIZONENKO,P. C., VAN DONSELAAR,W. & HEYDEN,T. (1976) Human melatonin changes during the menstrual cycle. J. Clin. Endocrinol. Metab. 42, 185-188. WURTMAN, R. J. & MosKowrrz, M. A. (1977) The pineal organ (first of two parts). New Engl. J. Med. 296, 1329-1333.
0306--4530/79/1001--0351$02,00/0
MELATONIN, PITUITARY FUNCTION AND STRESS IN HUMANS GEORGE M. VAUGHAN,* STEPHEN D. MCDONALD,'~ RICHARDM. JORDAN, JOHN P. ALLEN,~ RODNEY BELL and EDWIN A. STEVENS Audie Murphy Memorial Veterans Hospital, Department of Medicine, The University of Texas Health Science Center at San Antonio and Wilford Hall Medical Center, San Antonio, Texas, U.S.A.
(Received 23 April 1979) SUMMARY (1) The nocturnal rise in plasma melatonin concentration continued through two cycles with continuous light occlusion by blindfolds in normal subjects. A daytime nap did not disturb the rhythm. (2) The plasma melatonin rhythm was present in patients with pituitaryadrenal and pituitary-gonadal failure, but appeared diminished or absent in patients bearing lesions at different points in the pineal afferent pathway. (3) In other patients and subjects who showed normal response of cortisol, growth hormone or prolactin after stimuli including hypoglycemia, pneumoencephalography, exercise or administration of L-dopa during the daytime, there was no stimulation of plasma melatonin concentration. (4) Melatonin was not correlated with prolactin in blood or cerebrospinai fluid of patients with a wide range of plasma prolactin levels. (5) The adult human melatonin rhythm is relatively independent from pituitary, gonadal, and adrenal function, but may rely on a neural pathway similar to that controlling the rhythm in lower animals. The human melatonin rhythm may represent the output of a stable oscillator with a signal relatively free from acute perturbation by sleep, darkness, or stress sufficient to cause changes in other hormones.
Key Words--melatonin; human; stress; pituitary; L-dopa; cerebrospinal fluid; exercise; growth hormone; prolactin; cortisol. INTRODUCTION MELATONIN is considered a psychoactive hormone because its administration can produce oniric episodes and sleep (Ant6n-Tay, 1974; Cramer, Rudolph, Consbruch & Kendel, 1974), elevation of serotonin metabolite in urine (Papavasiliou, Cotzias, Diiby, Steck, Bell & Lawrence, 1972) and cerebrospinal fluid (Carman, Post, Buswell & Goodwin, 1976) in humans, and elevated brain serotonin levels in rats (Ant6n-Tay, 1974) and mice (Cotzias, Tang, Miller & Ginos, 1971). Although the melatonin rhythm is probably normal in depressed patients (Jimerson, Lynch, Post, Wurtman & Bunney, 1977), there is a controversey over whether nmlatonin treatment may be of benefit for neurologic (Ant6n-Tay, 1974; Papavasiliou et al., 1972) and psychiatric (Carman et ai., 1976; Baron, 1977) patients. Although very little has been published on the endocrine effects of melatonin in humans, a rich literature is available suggesting effects of melatonin on pituitary-dependent hormones * Address reprint requests to Dr. George M. Vaughan at the U.S. Army Institute of Surgical Research, Fort Sam Houston, Texas 78234, U.S.A. t Present address: Wright-Patterson Air Force Base, Ohio, U.S.A. ~/Present address: Department of Neurosciences, Peoria School of Medicine, Peoria, Illinois 61605, U.S.A. 351
352
GEORGEM. VAUGnANet al.
in animals (Reiter, Vaughan, Vaughan, Sorrentino & Donofrio, 1975; Reiter, Rollag, Panke & Banks, 1978). Furthermore, since several hormones manifest nyctohemeral rhythmicity, which is also a characteristic feature o f melatonin (Vaughan, Pelham, Pang, Loughlin, Wilson, Sandock, Vaughan, Koslow & Reiter, 1976; Arendt, Wetterberg, Heyden, Sizonenko & Paunier, 1977; Lynch, Jimerson, Ozaki, Post, Bunney & Wurtman, 1978), the discussion o f melatonin in h u m a n s has been couched in its possible relationship to hormones controlled by the pituitary. With few exceptions (Wetterberg, Arendt, Paunier, Sizonenko, van Donselaar & Heyden, 1976; Fevre, Segel, Marks & Boyar, 1978), this possible relationship depends experimentally on a small n u m b e r o f observations o f the in vivo (Nordlund & Lerner, 1977; Smythe, C o m p t o n & Lazarus, 1976) and in vitro (MacPhee, Cole & Rice, 1975) pharmacologic effects o f melatonin. One o f our goals has been to seek a possible link between naturally occurring levels o f h u m a n melatonin and pituitary hormones. Two approaches have been undertaken: (1) to look for correlation between levels o f melatonin and other hormones in normal subjects during periods o f spontaneous fluctuation in pituitary h o r m o n e concentration; and (2) to observe the relationship between melatonin and pituitary hormones when the latter have been altered by disease states or physiological stimuli. The first approach has uncovered no special relationship between melatonin and anterior pituitary hormones in adult men (Vaughan, Allen, Tullis, Siler-Khodr, de la Pefia & Sackman, 1978b). The aim o f the present report is to describe our effort at the second approach, and t o make initial observations on the possible neurological basis for the nocturnal melatonin surge. SUBJECTS AND METHODS The hormonal status of subjects and patients in this study was determined utilizing radio-immunoassays (RIA) for cortisol, New England Nuclear kits, Boston; corticotrophin (ACTH), Allen, Cook, Kendall & McGilvra (1973); growth hormone (GH), luteotrophin (LH), thyrotrophin (TSH), and follicuiotrophin (FSH), Rakoff, VandenBerg, Siler & Yen (1974); prolactin (PRL), Sinha, Selby, Lewis & Vanderlaan (1973); testosterone, Furuyama, Mayes & Nugent (1970); thyroxine, and tri-iodothyronine uptake (radioassay), Nuclear Medical Laboratories kits, Dallas; and melatonin, Vaughan et al., (1978b). Cyclic and prolonged darkness (Fig. l)
Two young healthy men were housed for 6 consecutive days (24-hr cycles) on a ward in a light cycle with darkness and sleep between 2300 and 0700 hr. Beginning at 0800 hr on day 3 and continuing for 48 hr without interruption, they wore blindfolds that totally occluded all light perception. For the final 2 days, they were again exposed to the normal light cycle. On days 2, 4 and 6, they took a 2 hr nap, during which they invariably reported normal sleep and this was also observed by experienced nursing personnel. Venous blood was sampled through a heparin lock for cortisol and melatonin assay at representative times of the day and night, including beginning, middle and end of the nap. Endocrine disease (Fig. 2)
Plasma was sampled from male patients at various times throughout the 24-hr cycle to determine melatonin levels. Ages and diagnoses are indicated in Fig. 2. The endocrine status of these patients is described below. ADX was bilaterally adrenalectomized and had pituitary irradiation for Cushing's disease 3 yr prior to the study. He was off all medications for 10 days, and received 50 mg cortisone acetate orally at midnight of the second of 2 consecutive 24-hr melatonin sampling cycles. Measurements on these samples (not shown in the figure) revealed undetectable cortisol (except after cortisone administration) and quite elevated ACTH levels clearly lacking the normal nyctohemeral rhythm and without suppression after the cortisone dose. Visual fields and polytomographic radiograms of the sella were normal. IAD (isolated ACTH deficiency) had low plasma cortisol which was stimulated in normal fashion by ACTH infusion. GH, but not the low levels of ACTH or cortisol, responded normally after administration of propranolol and glucagon and after induction of hypoglycemia by insulin. Plasma testosterone and free
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FIG. 1. Six consecutive days for each of 2 normal male subjects (NS). The numbers before and after the NS are age and month of sampling, respectively. The hatched area represents darkness. For the middle two days, they wore light-occluding blindfolds. Sleep occurred between 2300 and 0700 hr each night and during the times (nap) indicated by the boxes. thyroxine index were normal. He was off all medication for 7 previous days and during the 2 consecutive days of sampling for melatonin levels. A C R e had a pituitary tumor without suprasellar extension and active acromegaly with elevated (9-47 ng/ml) G H levels throughout the preoperative 24-hr period of sampling at 20 min intervals. Plasma cortisol rose in a normal progressive fashion from 2 ~tg/dl at 0100 hr to 20 ~tg/dl at 0900 hr. Because of tumor characteristics encountered during surgery, he underwent complete hypophysectomy. By 6 weeks following transsphenoidal surgery, he developed evidence of pan-hypopituitarism including new impotence; low testosterone, LH, and FSH levels; failure of cortisol and G H response to insulin hypoglycemia and of G H response to L-dopa; and complete diabetes insipidus verified by standard water deprivation testing. At this time, he underwent another 24 hr, 20 min interval sampling cycle during which G H remained undetectable and cortisol remained less than 2 Fg/dl throughout. His only medication was thyroxine replacement. The melatonin values for the postoperative 24 hr sampling cycle are presented in Fig. 2 for comparison with the preoperative cycle values. H H had ideopathic hypogonadotrophie hypogonadism (low testosterone, LH and FSH levels with small testes). He was off testosterone replacement therapy for a year prior to the time of study. LCT had a leydig cell tumor with normal plasma testosterone level, gynecomastia and elevated urinary excretion of estrogens. DEP had an endogenous depression without demonstrable endocrine abnormality. PT had a pineal tumor with large hypothalamic ectopic sites demonstrated by pneumoencephalogram that had almost completely regressed following irradiation one year prior to testing. There had been no biopsy taken. At the time of testing, he had residual hypothalamic damage expressed as hypogonadotrophic hypogonadism, low plasma cortisol responding to ACTH but not hypoglycemia and partial diabetes
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indipidus demonstrated on a formal water deprivation test. Sellar films remained normal. He was on no replacement therapy. SDS had the Shy-Drager syndrome with severe orthostatic hypotension and normal basal norepinephrine levels unresponsive to upright posture, characteristic of a preganglionie sympathetic lesion (Kopin, Lake & Ziegler, 1978). PSP had progressive supranuclear palsy, without evident endocrine abnormality. HT had a hypothalamic tumor (metastatic carcinoma from an undetermined primary site) replacing most of the hypothalamus, with a normal sella. He had pituitary-adrenal and pituitary-thyroid failure, although he was on thyroxine replacement at the time of testing. HD had hypothalamic damage from a pituitary tumor with suprasellar extension that had been partially resected and irradiated 4 yr prior to testing. At the time of testing, he was on replacement therapy for pituitary-adrenal and pituitary-thyroid failure, and his hypothalamic area was mostly replaced by recurrent tumor or cerebrospinal fluid visualized on contrast-enhanced computerized tomographie radiograms. Insulin tolerance test (1TT, Fig. 3) Insulin hypoglycemia was used as part of the regular evaluation of 3 women and 8 men referred for suspected pituitary abnormality. Plasma was sampled at 15 min intervals after i.v. bolus injection of insulin, 0.1 U/kg, for determination of cortisol, G H and PRL. Three patients were ultimately assessed to have no endocrine disease, 2 had empty sella syndrome and 6 had pituitary tumors. Pneumoencephalography (PEG) stress (Fig. 3) PEG was used when clinically indicated as part of the evaluaton of 16 women and 7 men referred for suspected pituitary disease. Plasma and lumbar cerebrospinal fluid (CSF) were sampled at the beginning of the PEG, near the middle, and at the end of the procedure for determination of PRL and melatonin. In
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Fie. 3. Insulin tolerance test (ITT) and pneumocncephalographic (PEG) test values for plasma glucose (GLU), cortisol (CORT), growth hormone (GH), melatonin (MEL) and prolactin (PRL). Average time zero was 0814 hr for the IT'I" and 0929 hr for the PEG. These tests were performed at various times throughout the year. The bottom portions represent data only from those patients with normal prolactin ( < 15 ng/ml) at time zero. One-way analysis of variance followed by a t-test for several means gave p values si~-,nitled by a, <0.05; b, <0.01; c, <0.001 compared with the mean marked by an asterisk. addition, cortisol and G H were determined on plasma. Four patients had the empty sella syndrome and 19 had pituitary adenomata. Eight patients were also in the group whose ITT's are reported. Melatonin levels on 6 of these 23 PEG patients have been reported in a previous rapid communication (Vaughan, McDonald, Jordan, Allen, Bohmfalk, Abou-Samra & Story, 1978c).
Plasma and cerebrospinal fluid (CSF) PRL and melatonin (Fig. 4, 5) Plasma and CSF from the PEG's mentioned above were analyzed for PRL (Fig. 4). Melatonin analysis was also performed on these samples and on paired plasma and lumbar CSF samples taken from 12 additional patients being evaluated for possible pituitary or pineal tumor (Fig. 5). All CSF samples were assayed in the same run that included the comparable plasma samples. Suprasellar extension (SSE) of a pituitary tumor was diagnosed on PEG by observing tumor mass above a line extending between the superior aspect of the posterior clinoids and the posterior ridge of the sphenoid. CSF/plasma ratios were calculated for PRL and melatonin and compared among the groups of patients as described in Figs. 4 and 5.
Exercise (Fig. 6) Two untrained, normal men, aged 21 and 24 yr, each ran 3 sprints of 100 yards at maximal speed in August. Plasma was sampled through a heparin lock before, between and after the sprints, and subjected to RIA of GH, cortisol, and melatonin.
L-dopa administration (Fig. 7) Five healthy subjects, 1 woman and 4 men, aged 21-35 yr, received 500 mg L-dopa orally in July or August. At the same time of day L-dopa was given, 4 of the same 5 subjects also received a placebo in single-blind fashion on the day preceding (2 subjects) or following (2 subjects) L-dopa administration. Plasma was sampled before and at various times after the tablet was administered.
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FIG. 4. Prolactin (PRL) concentrations in the initial sample of cerebrospinal fluid (CSF) and paired plasma from the 23 patients receiving PEG in Fig. 3. Open circles indicate patients with normal time-zero PRL in plasma ( < 15 ng/ml). Closed circles represent patients with basal plasma PRL ranging from 16 to 2740 ng/ml. Small arrows pointing down from a symbol indicate CSF PRL below the detectability limit. The separating line in the upper panel at 26 ng/ml was placed to include below the line all patients in the present group having pituitary (Pit) tumor without suprasellar extension (SSE). The line in the bottom panel at ratio 0.2 was placed at the level previously found to include below the line almost all patients with pituitary tumor without SSE (Jordan et al., 1979). Five patients were excluded from the bottom panel because their undetectable CSF PRL (using the detectability level of 2 ng/ml) would produce a CSF/plasma ratio >0.2. Arrows in the upper panel indicate whether the melatonin (M) concentration was above or below 11 pg/ml. The p value indicates the level of association between the group having pituitary tumor with SSE and PRL >26 ng/ml (Chi-square test). RESULTS
Cyclic and prolonged darkness (Fig. 1) B o t h subjects showed the n o r m a l n o c t u r n a l rise o f cortisol a n d m e l a t o n i n levels w h e t h e r o r n o t their eyes were e x p o s e d to light the previous day. A n a f t e r n o o n n a p h a d no consistent effect on levels o f these h o r m o n e s . W h e n sampling was m o r e frequent n e a r the m i d d l e o f the light period, occasional high levels o f m e l a t o n i n a n d cortisol indicated an episodic p a t t e r n o f b l o o d levels o f these h o r m o n e s .
Endocrine disease (Fig. 2) The n y c t o h e m e r a l r h y t h m a n d episodic p a t t e r n o f p l a s m a m e l a t o n i n levels was evident in the p a t i e n t with bilateral a d r e n a l e c t o m y with or w i t h o u t cortisone replacement. T h e r h y t h m was also present in A C T H deficiency a n d in acromegaly. Postoperatively, the acromegalic h a d p a n - h y p o p i t u i t a r i s m b u t retained his m e l a t o n i n r h y t h m . The m e l a t o n i n r h y t h m was also evident in the patients with h y p o g o n a d o t r o p h i c h y p o g o n a d i s m , leydig cell
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FIG. 5. Melatonin (MEL) levels in the initial sample of CSF of the patients receiving PEG, and in 12 additional patients receiving lumbar puncture for whom PRL levels are not available. The small arrows downward indicate CSF levels below the detectability limit. The separating line in both panels is placed at a level that includes below the line the group having pituitary tumor without SSE. Arrows in the upper panel indicate whether prolaetin (P) concentration was above or below 26 ng/ml. The p values indicate the level of association between the group having pituitary tumor with SSE and MEL > 11 pg/ml (Chi-square test).
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Fzo. 6. Two healthy young men ran a 100-yd dash at the times marked by the vertical lines. MEL, melatonin; CORT, cortisol; GH, growth hormone. t u m o r a n d e n d o g e n o u s depression. H o w e v e r , only a questionable r h y t h m with a n a t t e n u a t e d n o c t u r n a l rise (to < 6 0 pg/ml) was evident in patients with pineal t u m o r , S h y - D r a g e r syndrome, supranuclear palsy or hypothalamic tumor.
Insulin tolerance test (Fig. 3) S y m p t o m s o f h y p o g l y c e m i a a n d m e a s u r e d suppression o f glucose c o n c e n t r a t i o n s o c c u r r e d in each subject. Despite a significant s t i m u l a t i o n o f cortisol, G H a n d P R L c o n c e n t r a t i o n s , m e l a t o n i n levels d i d n o t rise.
GEORGEM. VAUGHANet al.
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Time (minutes) FIG. 7. Response of GH and melatonin to 500 mg oral L-dopagiven at time zero in young adults. Subject 2 was the only woman. Average time zero was 0957 hr.
Pneumoencephalography stress (Fig. 3) Despite a rise in cortisol and P R L levels, and elevated levels ( > 7 ng/ml) of G H throughout, melatonin showed no consistent change. Baseline plasma levels of melatonin and P R L at the beginning of the I T T ' s or P E G ' s showed no correlation on regression analysis.
CSF and plasma PRL and melatonin (Fig. 4, 5) Baseline P E G plasma P R L ranged from normal ( < 15 ng/ml) to over 2000 ng/ml. P R L in the CSF above 26 ng/ml was associated with suprasellar extention (SSE) of a pituitary tumor. However, this association lost its significance when CSF]plasma P R L concentration ratio was considered in this small group of patients (Fig. 4). Like PRL, melatonin concentration was always lower in CSF than in plasma (Fig. 5). However, the higher levels of CSF melatonin were associated with SSE of a pituitary tumor, whether melatonin concentrations were expressed directly for CSF or as the CSF/plasma ratio. In this small series, an elevated CSF/plasma melatonin ratio (>0.3) was the most specific CSF hormonal indicator for SSE, although it was only 50 ~o sensitive. During PEG, no change occurred in CSF levels of P R L or melatonin. A typical example was one patient in which plasma P R L rose from 100 to 418 ng/ml while P R L and melatonin in the CSF remained undetectable and plasma melatonin remained stable (41, 40, 38 pg/ml).
Exercise (Fig. 6) Although a dramatic rise in G H levels occurred after sprinting in both subjects, no change in melatonin or cortisol levels could be seen related to the exercise.
L-dopa (Fig. 7) Although an impressive rise of G H concentration occurred in 3 subjects after admini-
MELATONIN,PITUITARYANDSTRESS
359
stration of L-dopa, none of the subjects showed a melatonin rise following L-dopa beyond the melatonin levels observed following placebo. DISCUSSION Lack of an acute effect of daytime sleep (naps) on melatonin levels was consistent with previous reports (Vaughan et al., 1976; Jimerson et aL, 1977). The findings suggesting an episodic profile and occasional high levels of melatonin during the light phase also have precedent (Vaughan et al., 1976; Weinberg, D'Elleto, Weitzman, Erlich & Hollander, 1979). It is not yet known what factors exert moment to moment control over melatonin secretion. The presence of a nocturnal rise in melatonin in the patient without adrenal glands, with or without cortisol administration, conformed to the finding in rats that the nocturnal rise in pineal melatonin synthesis does not depend on the adrenals (Lynch, Ho & Wurtman, 1977). Independence of the melatonin rhythm from the ACTH-cortisol axis was confirmed by the melatonin rhythm in the patient with ACTH deficiency, and in the patient with acromegaly following removal of his pituitary. This latter patient also showed that the melatonin rhythm does not depend upon GH. The hypogonadal patient and the post-surgical acromegalic (HH and ACRO, Fig. 2) demonstrated that the melatonin rhythm can occur with a deficient pituitary-gonadal axis. Since all our measurements were taken in patients who were euthyroid, we cannot comment on any possible role of thyroid function in controlling the melatonin rhythm. It appears that an absent or attenuated melatonin rhythm was exhibited by the patient with Shy-Drager syndrome (SDS, Fig. 2). This disease is associated with failure of the preganglionic component of the sympathetic nervous system (Kopin et al., 1978). The patient with progressive supranuclear palsy (PSP) demonstrating brainstem involvement with severe cranial nerve deficits, also appeared to have an attenuated rhythm. The patient with an ectopic pinealoma (PT) could have had compromise of his melatonin rhythm as a result of hypothalamic damage or pineal irradiation. The patients with a hypothalamic lesion (HT and HD) also lacked a melatonin rhythm. It is possible that the above 5 patients had, in effect, lesions at various points in a pathway which normally would extend from the suprachiasmatic nucleus caudally through the hypothalamus and brainstem to the thoracic spinal cord, then through the sympathetic nervous system to the pineal. It is known that this pathway is responsible for the nocturnal rise of pineal melatonin synthesis in experimental animals (Moore, 1978). Kneisley, Moskowitz & Lynch (1978) have observed the absence of a nocturnal urinary melatonin surge in patients with cervical spinal cord transection. Our results with plasma melatonin tend to support their suggestion that the same afferent neural pathway to the pineal may also be operative in control of the human melatonin rhythm. Previous reports have indicated that stress applied to rats by inducing hypoglycemia (Lynch, Eng & Wurtman, 1973a), immobilization (Lynch et al., 1977), or ether anesthesia (Vaughan, Allen, Tullis, Sackman & Vaughan, 1978a) can result in increased synthesis of melatonin in the pineal. Administration of melatonin has reportedly resulted in stimulation of GH levels in humans (Smythe et al., 1976) and PRL levels in rats (Kamberi, Mical & Porter, 1971 ; Lu & Meites, 1973). Thus, it is reasonable to consider whether the rise in GH and PRL which follow stress are mediated by a rise in melatonin, We have shown that in
360
GEORGEM. VAUOHANet aL
humans this mechanism does not explain the responses of GH, PRL, or cortisol which variously occurred in the stresses of ITT, PEG and exercise, because we observed no increase in plasma melatonin concentration. Wetterberg (1978) has seen a similar dissociation between prolactin and melatonin levels after electrogenic convulsions. It is of interest that oral melatonin administration did not affect plasma prolactin in humans (Smythe et al., 1976). During the ITT, the melatonin levels tended to fall. Although this could have resulted from the test stimulus, we think it is more likely due to the normal tendency of melatonin to fall at that early time of the day. Although our studies indicate no stimulatory role for melatonin in GH responses, Smythe & Lazarus (1974) found some inhibition of the GH response to insulin after pre-treatment with melatonin. Whether the latter results indicate a pharmacologic effect or a physiologic role for melatonin in modulating brain serotonin is not known. The previously observed stimulation of plasma PRL in rats given melatonin into the blood (Lu & Meites, 1973) or into the cerebrospinal fluid (Kamberi et al., 1971; Iwazaki Kato, Ohgo, Abe, Imura, Hirata, Senoh, Tokuyama & Hayaishi, 1978) could raise the question of whether high PRL levels in patients with pituitary tumors might be related to high plasma or CSF melatonin levels. This seems not to be the case because baseline plasma prolactin levels over a wide range were not correlated with plasma or CSF melatonin in the PEG patients. Although high PRL and melatonin in the CSF seemed to be related to SSE of a pituitary tumor, the two hormones tended not to be high in the CSF of the same patients (top panels, Figs. 4, 5). Elevated PRL level in the CSF in SSE has already been reported. In a larger group of patients than reported herein, the CSF/plasma PRL ratio was greater in the presence than in the absence of SSE (Jordan, McDonald, Stevens & Kendall, 1979). We are not able to explain the association of SSE with elevated CSF melatonin levels. L-Dopa was observed to result in a striking elevation of pineal melatonin in rats (Lynch, Wang & Wurtman, 1973b), and was, therefore, suggested as the basis for a possible pineal function test in humans (Wurtman & Moskowitz, 1977). Despite GH stimulation by L-dopa in our subjects, there was no stimulation of melatonin levels. Arendt (1978) and Wetterberg (1978) also found no change in human melatonin after L-dopa. Our results suggest that it is unlikely that either stress or L-dopa would provide the basis for a human pineal function test, at least with respect to melatonin. In the rat, some authors (Lynch et aL, 1973a; Vaughan et aL, 1978a) have been able to produce a rise in pineal melatonin synthesis, using various forms of stress. Furthermore, pineal denervation potentiated the melatonin response to stress (of immobilization), whereas adrenalectomy prevented it (Lynch et al., 1977). Utilizing the same species, other workers found that stress (of swiming) failed to stimulate pineal melatonin synthesis. They provided evidence that this failure was due to protection of pinealocytes against stimulation by circulating catecholamines (presumably partly from the adrenal medulla), as a result of uptake of these amines in the intact pineal sympathetic nerve endings (Parfitt & Klein, 1976). Perhaps, such a mechanism is operative in humans and prevents stress-activated plasma melatonin increments. Whether a response in pineal melatonin could have occurred without a corresponding change in plasma melatonin is not yet known; nor is it known whether stresses other than those presently employed would have affected human pineal or plasma melatonin.
MELATON~q,PITUITARYAND STRESS
361
Wetterberg (1978); Wietzman, Weinberg, D'Eletto, Lynch, Wurtman, Czeisler & Erlich (1978); and L y n c h et al., (1978), have shown that after a prolonged (week or more) exposure to a shifted cycle o f light-dark and activity-sleep, the h u m a n melatonin r h y t h m shifts accordingly. Nevertheless, acute light exposure and wakefulness at night do not reduce melatonin levels in h u m a n s (Wetterberg, 1978; Arendt, 1978; Vaughan, Bell & de la Pefia, 1979). Similarly, the present results show that sleep, darkness or stress during the day do not elevate melatonin levels, and that the melatonin r h y t h m remains intact in altered h o r m o n a l states. This m a y allow the h u m a n melatonin r h y t h m to continuously reflect a stable oscillator which signifies at a given time the b o d y ' s exposure to a retrospective light-dark or sleep-activity schedule for the previous several days. The stresses o f day to day living probably would not produce acute perturbations in the melatonin signal. We c a n n o t exclude the possibility that a chronically shifted or modified melatonin r h y t h m resulting f r o m long-term exposure to an altered light-dark schedule might alter pituitary function. We thank the nurses of the Special Diagnostic and Treatment Unit of the V. A. Hospital; Dr. A. de la Pefia for use of the sleep laboratory; C. Ney and G. Ortiz for technical assistance; Dr. T. Siler-Khodr for GH assays; Dr. M. Ziegler for norepinephrine assays and informative consultation; Drs. G. Bohmfalk, W. Brown and M. Abou-Samra for surgical care of some of the patients; and Bess Mitchell for typing. This work was partially supported by NIH Grant No. P30 HI) 10202. REFERENCES
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