Melatonin and Growth Hormone Deficiency: A Contribution to the Evaluation of Neuroendocrine Disorders

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1 Melatonin and Growth Hormone Deficiency: A Contribution to the Evaluation of Neuroendocrine Disorders Fideleff G., Suárez M., Boquete HR, Azaretzky M., Sobrado P., Brunetto O*, Fideleff HL Endocrinology Unit, Hospital Alvarez; Endocrinology Division, Hospital Pedro Elizalde. CABA. Buenos Aires, Argentina. ABSTRACT Melatonin, a hormone secreted by the pineal gland, constitutes a landmark in neuroendocrine integration. The relationship between melatonin and different pituitary hormones and sex steroids has been studied; however, the relationship between growth hormone (GH) and melatonin remains unclear. Considering that melatonin is an essential component of the so-called biological clock, related to circadian rhythm, day-night cycle, and sleep-dependent hormonal alterations, and knowing that physiological GH secretion occurs predominantly at night, we decided to evaluate nocturnal melatonin secretion in a group of GH-deficient children and adults on and off replacement therapy. Patients and Methods: We studied 44 patients with GH deficiency (GHD), duly confirmed by pharmacological tests, divided into 4 groups: Group a (Ga): untreated GHD children; Group b (Gb): GHD children on GH replacement therapy (0.16 mg/kg/week, stable dose for at least 6 months); Group c (Gc): untreated GHD adults and Group d (Gd): GHD adults on GH replacement therapy ( mg/day, to maintain IGF1 between 0 and +2 SDS, stable dose for at least 6 months). All associated hormonal deficits were adequately replaced. Melatonin production was evaluated by measuring the excretion of its major urinary metabolite: 6-Sulphatoxymelatonin (6-SM). Urinary 6- SM was measured (radioimmunoassay, Stockgrand Ltd, Guildford, UK) in nocturnal samples (6PM to 8AM) in all patients. Results: Nocturnal 6-SM levels expressed as µg/unit of time were (mean ± SEM) for the pediatric group: Ga = 6.50 (± 5.10) and Gb = 8.21 (± 5.31) (Mann Whitney test, p = 0.82). For adults: Gc = 2.99 (± 1.17) and Gd = 6.60 (± 2.00) (Mann Whitney test, p = 0.35) Discussion and Conclusions: It is difficult to characterize the relationship between melatonin and GH in healthy individuals; however, the administration of intravenous melatonin stimulates GH secretion in normal adults. In some hypothalamic-pituitary alterations, changes in the secretory pattern of melatonin have been reported, but possible variations in GHD patients have not been thoroughly characterized yet. This led us to evaluate 6-SM concentrations in GH deficient children and adults on and off adequate replacement therapy. One of the major aspects of this study has been the evaluation of baseline 6-SM concentrations, with no physiological or pharmacological stimulation. Even if under the conditions of this study we found no differences in nocturnal excretion of 6-SM between untreated and treated GHD individuals in both groups, this does not rule out the potential existence of differences that might be detected by studying diurnal melatonin secretion and its difference with nocturnal secretion. Such studies may contribute to an understanding of potential chronobiological disorders involved in GH deficiency. Authors declare no conflict of interest. Study partially presented at the Eighth Congress of FASEN (2010). Key words: Melatonin, growth hormone deficiency, circadian rhythm, neuroendocrine disorders, growth hormone. INTRODUCTION Melatonin acts as a "neuroendocrine transducer of environmental information collected via the retina and passing through a retina-pineal gland neuronal circuit in which other structures are involved, including but not limited to, the retino-hypothalamic tract, the

2 supra-chiasmatic nucleus and the cervical ganglion 1. The relationship between melatonin and different pituitary hormones and sex steroids has been studied; however, the relationship between growth hormone (GH) and melatonin remains unclear. (2) Considering that melatonin is an essential component of the so-called biological clock, related to circadian rhythm, day-night cycle, and sleep-dependent hormonal alterations, and knowing that physiological GH secretion occurs predominantly at night, we decided to evaluate nocturnal secretion of melatonin by measuring the excretion of its major urinary metabolite, 6-Sulphatoxymelatonin (6-SM), in a group of GH-deficient children and adults on and off replacement therapy (3). MATERIAL AND METHODS Patients We studied 44 patients with GH deficiency (GHD), 16 children and 28 adults. In children, GH deficiency was confirmed by two stimulation tests with GH response below 5 ng/ml and in adults with an insulin tolerance test with a maximum response of < 3 ng/ml and/or arginine stimulation test < 1.8 ng/ml. The sample of pediatric patients was divided into the following groups: Group a (Ga): untreated GHD children; Group b (Gb): GHD children on GH replacement therapy at a dose of 0.16 mg/kg/week. Adults were divided into: Group c (Gc): untreated GHD adults and Group d (Gd): GHD adults on GH replacement therapy at a dose of mg/day, to maintain IGF1 between 0 and +2 SDS. All treated pediatric and adult patients maintained stable doses for at least 6 months. Patients with other associated hormonal deficits were receiving adequate replacement, with the exception of 3 females who were not receiving sex steroid replacement therapy because of their chronological age or gynecological contraindication. Clinical data of the patients enrolled are shown in Table I. Methods Urinary 6-SM was measured (radioimmunoassay, Stockgrand Ltd, Guildford, UK) in nocturnal samples (6PM to 8AM) in all patients. Intra- and interassay coefficients of variation (CVs) were 4% and 7%, respectively. Levels of 6-SM were expressed as µg excreted by time interval. Samples for IGF-I and stimulation tests for the diagnosis of GH deficiency were collected at 8.00 AM under fasting conditions. GH was measured by a solid-phase, 2-site chemiluminescent enzyme immunometric assay (IMMULITE, Siemens), calibrated against the first reference standard (WHO International Reference Preparation 80/505); as from batch 206 calibrated against the second reference standard (WHO International Reference Preparation 98/574). The detection limit was 0.08 ng/ml and the intra- and inter-assay CVs were below 3% for a dose of 1.8 ng/ml and below 5% for a dose of 9.6 ng/ml. IGF-I was measured by immunoradiometric assay (IRMA) after acid-alcohol extraction (Diagnostics Systems Laboratories, Webster, TX); the detection limit was 3.6 ng/ml. As from April 2010, IGF-I was measured by a solid-phase, enzyme-labeled chemiluminescent immunometric assay with sample pretreatment on the dilution step; the detection limit was 20 ng/ml. Intra- and interassay CVs were below 5.3% for a dose of 64 ng/ml and below 5.4% for a dose of 157 ng/ml for IRMA, while for the chemiluminescent assay, CVs were below 5.1% for a dose of 59 ng/ml and below 6.5% for a dose of 230 ng/ml. For both assays, standards were calibrated against WHO International Reference Reagent 87/518. The SDS for IGF-I was calculated according to previously published data. (4)

3 6-SM (µg/time interval) 6-SM (µg/time interval) Statistics A non-parametric statistical test (Mann-Whitney test) was used for the evaluation of nocturnal 6-SM results comparing untreated GHD children vs. GHD children on GH therapy and untreated GHD adults vs. GHD adults on GH therapy. The SPSS software, version 11 (SPSS Inc., Chicago, IL, USA) was used. Statistical significance was set at p< Values were expressed as mean and standard error (SE). RESULTS Nocturnal 6-SM levels expressed as μg/ time unit for the pediatric group were as follows: Ga = 6.50 (± 5.10) and Gb = 8.21 (± 5.31) (p = 0.82). For adults: Gc = 2.99 (± 1.17) and Gd = 6.60 (± 2.00) (p = 0.35). Individual values of patients in each group are shown in Figure 1. TABLE I: Clinical Data from patients in Group a (Ga): untreated GHD children, Group b (Gb): GHD children on GH therapy, Group C (Gc): untreated GHD adults, Group d (Gd): GHD adults on GH therapy. Ga Gb Gc Gd n Age (range) yr yr yr yr. Gender: F/M 1/5 2/8 5/5 9/9 Deficit: isolated / multiple 5/1 7/3 0/10 0/18 Deficit: idiopathic/ organic 6/0 9/0 5/5 4/14 Associated antidiuretic hormone deficit Figure 1. Individual values of 6-SM in patients of each group (Ga: untreated GHD children; Gb: GHD children on GH therapy; Gc: untreated GHD adults and Gd: GHD adults on GH therapy). The horizontal line in each point cloud indicates the mean for the group.

4 DISCUSSION Melatonin, generic name given to N-acetyl-5-methoxytryptamine, which was isolated by Lerner et al in 1958, occurs in most living organisms. (5,6) It is known that melatonin is the main product synthesized by the pineal gland, and is not only present in blood, but also in other body fluids including urine, semen, bile, saliva and cerebrospinal fluid (6). Melatonin has a circadian secretory rhythm with low concentrations during the daytime and high concentrations at night, with peak concentrations at approximately 3 AM. (7) In previous studies, it has been reported that maximum pineal activity occurs in childhood and progressively declines in the following decades of life (6,7). Even if the most well known function of melatonin in humans is its contribution to synchronization of biologic rhythms, with a key role in light-dark cycles, a large number of evidence in recent years has suggested that melatonin may be involved in other functions, including body temperature modulation, antiproliferative effects, antioxidant effects, interrelationship with the immune system. (8,9,10,11,12). In addition, it acts as a "neuroendocrine transducer of environmental information, as previously mentioned (13). It is difficult to establish the relationship between melatonin and GH in healthy individuals, since although it has been reported that intravenous melatonin administration stimulates GH secretion in healthy adults, other authors have reported a lack of effect or a decrease in GH following oral or intravenous administration of melatonin (14, 15, 16). It is also known that there are interrelations between the somatotropic axis and the sleep-wake cycle, with increased GH secretion during sleep. (17) However, in general, published studies assess melatonin concentrations under GH stimulation tests during daytime. In this respect, Muñoz-Hoyos et al. showed a decrease in melatonin concentrations in children at 30 minutes following clonidine administration (18). These authors also showed a decrease in melatonin concentrations at 120 minutes after the initiation of the propanolol plus exercise test. (19) Other authors observed this decreased during arginine, GH-RH and insuline intolerance tests (20,21). Some studies showed that melatonin administration produced spontaneous GH secretion or increased amplitude of GH secretion (21,22). In some alterations associated with hypothalamic-pituitary disorders, changes in the secretory pattern of melatonin have been reported, such as Prader Willi Syndrome (23). Some studies show that melatonin levels are higher in children with growth hormone deficiency than in patients with idiopathic short stature, with a negative correlation reported between GH peak after stimulation test and nocturnal melatonin concentrations. (24) However, possible variations in GHD patients have not been thoroughly characterized yet. This led us to evaluate 6-SM concentrations in GH deficient children and adults on and off adequate replacement therapy. Given the progressive decline in melatonin secretion from childhood to adulthood, we consider it appropriate to compare 6-SM values between the two groups. One of the major aspects of this study has been the evaluation of baseline 6- SM concentrations, with no physiological or pharmacological stimulation. Even if under the conditions of this study we found no differences in nocturnal excretion of 6-SM between untreated and treated GHD individuals in both age groups, this does not rule out the potential existence of differences that might be detected by studying diurnal melatonin secretion and its difference with nocturnal secretion. Administration of exogenous GH replacement therapy is not performed following the rhythm of physiological secretion of this hormone, which occurs mainly during the night with multiple peaks occurring approximately 90 minutes after the onset of sleep (stages 3 and 4 non-rem sleep). It could be hypothesized that, perhaps, treatment might allow for normalization of growth velocity in children and correction of associated metabolic disorders in adults, but it might not be enough to change the secretory pattern of melatonin. In this first study, we

5 preferred to evaluate only nocturnal secretion of 6-SM, given the higher concentrations of this metabolite during this period and the very low levels occurring during the morning and in the early afternoon. In addition, the relationship between 6-SM in treated and untreated GHD children and adults could also be conditioned by a large number of variables (age, gender, treatment duration, etc.). Our study would constitute a first approach to a deeper understanding of chronobiological disorders involved in GH deficiency. Furthermore, it might also contribute to an interpretation of the pathophysiologic mechanism underlying some GHD alterations associated with quality of life and metabolic disorders, which would put a new perspective to the design of future studies. REFERENCES.

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