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Biogenic Amine Assays for Pharmaceutical and Specialty
Research-summary product data: please request full insert for
current and more detailed information
The following Elisa kits are distributed by RDI Division of Fitzgerald Industries Intl for in vitro
research use only-not for use in or on humans or animals, not for use in
diagnostics.
 
MELATONIN SULFATE ELISA
 cat#RDI-RE54031, new cat#: 55R-RE54031   1395.00/kit
 
 




 
 
 
 Catalogue No : RDI-RE54031
 Product group : Tumor Markers 12 x 8 
 Product name : Melatonin-sulfate ELISA 96
 Method : ELISA 
 Incubation time: 20h, 3h, 15 min 
 Standard curve : 0.01 - 20ng/ml 
 Sample/Prep. : 10µl urine  
 Isotope/Substr.: TMB, 450nm 
 
 




 
 
 
 
 

CONTENTS

Introduction
Contents of the Kit
Principle of the Test
Test Procedure
Performance Characteristics
Expected Values
Alternative Applications
(More) Clinical Background
Sales Arguments
Product Literature
Miscellaneous
 
 

Introduction

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 The hormone melatonin, which is produced by the pineal gland, was first 
 discovered in 1958 by A. B. Lerner. The concentration of melatonin shows a 
 marked diurnal rhythm in the pineal gland and in the blood with high  
 levels normally occurring during the night and low levels during the day. 
 Maximal values of melatonin in the blood observed between midnight and 4 
 a. m. in the morning. 
  
 The biological half life of melatonin is 45 min. This implies that for 
 research purposes blood samples need to be collected in short time 
 intervals to determine the course of melatonin production. In addition, 
 waking up probands during the night for sample collection may affect the 
 melatonin levels in the blood. These problems are avoided, when 
 determining the melatonin metabolites 6-Sulfatoxy (MeSO4) and 
 6-Hydroxyglucuronide in urine. 80 - 90% of the melatonin is secreted as 
 6-Sulfatoxymelatonin in the urine. The concentration of 
 6-Sulfatoxymelatonin in urine correlates well with the total level of 
 melatonin in the blood during the collection period. 
 
 
 

Contents of the Kit

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 1. Assay Buffer 1 bottle 
 80 ml, ready for use. 
 Tris buffer with BSA and stabilizer,  
 
 2. Microtiter Strips 12 strips 
 each 8 wells, 
 coated with goat-anti-rabbit antibody. 
 
 3. Antiserum 1 vial 
 6 ml, ready for use, 
 antiserum from rabbit with stabilizers, 
 in Tris buffer with stabilizer. 
  
 4. Standards A - G 7 vials 
 0.1 ml each, ready for use, 
 in Tris buffer with stabilizers.  
 
 Concentrations: 
 Standard A B C D E F G 
 ng/ml 0 1.7 5.2 15.6 46.7 140 420 
 
 5. Enzyme Conjugate, Concentrate 1 vial 
 0,2 ml, MeSO4-Peroxidase-Conjugate 
 in Phosphate buffer with stabilizers. 
 
 6. Control 1 and 2  2 vials 
 
 0.1 ml each , ready for use 
 for concentration see quality control certificate. 
  
 7. Wash Buffer 1 bottle 
 50 ml, concentrate, 
 phosphate buffer with Tween and stabilizers. 
 
 8. TMB Substrat Buffer 1 bottle 
 30 ml, ready for use,  
 contains H2O2 in citrate buffer with stabilizers. 
 
 9. TMB Substrate Solution, Concentrate 1 vial 
 1 ml, 
 contains Tetramethylbenzidine (TMB) 
 with stabilizers. 
  
 10. TMB Stopping Solution 1 vial 
 15 ml, ready for use, 
 contains 1 M H2SO4  
 Corrosive, avoid skin contact. 
 
 11. Adhesive Foil  3 pieces 
 
 

Principle of the Test

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 The 6-Sulfatoxymelatonin ELISA kit provides material for the quantitative 
 measurement of MeSO4 in urine. The assay procedure follows the basic 
 principle of competitive ELISA: the competition between MeSO4 konjugated 
 to horseradishe peroxidase and MeSO4 in the sample for a fixed number of 
 antibody-binding sites. The amount of complexes bound to the microtiter 
 plates is inversely proportional to the analyte concentration of the 
 sample. 
  
 After 2 hours of incubation, the non-fixed antigens are removed by washing 
 and the bound antibodies are determined by use of TMB as substrate. 
  
 Quantification of unknowns is achieved by comparing the enzymatic activity 
 of unknowns with a response curve prepared by using known standards. 
 
 

Test Procedure

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 A Summary  
 ========= 
 1. Pipet 50 µl each of diluted standards, diluted controls and diluted 
 sample.  
 2. Pipet 50 µl peroxidase conjugate. Add 50 µl antiserum. 
 3. Incubate 120 min. at room temperature on an orbital shaker. 
 4. Wash four times with wash buffer.  
 5. Pipet 200 µl of TMB substraze solution. Incubate at room temperature 
 for 30 min. 
 6. Add 100 µl of TMB stop solution. 
 7. Read the optical density at 405 nm. 
 
 
 B Detailed Instructions  
 ======================= 
 
 Materials required but not provided  
 
 - Pipet 10, 20, 25, 50, 100, 1000 µl; Mutlipette Eppendorf or similar 
 product  
 - Polystyrene test tubes (12 x 75 mm) 
 - Vortex mixer 
 - ELISA reader capable of reading absorbance at 450 nm. 
  
 Sample Preparation 
 
 NOTE: Avoid direct sun light.  
 
 Melatonin sulfate is stable without preservative in urine for up to four 
 days at 4 °C and for up to two years when stored at - 20 °C. 
 
 Dilute standards, controls and samples 1 : 51 : 
 Pipet 10 µl of standards, controls and patient urine into polystyrene test 
 tubes, add 500 µl of assay buffer and vortex mix. 
 
 Withdraw 50 µl aliquots for ELISA! 
  
 Test Procedure 
 
 1. Pipet 50 µl each of diluted standard, diluted control and diluted 
 sample into the appropriate wells. 
 
 2. Add 50 µl diluted peroxidase conjugate and 50 µl antiserum.  
 
 3. Cover the plate with the adhesive foil and incubate 120 min. at room 
 temperature on an orbital shaker (500 U/min) 
  
 4. Wash each well four times with wash buffer (the use of a washer is 
 recommended!). Remove the wash buffer carefully. Invert plate to 
 remove any remaining liquid by tapping plate on clean blotting paper. 
 
 NOTE: The correct performance of the washing procedure is of vital 
 importance for the sensitivity and precision of this assay! 
 
 5. Pipet 200 µl TMB substrate solution into each well. 
 
 6. Incubate at room temperature on a shaker for 30 minutes. 
 
 7. Stop the substrate reaction by adding 100 µl of TMB stopping solution 
 to each well.  
 
 8. Briefly mix contents by gently shaking the plate. 
  
 9. Read the optical density at 450 nm (reference wave length 600 - 650 
 nm) with a microtiter plate reader within 60 minutes after stopping. 
 ---------------------------------------------------------------------------
 
 
 Preparation of Reagents 
  
 The contents of the kit can be divided into three separate runs. The 
 volumes stated below are for one test procedure with 4 strips (32 
 determinations). If a larger number of strips is to be used, the volumes 
 have to be changed accordingly. 
 
 1. Wash Buffer  
 Phosphate precipitates, which may form during storage at 4°C, 
 redissolve at room temperature. 
 15 ml of the concentrate have to be diluted 1:20 with bidistilled 
 water up to 300 ml. The wash buffer is now ready for use. Store at 
 2-8°C for 4 weeks. 
 
 2. Enzyme Conjugate 
 Dilute 50 µl of the concentrate with 2.0 ml of assay buffer. Prepare 
 freshly before use and use only once! 
  
 3. TMB Substrate Solution 
 Add 300 µl TMB substrate solution, concentrate to 9 ml TMB substrate 
 buffer and mix. Prepare TMB substrate solution just before use and use 
 only once. 
 
  
 
 
 
 

Performance Characteristics

Back to Contents
  
 1. Specificity 
 
 The cross reactivity of the anti-6-Sulfatoxymelatonin antiserum has been 
 measured against various compounds. 
 
 Compound Cross reactivity (%) 
 6-Sulfatoxymelatonin 100.0 
 Melatonin 0.002 
 6-Hydroxy-Melatonin 0.001 
  N-Acetyl-L-Hydroxytryptamin 0.0005 
 N-Acetyl-L-Tryptophan <0.0001 
 5-Methoxytryptamin <0.0001 
 Tryptamin  <0.0001 
 5-Methoxytryptophol <0.0001 
 5-Methoxyindol-3-acetic acid <0.0001 
 DL-5-Methoxytryptophan <0.0001  
 5-Hydroxyindol-acetic acid <0.0001 
 5-Hydroxy-L-Tryptophan <0.0001 
 6-Methoxytryptamin Hydrochlorid <0.0001 
 DL-Tryptophan <0.0001 
 
 2. Sensitivity 
  
 The lowest detectable level that can be distinguished from the zero 
 standard is 0.1 pg per well resp. 1 ng/ml in the undiluted sample. 
  
 3. Recovery 
 
 1000 µl patient urine was mixed with 10 µl each of different 
 6-Sulfatoxymelatonin stock dilutions. 
 (data in ng/ml) 
 
 Basic Added Actual  Expected Recovery (%) 
 Conc. Value Value Value 
 41.6 6.2 50.8 47.8 106 
 41.6 18.5 67.9 60.1 113  
 41.6 55.5 103.3 97.1 106 
 41.6 166 212.9 207.6 103 
 67.7 6.2 81.4 73.9 110 
 67.7  18.5 87.3 86.2 101 
 67.7 55.5 122.3 123.2 99 
 67.7 166 242.3 233.7 104 
 87.2 6.2 99.4 93.4 106 
 87.2 18.5 107.9 105.7 102 
 87.2 55.5 145.6 142.7 102 
 87.2 166 258 253.2 102  
 
 4. Precision 
 Intra Assay Variation (in ng/ml) 
  
 Mean Standard deviation CV (%) n 
 7.27 0.39 5.4 10 
 45.2 1.7 3.8 10 
 
 Inter Assay Variation (in ng/ml) 
  
 Mean Standard deviation CV (%) n 
 6.21 0.54 8.7 10 
 41.28 3.51 8.5 10 
  
 5. Dilution 
 
 Unknown samples have been diluted with Assay Buffer and then measured. The 
 following table shows the calculated results (in ng/ml): 
 
 Dilution undiluted 1/2 1/4 1/8 1/16  
 Urine 1 67.7 67.1 66.7 63.2 69.1 
 Urine 2 84.9 84.0 82.6 85.8 77.9 
 Urine 3 219.5 226.9 238.0 226.8 213.4 
 
 

Expected Values

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 Expected values 
  
 The serum levels of melatonin in humans show a marked circadian rhythm and 
 are age-dependent. Daytime concentrations of serum melatonin are at their 
 lowest level around 2 - 4 p. m. and reach their peak around 2 - 4 a. m. 
 Circadian rhythms similar to those of serum melatonin were found for 
 melatonin sulfate in urine excretion. 
 
 

Alternative Applications

Back to Contents
 
 

(More) Clinical Background

Back to Contents
 Melatonin RIA/ELISA 
 
 I. Clinical indications of Melatonin published in: 
 
 1. Source: Anticancer Res. 1995 Nov.-Dec., 15 (6B): 2633-7 
  
 Autor:Tarquini R., Perfetto F., Zoccolante A., Salt 
 F., Piluso A, De-Leonardis V., 
 Lombardi V., Guidi G., Tarquini B.  
 
 Titel:Serum Melatonin in multiple myeloma: Natural 
 brake or epiphenomenon? 
  
 Melatonin, the main hormone produced by the pineal gland, seems 
 to exert antineoplastic activity bith in vitro and in vivo. 
 Moreever, several studies reported increased Melatonin blood 
 levels in cancer patients. Plasma Melatonin concentrations were 
 determined in 46 patients with multiple myeloma and in 31 age 
 matched healthy subjects (57.8 6.9 versus 55.2 8.9 years). 
 Venous blood was drawn between 7.30 and 9.30 a.m. and Melatonin 
 was assayed using a commercially available radioimmunoassay. 
 The data were analysed by sudent's t-test and results reported 
 as mean values standard deviation. The patients with multiple 
 myeloma showed significantly higher mean Melatonin serum levels 
 than healthy subjects (21.6 13.5 versus 12.1 4.8 pg/ml; p < 
 0.001). This behaviour could actually represent a phenomenon 
 secondary to an altered endocrine-metabolic balance caused by 
 an increased demand of the developing tumor. On the other hand, 
 the increased Melatonin secretion might be considering as a 
 compensatory mechanism due to this antimitotic action and 
 therefore as an effort to sevrete substances caple of 
 regulation neoplastic growth.  
 
 2. Source: J. Pineal Res. 1996 Jan; 20 (1): 21-3 
 
 Autor:Clemons A.A., Geffen J.F., Otto J.M., Pratt 
 K.L., Harker C.T.. 
 
 Titel:Dithiothreitol treatment permits measurement of 
 Melatonin in otherweise unusable salvia samples. 
 
 Melatonin research has primerily utilized blood as the source 
 of samles, but there is now increasing interest in measuring 
 levels of the hormone found in saliva. One impediment to this 
 approach is that several Melatonin assays involve a colum- 
 extraction step that can prove very time-consuming or even 
 impossible when salivary samples are excessively viscous. We 
 have treated 67 samples with dithiothreitol to enhance their 
 passage through the column. Following this treatment, all 
 samples passed freely through the columns. The minimum and 
 maximum values measured were 0.7 - 50.0 pg/ml for the untreated 
 controls and 0.1 - 51.9 pg/ml for the treated samples. The 
 means ( SEM) for these groups were 9.5 1.6 and 9.9. 1.7, 
 respectively, and were not significantly different from one 
 another as assessed by student's t-test (P = 0.08). In summary  
 we have found that this technique permits us to obtain values 
 on samples which would otherwise be unusable and that such 
 treatment does not alter the Melatonin values yielded by RIA 
 analysis.  
 
 
 3. Source: In Vitro. 1995 Jul.-Aug.; 9 (4): 375-8 
 
 The longitudinal follow-up of a patient with an advenced 
 adenocarcinoma of the ovary sheds new light on the involvement 
 of the pineal in carcinogenesis. The changes in the circadian 
 MESOR of 6-sulfoxy-Melatonin following a course of chemotherapy 
 may differ in relation to the success or failure of treatment, 
  yet the MESOR does not correlate with tumor burden assessed by 
 circulating CA 125. By contrast, the ratio of circaseptan-to- 
 circadian amplitudes involving two chronome components 
 correlates with the cancer marker. To that extent, the study 
 reveals a critical about 7-day (circaseptan) aspect of the 
 pineal involvement in cancer progression. This information 
 could be exploited in designing schedules of Melatonin 
 administration to cancer patients. 
 
  
 4. Source: Am. J. Perinatol. 1995 Jul; 12 (4): 299-302 
 
 Autor:Katz V.L., Ekstrom R.D., Mason G.A., Golden R.N. 
 
 Titel:6-sulfatoxymelatonin levels in pregnant women 
 during workplace and nonworkplace stresses: A potential  
 biologic marker of sympathetic activity. 
 
 Melatonin production is regulated by both catecholamines and 
 sympathetic activity. Urine levels of the major metabolite of 
 Melatonin, 6-Sulfatoxymelatonin, correlate well with serum 
 Melatonin levels and have been used to evulate sympathetic 
 output. We tested the hypothesis that urinary levels of 6- 
 Sulfatoxymelatonin would reflect the change in adrenergic 
 activity on working days compared with nonworking days during 
 pregnancy. Twenty-three healthy pregnant women, employed in a 
 variety of occupations, including physicians, nurses, 
 secretaries, salespeople and laboratory workers were recruited 
 from the clinics of the University of North Carolina School of 
 Medicine. We measured 6-Sulfatoxymelatonin levels, in first 
 morning voids and for the subsequent 10 hours at 24, 28, 32 and 
 36 weeks gestation. Urine was collected in sets during working 
 days and during nonworking days. 6-Sulfatoxymelatonin was 
 measured by radioimmunoassay. In 11 women we also measured 
 urine catecholamines by high-perfomance liquid chromatography. 
 Levels of 6-Sulfatoxymelatonin output did not change across 
 gestation, although they tended to drift down as pregnancy 
 progressed. Median levels at first morning void were 6.3 
 micrograms on workdays and 4.6 micrograms on nonworkdays. 
 Although all values were skewed toward work being greater than 
 nonwork, there were large interindividual variations. We 
 therefore compared subjects against themselves and compared 
 work levels for each subject to the corresponding gestitional 
 age-matched nonwork value. Among the 23 women, median 6- 
 Sulfatoxymelatonin levels were 81% greater during work than  
 nonwork (p < 0.0002) when first morning collections were 
 compared. Daytime urinary excretion of 6-Sulfatoxymelatonin on 
 workdays was 38% (p < 0.0005) greater than during nonworkdays. 
 (Abstract Truncated at 250 words) 
 
 
 5. Source: J. Clin. Endocrinol. Metab. 1996 May; 81 (5): 
 1877-81 
 
 Autor:Ozata M., Bulur M., Bingol N., Beyhan Z.,  
 Corakci A., Bolu E., Gundogan M.A. 
 
 Titel:Daytime plasma Melatonin levels in male 
  hypogonadism. 
 
 It has previously been shown that increased nocturnal Melatonin 
 (MT) secretion exists in male patients with hypogonadotropic 
 hypogonadism. However, little is known about the effects of 
 gonadotropin and testosterone (T) treatment on early morning 
 plasma MT levels in male hypogonadism. Also, the impact of 
 gonadal steroids on plasma MT levels is an open question. We, 
 therefore, determined early morning plasma MT levels at the 
 same hour before and 3 months after treatment in 21 patients 
 with idiopathic hypogonadotropic hypogonadism (IHH), 10 
 patients with primary hypogonadism and 11 male controls. Plasma 
 FSH, LH, PRL, T and estradiol levels were also determined 
 before and 3 months after treatment. Patients with IHH were 
 treated with hCG/human menopausal gonadotropin, whereas 
 patients with primary hypogonadotropism received T treatment. 
 Short term treatments did not achieve normal T levels, although 
 significant increases in T were observed in both groups. Plasma 
 MT levels were measured by a RIA with a sensitivity of 10.7 
 pmol/L. Mean plasma MT levels before treatment were 
 significiantly higher in IHH (41.8 24.4 pmol/L compared with 
 those in the controls (21.7 10.8 pmol/L; P < 0.05). However, a 
 slight, but not significant, increase in MT (34.2 21.1 pmol/L)  
 was found in primary hypogonadism. Mean MT levels did not 
 change significantly 3 months after the initiation of 
 gonadotropin (41.7 22.8 pmol/L) or T (28.4 12.6 pmol/L) 
 treatment in either IHH or primary hypogonadism, although a 
 tendency for MT to decrease was observed in both groups. No 
 correlation was found between MT and circulation FSH, LH, PRL 
 and gonadal steroids either before or after therapy. We 
 conclude that male patients with IHH have increased early 
 morning MT levels, although the pathophysiological mechanism is 
 not clear. Furthermore, our study demonstrated that mean plasma 
 MT levels are not influenced by short term gonadotropin or T 
 treatment in male hypogonadism, although a longer time effect 
 of gonadotropins ot T treatment my not be excluded. The lack of 
 cerrelation between plasma MT and circulation gonadal steroids 
 before and after treatment suggests that there is no classic 
 feedback regulation between the pineal gland and the testes. 
  
 II. Role of Melatonin in health and diseases 
 
  
 Susan M. Webb and Manuel Puig-Domingo 
 
 Department of Endocrinology, Hospital de la Santa Creui sant  
 Pau, Autonomous University of Barcelona, Spain. 
 (Received 11 August 1994; returned for revision 21 September 
 1994; finally revised 31 October 1994; accepted 13 December 
 1994).  
 
 Pineal function and its main hormonal product Melatonin has 
 often been ignored by many clinicans. In this review, the 
 evidence pointing towards an undeniable role of Melatonin in 
 certain clinical instances will be presented and discussed. 
  
 In the last 3 decates tremendous advances in the understanding 
 of the biochemistry and physiology of the pineal gland have 
 occured. It is now evident that the pineal interacts with many 
 endocrine as well non-endocrine tissues to influence their 
 metabolic activity. The most extensively studied pineal effect 
 on the neuroendocrine-reproductive axis is by no means the only 
 or necessarily the most important role of this gland, which 
 through its hormone, Melataonin, is able to modulate many 
 organs and functions. 
  
 Regulation of pineal function 
 
 The synthesis and secretion of the best studied pineal hormone,  
 Melatonin (MEL; N-acetyl-5-methoxytryptamine), is principally 
 controlled by the prevailing lightdark environment, acting via 
 the hypothalamic suprachiasmatic nuclei (SCN). It is 
 independent of sleep. While in mammals it is the light and 
 darkness perceived by the eyes which synchronised the circadian 
 activity of the gland, in birds light directly penetrating the 
 skull influences pineal function (Reiter, 1980). Pineal MEL is 
 inhibited by light and stimulated during darkness via a multi- 
 synaptic neutral pathway which connects the retina, through the 
 SCN of the hypothalamus, preganglionic neuroses in the upper 
 thoracic spinal cord and post-ganglionic sympathetic fibres 
 from the superior cervical ganglia, to the pineal gland. The 
 importance of an intact sympathetic innovation in determining 
 the nyctohemeral MEL rhythm is illustrated by our recent 
 experience in diabetic with automatic neuropathy. In these 
 patients, 24-hour MEL values are lower, and exhibit a 
 significantly lower nocturnal peak, than in diabetics with an 
 intact automatic system, confirming previous studies (O’Brien 
 at al., 1986) of patients with preganglionic (Shy-Drager 
 syndrome) or post-ganglionic sympathetic nervous system 
 involvement such as idiopathic orthostatic hypotension (Testsuo 
 et al., 1981; Vaughan 1984). Similar results are found in 
 patients with sympathetic dysfunction and quadriplegia due to  
 cervical spinal cord transection (Kneisley et al.; 1978: Li et 
 al., 1989). 
 
 The endocrine cells of the pineal gland (the pinealocytes) 
 receive sympathetic nerve endings which release the 
 neurotransmitter noradrenaline during darkness; by acting on - 
 adrenergic receptors, this neurotransmitter determines the 
 uptake of tryptophan and the synthesis of MEL from the 
 precursor serotonin, after different enzymes have been 
 activated -Adrenergic receptors have been shown to potentiate 
 the -adrenergic receptors (Klein et al.; 1983), which are 
 linked to adenylate cyclase via a stimulatory guanine- 
 nucleotide-binding protein (Gs protein). After noradrenaline  
 stimulation, the synthesis of the intracelluar second-messenger 
 cAMP is amplified, leading ultimately to MEL synthesis (Reiter, 
 1991). Although the mechanism involved in this signal 
 amplification have not been totally elucidated, the calcium 
 activated, phospholipid dependent enzyme protein kinase C is 
 involved (Chik & Ho, 1989). At the molecular level, (Stehle et 
 al.; 1993) have recently identified an inducible cAMP early 
 repressor (ICER), an isoform of the cAMP responsive element 
 modulator (CREM), which in rat pineal gland is under neural, 
 adrenergic control. This ICER shows a circadian fluctuation 
 with maximal mRNA expression at night, similary to MEL, but 
 represses cAMP induced transcription in pineal cells. It 
 constitutes the first example where concomitance of Melatonin 
 synthesis and inducibility of a specific gene can be 
 dissociated. These findings could lead to speculation that the 
 repressing effect of ICER on cAMP dependent synthesis, 
 including that of MEL, could explain the fall in pineal MEL 
 towards the end of the dark period. 
 
 Circulating MEL is almost exclusively of pineal origin, since 
 plasma concentrations, particularly at night, are depressed and 
 in some cases reportedly undetectable after pinealectomy. 
 Furthermore, practically no storage takes place, so that 
 circulation Melatonin levels clearly parallel pineal content of 
 the indole. Apart from its major nocturnal peak. MEL exhibits a 
 pulsatile episodie secretion superimposed on the nyctohemeral 
 rhythm, which appears to be independent of the endogenous LH 
 pulses (de Leiva et al.; 1990). 
  
 Besides the sympathetic innervation, the pineal also receives 
 nerve fibres from the central nervous system; in this case the 
 neurotransmitters are either peptides (VIP, AVP, Somatostaion, 
 Neuropeptide Y, TRH etc.) or acetyl choline and the cell bodies 
 of these neurons are located in different nuclei of the brain 
 (Korf & Moller, 1983). The function of this central innervation 
 is only beginning to be realized and is probably related to 
 additional brain modulation of pineal function (Moller et al., 
 1992). 
  
 MEL is metabolized in the liver to 6-hydroxy-MEL, which is 
 conjugated to either glucuronide (20-30%) or sulphate (60-70%). 
 The main metabolite excreted is urinary 6-sulphatoxy-MEL. An 
 excellent correlation has been observed between pineal and 
 circulating MEL, urinary 6-sulphatoxy-MEL (Arendt et al., 1985; 
 Matthews et al.; 1991) and even salivary MEL (Nowak et al., 
 1987; Deacon & Arendt, 1994). Given the circadian nature of MEL 
 secretion, which reders isolated measurements of circulating 
 MEL uninterpretable, the possibility of using a non-invasive 
 method such as urinary excretion of 6-sulphatoxy-MEL to study 
 pineal function throughout 24 hours is very attractive, 
 especially when investigating children. 
  
 Melatonin and circadian rhythmicity 
 
 The two major physiological roles for MEL identified up to now 
 are its influence on circadian rhythmicity and the induction of 
 seasonal responses to changes in day length (Reiter, 1980; 
 Redman et al., 1983; Tamarkin et al.; 1985; Bartness et al.,  
 1993). The former is most pronounced in certain birds and 
 reptil and more subtle in mammals; however, convincing evidence 
 that MEL affects circadian rhythmicity is now available in 
 rats, fetal hamsters and humans (Vaughan, 1984; Arendt et al., 
 1985, 1988; Vanecek et al., 1987; Davis & Mannion, 1988; 
 Cassone, 1990; Dahlitz et al., 1991; Tzischindky et al., 1993). 
 For example, timed exogenous MEL administration to subjects who 
 are blind and therefore unable to synchronize to the light-dark 
 cycle, or to individuals who suffer from delayed sleep phase 
 insomnia, support an influence of MEL on circadian rhythmicity 
 (Arendt et al., 1988; Dahlitz et al., 1991; Tzischinsky et al., 
 1993). In patients with delayed sleep phase syndrome, 5 mg of 
 MEL administered orally 2 hours before the desired sleep time, 
 for several weeks, successfully advanced sleep; consequently, 
 patients woke up earlier and were able to resume a normal 
 working life (Tzischinsky et al., 1993). Given the short half- 
 life of MEL (20-30 minutes, depending on whether the t ½ is 
 calculated on endogenous (de Leiva et al., 1990) or exogenous 
 MEL (Waldhauser et al., 1984a), it is improbable that this 
 indole acts solely through a direct hypnotic effect (Waldhauser 
 et al., 1987b), but favours the hypothesis that once 
 administered, MEL initiates a cascade of events, leading within 
 2-3 hours to the opening of the “sleep-gate”. Furthermore, 
 during and immediately after a change to night-shift work or 
 travel across time zones, secretion of MEL depends more on the 
 clock than on the light-dark cycle; jet lag caused by this 
 transmeridian travel is attenuated by timed MEL administration 
 (Arendt, 1988), a result of resetting of the biological clock 
 to match the new environmental time. Studies are being  
 conducted to investigate the potential use of exogenous MEL, or 
 alternatively inducing a timed endogenous MEL peak by adequate 
 bright light exposure, to improve performance and synchronize 
 the onset of sleep in shift workers (Lewy & Sach, 1993). A 
 correlation between self-rated alterness and endogenous MEL has 
 been observed in young volunteers exposed to various periods of 
 bright light on consecutive days. These periods were designed 
 to produce a shift in their endogenous hormonal circadian 
 rhythms by delaying for 2 hours the 6 hours of bright light 
 exposure during the evening/night period. The authors  
 demonstrated that this shift in MEL rhythm paralleled the shift 
 observed in a major behavioural rhythm, namely alertness rated 
 on a visual analouge scale (Deacon & Arendt, 1994). 
  
 The recent demonstration of MEL receptors in human SCN of the 
 hypothalamus, suggests a direct action of MEL on this nucleus 
 to influence circadian rhythms (Weaver et al.. 1993). the role 
 of MEL in the seasonal responses to changes in day lenght is 
 most abvious in seasonally breeding mammals (Reiter, 1980). As 
 days get shorter in the autumn, the nocturnal MEL peak is 
 prolonged; this signal informs the animal of the time of year 
 and is sensed by neuroendocrine axes, which control seasonal 
 changes in coat-hair colour and quantity, growth and 
 metabolism, in addition to reproduction. Prolonged nocturnal 
 MEL secretion, acting via inhibition of GnRH secretion, is 
 responsible for the winter induced regression in gonadotrophin 
 secretion in long-day breeding animals such as various rodents. 
 However, in short-day breeders, such as sheep, prolonged MEL 
 exposure is stimulatory to reproduction, while short exposure  
 to MEL, such as that which occurs in spring, inhibits the 
 gonadal axis (Karsch et aql., 1986). Artificially exposing both 
 long and short-day breeding animals to timed Melatonin 
 infusions of different durations has permitted investigation of 
 the importance of intensity, duration and frequency of the MEL 
 signal to the induction of photoperiodie changes and seasonal 
 responses (Bartness et al., 1993). The integrity of the 
 ventromedial hypothalamus is vital for the inhibitory effect of 
 MEL on gonadotrophin secretion (Hastings et al., 1994) 
 Additionally, sensitivity of different tissues to MEL may vary 
 throughout the year and only when the MEL rhythm and the 
 sensitivity are synchronous is there a maximal response to MEL. 
 The magnitude of the nocturnal MEL peak, as well as the 
 duration of the MEL signal, may also indicate the season of 
 year to the animal, as well as the frequency of this MEL 
 signal, which has recently been found to be crucial in order to 
 induce gonadal regression. Very frequent exposure to MEL 
 (approximately every 6 hours) or too infrequent exposure to the 
 indoleamine (less than every 28 hours) cannot induce gonadal 
 regression, since they are outside the sensitivity window to 
 MEL. Thus, the accurate generation of a nocturnal MEL signal 
 with a duration appropriate to the length of night is mandatory 
 (Hastings et al., 1994).  
 
 Even though MEL influences seasonal reproduction by altering 
 hypothalamie neurosecretion, MEL receptors have not found 
 consistently in the hypothalamus of these photoperiodic 
 breeders, but have been demonstrated in the portion of the 
 pituitary gland that covers the surface of the stalk known as 
 the pars tuberalis, in all seasonal breeding species studied 
 (Weaver et al., 1993; Gauer et al., 1993). The relative absence 
 of MEL receptors in human pars tuberalis suggest that the 
 neuroendocrine responses to MEL in humans (who do not exhibit 
 seasonal reproductive activity such as that seen in hamsters or 
 sheep, but in whom residual seasonality may be evidenced in 
 extreme conditions of climate (Rojansky et al., 1992) occur by 
 fundamentally different mechanisms from those which underly the 
 photoperiodie regulation of reproduction in seasonally breeding 
 species. Nevertheless, in humans the duration of MEL secretion  
 also varies with the duration of darkness, so that 24-hour MEL 
 secretion has been reported to be greater during the winter 
 than during the summer, in women living in Finland in regions 
 with considerable seasonal variation in light exposure, despite 
 no differnece in the lenght of their menstrual cycles (Kauppila 
 et al., 1987). These authors concluded that, although social 
 factors determine family planning, a biological background 
 probably does exist for seasonal variations in birth and 
 conception rates. This has not been the experience of other 
 authors who observed no seasonal change in the excretion of 6- 
 sulphatoxymelatonin in volunteers living in Antarctiva, 
 although there was some evidence of a phase shift (Griffiths et 
 al., 1986). A phase delay of approximately one and half hours 
 in winter as compared to summer circadian rhythmus of 
 circulating MEL, with no differences in the duration of 
 elevated night-time MEL, has also been observed in city 
 dwellers in Central Europe (Illnerova et al., 1985). A major 
 breakthrough is the discovery of a high affinity receptor for 
 MEL, cloned and expressed from amphibian dermal melanophores  
 (Ebisawa et al., 1994). It is to be expected that now the 
 structure of this MEL receptor gene has been defined, the 
 answers to many of these questions may be nearer. 
  
 Melatonin throughout life 
 
 Nocturnal secretion of MEL and of its main urinary metabolite 6- 
 sulphatoxy-MEL both of which are highly correlated is highest 
 in young children and falls with age, leading to speculation 
 that decreases in such secretion may be related to the pubertal 
 maturation of the neuroendocrine reproductive axis (Attanasion 
 et al., 1985; Garcia-Patterson et al., 1994a). Despite the lack 
 of correlation of threshold or sudden change in MEL with any 
 stage of pubertal development in normal children, a negative 
 correlation has been reported between nocturnal plasma 
 concentrations of LH measured at various stages of puberty and 
 serum MEL in a group of 89 children, adolescents and young 
 adults (Waldhauser et al., 1984b). However, as will be 
 discussed later, in other circumstances no correlation has been 
 observed between LH and MEL (Penny, 1985; de Leiva et al., 
 1990). 
 
 Melatonin continues to fall from adulthood to old age at which  
 time virtually no MEL rhythm can be observed (Iguchi et al., 
 1982; Attanasio et al., 1985; Sach et al., 1986; Fernandez et 
 al., 1990). The mechanism of this progressive fall in pineal 
 MEL with aging is unknown, but may be related to very long- 
 acting endogenous rhythms which extend over years or decades, 
 and which determine reduced activity of the genes which encode 
 for the enzymes involved in the synthesis of MEL. In aged 
 rodents, pinealocyte -adrenergic receptors decrease in number 
 and in their capacity to respond to noradrenaline (Greenberg & 
 Weiss, 1978). A similar mechanism might explain the low  
 concentrations of MEL in elderly humans. Alternatively, aging 
 has been considered as a state of supersensitivity to light, in 
 which the centres in the supraichiasmatic nucleus controlling 
 circadian rhythmicity remain predominantly inhibited (Touitou 
 et al., 1985). 
 
 Relation of Melatonin to the neroendocrine-reproductive axis 
 
 Data pointing towards a relation between MEL and the 
 neuroendocrine-gonadal axis are becoming available in children 
 and adults of both sexes. Such evidence was already present 
 experimentally, supporting a relation between pineal MEL and 
 pubertal development. Both pinealectomy and its sympathetic  
 denervation greatly decrease circulation MEL concentrations and 
 hasten pubertal development. Conversely, short-day exposure, 
 which is known to exaggreate the antigonadotrophic potential of 
 the pineal gland, or exogenous MEL administration, delays 
 sexual maturation in experimantal animals (Rivest et al., 1985; 
 Arendt, 1988; Reiter, 1986; Utiger, 1992). There are two lines 
 of evidence which suggest similar relations in the human. 
 Pineal tumours have been associated with pubertal maturational 
 abnormalities and these were thought to result from pressure of 
 the tumour on the hypothalamic centres which determine 
 gonadotrophin secretion. However, both advancement and delay of 
 puberty have been experienced, depending on the patient’s age 
 and the parenchymal or non-parenchymal nature of the tumour,  
 irrespective of this size. Pinealocyte tumours secrete 
 excessive amounts or alter the rhythm of MEL causing delayed 
 puberty in adolescents, while non-parenchymal tumours such as 
 teratomas destroy the gland and reduce its potential 
 antigonadotrophic function, which could explain precocious 
 puberty in prepubertal children. As mentioned, MEL falls 
 throughout childhood, reaching adult leveld at puberty. A rapid 
 decrease in MEL has been observed during successful treatment 
 of delayed puberty (Arendt et al., 1989). Furthermore, in 
 patients with precocious puberty MEL concentrations tend to 
 fall (Cohen et al., 1982; Low et al., 1989; Waldhauser et al., 
 1991), supporting a probable inverse relation between pineal 
 and gonadal functions. After successful treatment with long- 
 acting GnRH analogues, which inhibited gonadotrophics and sex 
 steroids and caused regression of pubertal development, no 
 recovery of night-time prepubertal MEL levels was observed 
 (Waldhauser et al., 1991). All these findings could indicate 
 that the reduction in circulating MEL amplitude plays an 
 initiating role for pubertal development or, alternatively, may 
 reflect only the degree of central maturation of the 
 hypothalamic-pituitarygonadal axis. Additionally, no role has 
 been suggested for adrenarche in pineal-pubertal relations in 
 humans (Cavallo, 1992).  
 
 Conflicting results on a possible relation between MEL and 
 onset of puberty are not surprising. The research methods 
 applied have not always been adequate; factors such as previous 
 light exposure history, isolated and therefore incomplete 
 sampling throughout the 24 hour light dark cycle, inadequately 
 small study groups, lack of precise markers of pubertal stage 
 (which is usually defined by broad clinical features) and the 
 cross-sectional nature of most of the studies, complicate the 
 identification of a possible relation (Cavallo, 1993). 
 
 Given the effect of MEL on hypothalamic GnRH in experimental 
 studies, interest in investigating a possible correlation  
 between serum LH and MEL in humans has emerged (Waldhauser et 
 al., 1984b). LH is secreated in rather regular pulöses and MEL 
 also exhibits pulsatile variations. 
  
 However, when both parameters were simultancously analysed in 
 young healthy individuals, no correlation could be observed 
 between the two hormones because the MEL pulses were irregular 
 and acyclic (Penny, 1985; de Leiva et al., 1990). 
 
 Further evidence of pineal hypothalamic interplay through 
 circulating MEL is strongly supported by our observation of a 
 young man with massively elevated circulating levels of MEL 
 related to pineal hyperplasia, in whom hypogonadotrophic 
 hypogonadism resulting from GnRH deficiency was reserved when 
 MEL levels fell (Puig-Domingo et al., 1992). However, he became 
 fertile when his circulating MEL levels were still more than 
 twice normal, suggesting that fall in MEL rather than the 
 absolute concentration triggered some mechanism which permitted 
 hypothalamic activitation of GnRH and consequently sexual  
 maturation. This observation is in keeping with other situation 
 in which the increase or decrease in amplitude of the MEL 
 circulation rhythm, or the lengthening or shortening of the 
 duration of elevated circulating MEL concentrations, is more 
 important for adaptive changes to the environment than the 
 actual concentration of the indole. This patient showed further 
 interesting features, namely, his pineal was heavily calcified 
 supporting the concept that premature calcium deposits may be 
 indicative of a hyperfunctional gland (Trapp & Huxley, 1972; 
 Lukaszyk & Reiter, 1975). Since the deposition and maintenance 
 of calcareous deposits within the gland are known to require 
 sympathetic innervation, it is feasible that pineal 
 calcification was a result of abnormal responses to 
 noradrenergic stimulation. This is further supported by 
 unexpected changes in MEL on exposure to light and dark, 
 suggesting inappropriate neural responses to information about 
 light and darkness transmitted from the eyes to the pineal via 
 the hypothalamus. Despite these abnormalities, this patient’s 
 circadian MEL rhythm was preserved, supporting a 
 hyperfunctional rather than an autonomous, pineal tumour. This 
 patient’s investigations also support a possible relation 
 between MEL and circulation gonadal steroids, since treatment 
 with gonadotrophins and later testosterone for several years 
 have contributed to the later decline in MEL secretion. In 
 keeping with the concept of an interplay between MEL and 
 testosterone, Anderson et al., (1993) observed an increase in 
 sensitivity of the hypothalamic-pituitary axis to testosterone 
 feedback, when treating young healthy men with 100 mg of oral 
 MEL for 2 weeks. 
  
 Supranormal nocturnal plasma MEL concentrations have also been 
 found in women with stress induced (Berg et al., 1988), 
 exercise induced (Laughlin et al., 1991) or functional 
 hypothalamic hypogonadism (Brzezinski et al., 1988) or anorexia 
 nervosa (Brambilla et al., 1988; Tortosa et al., 1989; Ferrari 
 et al., 1989; Arendt et al., 1992), all with low donadotrophins 
 and in men suffering from primary hypogonadism with elevated 
 gonadotrophins (O. Rajmil et al, unpublished), or infertility 
 with oligozoospermia or azoospermia (Karasek et al, 1990).  
 Substitution treatment of these males with testosterone induced 
 a fall in circulating MEL, which however, did not reach control 
 values. Similar findings have recently been described in women 
 with functional amenorrhoea, where a relation between 
 oestrogens and MEL has been found; MEL rose when oestrogens 
 were low and after exposure to oestrogens MEL fell but did not 
 reach normal values (Okatani & Sagara, 1994). After oestrogen 
 deprivation with a GnRH analouge, these authors also observed 
 that MEL fell to normal within 3-4 months of stopping 
 treatment.  
 
 Some recent controversial results indicate that further studies 
 are still required before the role of MEL in the gonadal axis 
 is completely elucidated; 7 women with anorexia nervosa and 8 
 with bulimia nervosa and normal weight (two of whom were 
 cycling), in whom endogenous depression had been ruled out, 
 were found to exhibit a MEL rhythm (with blood sampling at 20 
 minute intervals throughout 24 hours) which did not differ from 
 a group of 21 normal cycling controls (Mortola et al., 1993). 
 Oestrogens were not reported in this study and, naturally, body 
 mass index was much lower in anorectics than in bulimics and 
 controls. 
  
 Taken together, most of these examples would seem to suggest 
 the existence of a relation between MEL and the neuroendocrine 
 gonadal axis. The nature and direction of this relation is 
 still a matter of debate but two hypotheses, which are not 
 necessarily mutually exclusive, can be considered. In some 
 instances it would seem that increased circulating MEL is 
 capable of inhibiting GnRH as exemplified by our young man with 
 a hyperplastic pineal, or children with parenchymal pineal 
 tumours. In other situations it would seem that sex steroids 
 modulate MEL levels, as seen after treating our patient with 
 pineal hyperplasia or men with primary hypogonadism with 
 testosterone, in children with precocious puberty, or in women 
 with secondary functional amenorrhea (Okatani & Sagara, 1994). 
 However, this is still an open question, with many answers 
 awaited. This latter hypothesis would not explain the situation 
 observed in old age, where both gonadal function and MEL are 
 low; however, in these elderly subjects other dominant factors, 
 probably specific to old age, may affect MEL, which makes 
 interpretation of a possible relation between the parameters 
 difficult.  
 
 Melatonin in hypothalamic-pituitary disorders 
  
 Several authors have attempted to analyse MEL secretion in 
 patients with pituitary tumours (Vaughan et al., 1979; Werner 
 et al., 1980; Young, 1981; Dempsey & Chandler, 1984; Soszynski  
 et al., 1989; Piovesan et al., 1990; Lissoni et al., 1992). 
 Most report the presence of a normal pattern of MEL secretion, 
 that is, a nocturnal rise and low daytime values, independent 
 of the secretory or non-secretory nature of the adenoma (ACTH, 
 PRL, GH or non-functional) and the presence or absence of 
 treated or untreated hypopituitarism. However, an abolished MEL 
 rhythm has also been described in patients with Cushing’s 
 syndrome (of pituitary or adrenal origin) (Soszynski et al., 
 1989) and with pituitary PRL and ACTH-secreting adenomas 
 (Werner et al., 1980; Young, 1981). Alternatively, in  
 acromegalic patients abnormally high diurnal variations of MEL 
 (Piovesan et al., 1990) associated with low nocturnal levels 
 have been reported, a pattern also described in prolactinomas 
 (Lissoni et al., 1992). Vaughan (1984) suggested that the lack 
 of a MEL circadian rhythm could be a result of hypothalamic 
 involvement by large invasive lesions of the pituitary- 
 hypothalamic region, which would interrupt the neural 
 connections between the hypothalamus and the spinal cord. This 
 view would be supported by the findings in patients with large 
 invasive prolyatinomas associated with panhypituitarism, but 
 not in those with primary empty sellas, who exhibited a normal 
 MEL circadian pattern (Werner et al., 1980). 
  
 A relation between the hypothalamic pituitary adrenal axis and 
 MEL seems highly unlikely, since patients without ACTH can 
 exhibit a normal MEL rhythm and others with no MEL rhythm can 
 show normal ACTH cortisol circadian changes (Vaughan, 1984). 
 Furthermore, children with congential adrenal hyperplasia 
 exhibited comparable MEL circadian rhythms, both on and off 
 dexamethasone and fludrocortisone, which were no different from 
 those observed in a control group of age-matched normal 
 children (Waldhauser et al., 1986). Even though 1 mg of 
 dexamethasone at 2300 h inhibited early morning cortisol and 
 attenuated the nocturnal secretion of MEL in 9 out of 11 
 subjects, no causal relation between the pineal gland and the 
 hypothalamic-pituitary adrenal axis be demonstrated. In a  
 further study of children with weight problems who were given a 
 single evening dose of 2 mg dexamethasone, a stimulatory effect 
 on nocturnal MEL was observed only when cortisol was suppressed 
 (Lang et al., 1986). Variability in dexamethasone availability, 
 which is known to influence post-dexamethasone cortisol levels, 
 might explain the presence or absence of MEL attenuation after 
 dexamethasone, but this has not been conclusively demonstrated 
 (Demisch et al., 1988). Furthermore, MEL does not seem to act 
 as a tonic inhibitor of the hypothalamic-pituitary-adrenal axis 
 on an acute basis (Demitrack et al., 1990).  
 
 An attempt to elucidate possible MEL abnormalities in patients 
 with intrasellar pituitary tumours was undertaken by Dempsey 
 and Chandler 8!) in a heterogeneous group of 5 patients (1 
 prolactinoma, 2 Cushing’s diseases, 1 non-functioning adenoma 
 and 1 small intrasellar crainiopharyngioma); unfortunately, the 
 “control” group included patients with spinal problems and 
 supratentorial lesions which included large crainiopharyngiomas 
 with probable hypothalamic involvement, since two of them were 
 hyperprolactinaemic. With 4 samples from each patient (during 
 the day and night, pre and postoperatively) they reported lower 
 night-time and higher daytime MEL preoperatively which tended 
 to normalize post-operatively. Their so-called controls showed  
 opposite effects and seemed to lose their normal MEL pattern 
 post-operatively. Since these patients were only 4-6 days after 
 leaving intensive care units with 24-hour continuous light 
 exposure, which is known to disrupt the MEL rhythm, the results 
 are inconclusive. 
 
 In summary, no definite pattern of MEL secretion has been 
 identified in patients harbouring a pituitary tumour. However, 
 in large invasive lesions which involve the hypothalamus and 
 its neural connections, a loss of the nocturnal rise of MEL, is 
 frequently encountered. 
 
 Effects of exogenous Melatonin on the endocrine system 
  
 When trying to investigate the effect of exogenous MEL on 
 different hormones, it should be recalled that this indole has 
 a short half-life (less than an hour) after oral 
 administration. Circulating blood levels will therefore exhibit 
 a profile which is quire different from the endogenous one, 
 that is, a rapid, marked, sharp initial peak will be followed 
 by an equally rapid fall with practically undetecable 
 concentrations within 4 hours. Consequently, the effect of 
 oral, exogenous MEL on different hormones may be 
 pharmacological rather than physiological. A submucosal patch 
 which is easily applied and removed has recently been developed 
 (Bene et al., 1993). In comparison to oral administration, 
 submucosal MEL can simulate the endogenous rhythm quite well, 
 since it produces a gradual rise in MEL following application, 
 followed by a slightly ascending plateau, and a rapid decrease  
 as soon as the patch is removed. It can be applied at bedtime 
 and removed upon waking, thus constituting a promising MEL 
 delivery system for future research and therapy. 
  
 Early studies using acute doses of oral MEL observed a 
 stimulatory effect on PRL, a slight, but insignificant rise in 
 GH, but none on LH, FSH, testosterone or TSH (Wright et al., 
 1986; Waldhauser et al., 1987a). These findings have recently 
 been confirmed (Terzolo et al., 1993). In relation to this 
 finding, it is of interest that photoperiod has been found to 
 influence PRL secretion through its effects on the secretion of 
 Melatonin, being higher in spring and summer and exhibiting a 
 nadir in autumn and winter (Curlewis, 1992). These seasonal 
 variations in PRL have been found in both seasonal and non- 
 seasonal breeders, implying changes in neuroendocrine 
 sensitivity to changing photoperiod. The discovery of Melatonin 
 receptors in the pars tuberalis of the hypothalamic-pituitary 
 unit would suggest a common site of action for Melatonin on 
 both gonadotrophins and PRL. Furthermore, variations in 
 response to the light-dark cycle could be the result of 
 differences in processing and/or interpretation of the 
 Melatonin signal. 
  
 Robinson (1994) had also reported little effect on reproductive 
 or other endocrine functions in adults treated for several 
 weeks with high doses of oral MEL. This has been suggested to 
 result from down-regulazion of MEL receptors at the 
 hypothalamic-pituitary level. Even though these findings would 
 provide little support for the notion that decreases in MEL 
 secretion initiate puberty or that MEL inhibits reproductive 
 function, it should be emphasized again that endogenous 
 increases in MEL should not be expected always to exert 
 identical effects to those produced by pharmacologically 
 administered exogenous MEL. Additionally, senitivity to MEL 
 probably depends on age and even though the hypothesis has not 
 been demonstrated, it could be that younger people are more 
 sensitive than older to MEL. 
 
 The dose of oral MEL given in another variable to consider.  
 Since it is well tolerated, non-toxic and highly diffusable due 
 to ist lipophilic nature, extremely high doses of up to 100 mg 
 (Cagnacci et al., 1991) or even 300 mg per day (Voordouw et 
 al., 1992) have been used. At these doses, MEL remains present 
 in the circulation over 24 hours. However, behavioural effects 
 have recently been observed after doses as low as 0.1 mg, after 
 which circulating concentrations of MEL are in the 
 physiological range, below 100 ng/l (430 pmol/l) (I. Zhdanova, 
 unpublished observation). These authors administered MEL as 
 oral gelatin capsules, which may explain a more progressive and 
 slower release than after other oral preparations. With these 
 low doses they observed a significant fall in sleep latency, 
 which led them to suggest that sleep may be influenced by the 
 physiological secretion of MEL: 
 
 MEL has been reported to enhance basal GH and decrease the GH 
 response to insulin-induced hypoglycaemia in adults (Smythe & 
 Lazarus, 1974; Valcavi et al., 1987; Esposti et al., 1988). An 
 acute oral administration of MEL was followed by a fall in GH  
 in prepubertal, but not in the majority of pubertal, children 
 (Lissoni et al., 1986), suggesting that age and sexual 
 development modulate the sensitivity of the hypothalamic- 
 pituitary axis to MEL (Garcia-Patterson et al., 1994b). The 
 stimulatory effect of MEL on GH is believed to result from an 
 inhibition of hypothalamic somatostatinergic tone; this 
 hypothesis is supported by further studies by Valcavi et al. 
 (1993), who showed that a second release of GH by GHRH, 120 
 minutes after the first i.v. administration, could be elicited 
 only if MEL was administered at 60 minutes, but not after  
 placebo. 
 
 Melatonin as an oncostatic neurohormone 
  
 Melatonin has been observed to exert potent inhibition on 
 cancer growth. This has been demonstrated in certain human 
 breast cancer cell lines such as MCF-7, with additional in-vivo 
 effects on breast oncogenesis in various rat models (Blask et 
 al., 1991). Dosage and timing of MEL injections are important; 
 the most effective concentration of MEL as an antiproliferative 
 drug on growth of MCF-7 cells has been shown to be 10 9 M, 
 which is the physiological range. As far as the time of day is 
 concerned, the indole is most effective when administered in  
 the evening. 
 
 Circulating nocturnal MEL has been found to be lower in women 
 with oestrogen positive/progesterone positive receptors in 
 their breast carcinoma (Cohen et al., 1978; Tamarkin et al., 
 1982). Furthermore, there are data which indicate that MEL 
 antagonizes the mitogenic effects of oestrogens (Blask et al., 
 1991, 1992; Wilson et al., 1992), suggesting that the 
 antiprofilerative effect of MEL is exterted on oestrogen 
 regulated pathways (Hill et al., 1992). Recent work by these 
 authors has demonstrated that MEL suppresses oestrogen receptor 
 mRNA expression by inhibiting transcription of the oestrogen 
 receptor gene. This is believed to be due to destabilization of 
 estrogen receptor transcripts (S.M. Hill et al., unpublished). 
 
 A further recently discovered mechanism is the maintenance of 
 the intracelluar redox state. Inhibition of antioxidants by 
 reducing agents such as glutathione eliminates the oncostatic 
 effects of MEL in certain human breast cancer cell lines (Blask 
 et al., 1994). This effect would not be mediated via the MEL 
 receptor. MEL has been found to be the most effective scavenger 
 of highly toxic free radicals (Tan et al., 1994), which induce 
 DNA damage. Concomitant exposure of rats to the indirectly  
 acting cacinogen safrole and MEL, markedly reduced hepatocyte 
 DNA damage in a dose-dependent manner (Poeggeler et al., 1993; 
 Hardeland et al., 1993). 
  
 Another potentially useful aspect of MEL treatment in cancer 
 patient is ist reported stimulatory effect on circulating 
 natural killer cells (Maestroni et al., 1989) and antagonism of 
 stress-induced immunosuppression (Pierpaoli & Meastroni, 1987). 
 This latter effect is believed to be mediated via the 
 endogenous opioid system since naltrexone, an opioid  
 antagonist, completely abolishes the immunoenhancing effects of 
 MEL in mice. Finally, MEL binding sites have been reported in 
 lymphocytes, which could mediate indole effects on 
 immunocompetent cells (Martin-Cacao et al., 1993). 
 
 Research in cancer is still at an early stage and controversial 
 and inconclusive results have frequently been presented. This 
 is not altogether surprising since age, weight, height, 
 reproductive status and season of the year, all of which may 
 modify MEL secretion, as well as dose and time of 
 administration of exogenous MEL, have not always been 
 adequately controlled. However, the described results hold 
 promise that MEL may be helpful in some of these neoplastic  
 conditions. 
 
 Melatonin as an oral contraceptive 
  
 Secretion of LH was found to fall in women given exogenous MEL 
 together with a synthetic progestagen as a contraceptive 
 (Voordouw et al., 1992). Recently these Dutch investigators 
 have pursued studies combining MEL with norethisterone as an 
 oral contraceptive, and report a synergistic effect between the 
 two drugs (Cohen et al., 1993). In a group of 42 women studied 
 throughout a treated and a control cycle, no LH surges were 
 observed at midcycle in the treated cycles. Furthermore, FSH 
 did not change significantly, and consequently no ovulation or  
 lutela increase in progesterone was observed, despite certain 
 follicle development. Oestradiol was not inhibited since 
 gonadotrophins were present, but the midcycle surge was delayed 
 compared to control cycles. Potential advantages of MEL 
 compared to oestrogen in such a contraceptive preparation would 
 be no deleterious vascular effects, and possible protection 
 against breast cancer through oncostatic and immunostimulant 
 effects. The mechanism involved in the suppression of the 
 hypothalamic-pituitary-ovarian axis could involve alterations 
 in the hypothalamic pulsatile secretion of GnRH (as  
 demonstrated in experimental models) (Karsch et al., 1986), a 
 possible effect on pituitary synthesis and/or release of LH (as 
 documented in animal and pituitary tissue culture studies) 
 (Martin & Sattler, 1982), and/or a direct effect on the ovary. 
 This latter possibility is supported by the finding that MEL 
 accumulates in the follocular fluid of women (Brezezinski et 
 al., 1987).  
 
 Melatonin and psychiatric diseases 
  
 An association between diurnal and seasonal rhythmicity of MEL 
 production and seasonal affective disorders and various types 
 of endogenous depression has been described (Lewis et al.,  
 1990). The recurring winter depression known as seasonal 
 affective disorder, also charakterized by weight gain, 
 carbohydrate craving and hypersomnia, was found to improve 
 dramatically after treatment with bright light, which 
 artificially extended the natural photoperiod (Rosenthal et 
 al., 1984). However, it appeard that light itself was more 
 important than ist inhibitory effect on MEL, 
 sincepharmacological suppression of MEL in these patients did 
 not improve their depression (Rosenthal et al., 1986). 
  
 MEL has been reported to be low in depressed subjects (Melntyre 
 et al., 1986). This is also the case in patients with unipolar 
 or bipolar affective disorders (Beck-Friis et al., 1985), but 
  can be normalized by antidepressant drugs such as noradrenaline 
 re-uptake blockers (Brown, 1989). Furthermore, an increase in 
 the cortisol/melatonin ration together with an inverse relation 
 between MEL and cortisol rhythms have been reported in 
 depressed individuals when compared to control (Claustrat et 
 al., 1984). Additionally, in non-affective disorders such as 
 chronis schizophrenia, abnormally low Melatonin levels have 
 also been observed (Ferrier et al., 1982). An absent circadian 
 rhythm for MEL with a preserved plasma cortisol profile and 
 consequently an increase in the MEL/cortisol ration, has also  
 been observed in drug free male paranoid schizophrenics 
 (Monteleone et al. 1992). Low nocturnal MEL has been proposed 
 to be a trait marker for major depressive disorders and 
 depressive states with abnormalities in the hypothalamic 
 pituitary adrenal axis, as demonstrated by the overnight 
 dexamethasone suppression test (Beck-Friis et al., 1985). 
  
 In rodents and humans, therapy with antidepressants such as 
 monoamine oxidase (MAO) inhibitors, increases the pinal content 
 of the Melatonin precursors serotonin, N-acetylserotonin and 
 consequently MEL in both blood and cerebrospinal fluid, by 
 enhancing N-acetyltransferase activity; in contrast, tricyclic 
 antidepressants reduce MEL production and secretion in rodents 
 (Lewis et al., 1990). Other psychotropic drugs which interfere 
 with monoamine pathways also affect pineal MEL, which is 
 normally stimulated by -adrenergic stimulation, as already 
 mentioned. Finally, receptors for benzodiazepines have been 
 reported to exist in the pineal gland of several animal species 
 (Lowenstein et al., 1984). Their function is unclear, but one 
 can speculate that thea may respond to benzodiazepine treatment 
 in psychiatric patients. In humans, the benzodiazepine drug 
 alprazolam given prior to lights out suppressed both nocturnal 
 MEL and cortisol concentrations, which again could point  
 towards the causal involment of binding sites for this drug or 
 of GABA neurotransmission in the human pineal, suprachiasmatic 
 nuclei and retina (McIntyre et al., 1993). As already 
 discussed, these findings do not suggest a simple, direct 
 relation between MEL and the hypothalamic-pituitary adrenal 
 axis in humans, even though MEL has been proposed to inhibit 
 CRH during major depression (Beck-Friis et al., 1985). 
 
 The multifactorial nature of both endogenous and enviromental 
 factors which converge in psychiatric patients and can 
 potentially alter MEL secretion and metabolism complicates the 
 identification of a possible relation between this pineal 
 indole and these diseases.  
 
 Melatonin and sudden infant death syndrome 
 
 the sudden infant death syndrome (SIDS) is diagnosed following 
 autopsy, as an unexplained death in an apparently healthy 
 infant, in whom non-specific post-mortem findings compatible 
 with respiratory distress such as petechial haemorrhages in the 
 pleura, heart and thymus are found (Campbell & Read, 1980; 
 Valdes-Dapena, 1982). This syndrome exhibits circannual, 
 circadian and ontogenetic features compatible with an impaired 
 maturation of the photoneuroendocrine system caused by a 
 genetic absence or mutation of the rate-limiting enzyme for the 
 biosynthesis of Melatonin, N-acetyltransferase (Weissbluth & 
 Weissbluth, 1994). Additionally, these infants often have a 
 history of sleep distrurbances and apnoeie episodes, which may 
 reflect failure of the automatic control of respiration during 
 sleep (Shannon et al., 1977; Watanabe et a., 1983). There is a 
 temporal coincidence between the apperance of the normal 
 pattern of predominant nocturnal sleep and the maturation of 
 the nocturnal MEL peak around the age of 2-3 months. With this 
 in mind, Sturner et al. (1990) investigated MEL concentration 
 in blood, CSF and vitreous humour from 32 infants who died of 
 SIDS and compared the values with another group of 36 infants  
 who died from other causes. After adjustment for age, mean CSF 
 and blood MEL was significantly lower in SIDS (91 29 vs 180 
 27 pmol/l in CSF; 97 29 vs 144 22 pmol/l in blood) and these 
 differences were maintained when infants aged 3 months or less 
 were considered (62 11 vs 173 42 pmol/l in CSF and 67 14 vs 
 155 44 in blood) (Wurtman et al., 1991). Similar trends, which 
 did not attain statistical significance, were also observed in 
 vitreous humour. These findings led these authors to suggest 
 that a deficient maturation of the endogenous MEL rhythm, 
 reflecting an abnormal maturation of the sympathetic nervous 
 system, might be pathogenetically related to SIDS. This view is 
 supported by Weissbluth and Weisbluth (1994), who considered 
 that the failure of normal pineal gland development and 
 Melatonin secretion may cause a lethal chemical imbalance 
 between serotonin, progesterone and catecholamines, culminating 
 in SIDS as a result of neurotoxic and cardiomyotoxic effects of  
 abnormally elevated catecholamines and intracelluar calcium 
 ions. 
 
 Magnetic field effects on Melatonin 
 
 as mentioned in earlier sections of this review, light inhibits 
 pineal MEL synthesis and secretion. This magnitude of this 
 inhibition depends on the animal species (nocturnal and 
 pigmented animals are sensitive to light than diurnal and 
 albino species (Webb et al., 1985), as well as on intensity or 
 brightness of the light and ist colour or wavelength (Brainard 
 et al., 1982, 1983). Apart from visible light, certain non- 
 visible ultraviolet wavelengths and extremely low frequency 
 electric and magnetic fields may influence MEL rhythm (for 
 review see Olcese, 1990; Reiter, 1992). Severe attenuation of 
 the circadian MEL rhythm was first demonstrated by Wilson et 
 al. (1989) in adult rats, and was found to be reversible. The 
 inhibitory effect of pulsed, intermittent static magnetic 
 fields on the nocturnal metabolism of serotonin to Melatonin in 
 the mammalian pineal was subsequently observed (Reiter & 
 Richardson, 1992). The physiological relevance of these 
 phenomena in man is still unknown; however, the potentially 
 hazardous effects of electromagnetic fields on MEL rhythm 
 disturbances should not be forgotten, given the ever increasing 
 exposure to man-made magnetic fields in industry, offices and 
 homes, as well as natural electric and magnetic fields. 
 
 Conclusions 
  
 Clearly much remains to be elucidated in defining the precise 
 involments of MEL in all these clinical situations. However, 
 even the most sceptical would be unwise to continue ignoring 
 the pineal gland and ist main hormone, Melatonin, when 
 confronted with a patient with abnormal neuroendocrine gonadal 
 function. Potential uses of exogenous Melatonin in cancer 
 patients and in oral contraceptives have been described, 
 although the chronrobiological aspects of such treatments, as 
 well as optimal doses of the indole, must be clarified. 
 Furthermore, the chronobiological properties of Melatonin are 
 only beginning to be understood but can constitute an important 
 means of synchronizing and improving performance in this age of 
 increasing international travel within a highly competitive 
 society. 
 
 
 

Sales Arguments

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 Potential costumers and indications 
 
 The clinical indicationsare in the area of depression, schizophrenia, 
 sleep disorders, jet lag, some forms of cancer and control of sexual 
 maturation during puberty (see Guide- line to Biogenic Amines). 
 
 - Private labs, universities, sleep centers, pharmaceutical industry e.g. 
  testing of drugs in connection with management of wakefulness, sleep and 
 jet lag 
 - A lot of studies will start in the near future concerning clinical 
 trials with melatonin derivatives ( e.g. Eli Lilly and Company, SERVIER, 
 France) ---- see EPS 96 Abstracts 
 
 Arguments  
 
 - Publication in: J. Pineal Research, 21, (1996) pp 91 - 100. Assay 
 successfully used to investigate the influence of magnetic fields on 
 humans. Literature available on request. 
 - Break apart strips for short assay runs 
 - Direct assay without extraction of urine 
 - Wide standard range, applicable to therapeutic drug monitoring. 
 - Only 2 hours incubation time 
 - Calibrated against the "Golden Standard" RIA of J. ARENDT. Univ. 
 Gilford, UK  
 - Excellent correlation with the "Golden" RIA standard with r = 0,97 
 - Adaption protocol available for the analyzer "PersonalLab" from Biochem. 
  
 
 General arguments HPLC vs. immunoassays 
 
  After switching to immunoassays, the HPLC equipment may be used e.g. for 
 therapeutic drug monitoring or for other analytes for which no routine 
 test kits are available. 
  
 
 

Product Literature

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 Reference labs of Melatonin and Melatonin sulfate  
 
 1. Melatonin RIA RE29301 
  
 Charles A. Czeisler: Section on sleep Disorders and Cicadian Medicine, 
 Division of Endocrinology, Department of Medicine, Harvard Medical 
 School and Brigham and Women's Hospital, 221 Longwood Ave., 
 Boston, MA 02115 
 
 
 Dubbels R.: Center of human Genetics and Genetic Counselling, 
 University of Bremen, D-28359 Bremen, Germany 
 
  
 Follenius M.:L.P.P.E., CNRS, 21, rue Becquerel, F-67087 Strasbourg 
 Cedex, France 
 
  
 2. Melatonin-Sulfate Elisa RE 54031 
 
 Pfluger DH: Department of Social and Preventive Medicine, University 
 of Berne, Finkenhublweg 11, CH-3012 Berne, Switzerland. 
 
  
 3. Product Literature 
 
 3.1. Product literature references  
 
 3.1.1. Pfluger, D.H., Minder C.E., Effects of exposure to 16.7 Hz magnetic 
 fields on urinary 6-hydroxymelatonin sulfate excretion of swiss 
 railway workers, J. Pineal Res., 21, 91-100 (1996) 
 
 3.2. Other literature references 
  
 3.2.1. Arendt, J. et al., Immunoassay of 6-Hydroxymelatonin Sulfate in 
 Human Plasma and Urine: Abolition of the Urinary 24-Hour Rhythm 
 with Atenolol, Journal of Clinical Endicrinology and Metabolism, 
 60 (6): 1166-1173 (1985) 
 
 3.2.2. Fellenberg, A.J. et al., Urinary 6-sulphatoxymelatonin excretion 
  and production rate: studies in sheep and man, In: Matthews C.D., 
 Seamark R.F., eds., Pineal Function. Amsterdam, Elsevier, 143-150 
 (1981) 
  
 3.2.3. Fellenberg, A.J. et al., Urinary 6-sulphatoxymelatonin excretion 
 during the human menstrual cycle, Clin. Endocrinol., 17: 71-75 
 (1982)  
 
 3.2.4. Bojkowski et al., Annual changes in 6-sulphatoxymelatonin excretion 
 in man, Acta Endocrinol. (Copenh), 117: 470-476 (1988)  
 
 3.2.5. Fellenberg, A.J. et al., Specific Quantitation of Urinary 
 6-Hydroxymelatonin-sulphate by GCMS, Biomedical Mass Spectrometry, 
 7 (2): 84-87 (1980) 
 
 3.2.6. Webley, G., Leidenberger, F., The Circardian Pattern of Melatonin 
 and its Positive Relationship with Progesteron in Women, Journal 
 of Clinical Endocrinology and Metabolism, 63 (2) (1986) 
 
 3.2.7. Short Reports, Alleviation of Jet Lag by Melatonin, British 
 Medical Journal, 292: 1170 (1986) 
 
 3.2.8. Bartsch, Ch. et al., Melatonin and 6-sulphatoxymelatonin 
  circardian rythms in serum and urine of primary prostate cancer 
 patients: evidence for reduced pineal activity and relevance 
 of urinary determinations, Clinica Chimica Acta, 209: 153-167 
 (1992)  
 
 

Miscellaneous

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