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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   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

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 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

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(More) Clinical Background

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 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 
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