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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 manufactured by IBL (Immuno Biological Laboratories, Hamburg Germany) and distributed by RDI Divison of researchd Industries Intl for in vitro research use only-not for use in or on humans or animals, not for use in diagnostics.

Melatonin ELISA  cat#RDI-RE54021  $594.00/1kit    $562.00/kit  5-9

 Catalogue No   : RDI-RE54021
 Product group  : Tumor Markers                       12 x 8      
 Product name   : Melatonin                     ELISA   96
 Method         : ELISA                                                       
 Incubation time: 18h, 2h, 0,5h                                               
 Standard curve : 0.0 - 300 pg/ml                                             
 Sample/Prep.   : 50�l serum, plasma                                          
 Isotope/Substr.: PNPP, 405nm                                                 


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


Back to Contents The pineal gland ("corpus pineale") has been called a neuroendocrine transducer because of its important role in photoperiodism. The major hormone of the pineal gland is N-acetyl-5-methoxy-tryptamine or melatonin which is synthezised from the amino acid tryptophane. Melatonin has its highest levels in plasma during nighttime. The circulatory melatonin is primarily derived from the pineal gland. Its characteristic nocturnal surge appears to encode temporal information such as length of night. Human plasma melatonin levels show marked individual variation. Daytime levels are usually in the range of 20 pg/ml in the morning hours whereas nighttime levels generally exceed 55 pg/ml. However, the interindividual ranges are significant. Regulation of the melatonin secretion is under neural control. Sympathetic innervation seems to play a major role via its release of noradrenaline. High affinity binding sites specific for melatonin have been identified in the hypothalamus of the human and many other mammalian species. Altered patterns and/or levels of melatonin secretion have been reported to coincide with sleep disorders, "jet lag", depression, stress, schizophrenia, hypothalamic amenorrhea, pregnancy, anorexia nervosa, some forms of cancer, immunological disorders as well as control of sexual maturation during puberty. The physiological and pathopysiological role of melatonin is under intensive investigation in the last years. The half-life of melatonin in human circulation is approximately 47 minutes. Most of the circulating melatonin is metabolized in the liver to 6-hydroxymelatonin and subsequently to 6-sulfatoxymelatonin which is excreted into the urine. The melatonin concentrations in saliva are approximately 30 % of plasma levels showing the same typical circadian pattern.

Contents of the Kit

Back to Contents 1. Contents of the Kit The quantity supplied is sufficient for the determination of 48 samples, which can be performed in duplicate. 1.1. Extraction Columns 20 pieces 1cc/100mg, ready to use C18 reversed phase, after elution with methanol use for extraction the next samples or store at 4 - 8�C protected from dust, re-use up to 4 times. Note: Additional extraction columns may be ordered from IBL under Cat.-No. RE 540 23. 1.2. Assay Buffer, Concentrate 1 bottle 50 ml, phosphate buffer with tween and stabilizer; dilute 1:10 with bidist. water. 1.3. Antiserum 3 vials lyophilized, stabilized, dissolve contents of each vial in 2 ml of bidist. water. 1.4. Microtiter Strips 12 strips each 8 wells, coated with goat-anti-rabbit antibodies. 1.5. Melatonin-Biotin 3 vials lyophilized, stabilized, dissolve contents of each vial in 2 ml of assay buffer. 1.6. Standard A - F 6 vials lyophilized, stabilized, dissolve contents of each vial in 2 ml of bidist. water, store aliquotes at -20 �C. For each lot of standards, refer to vial labels as well as to quality control certificate for exact concentrations. 1.7. Control Serum 1 and 2 2 vials lyophilized, stabilized, dissolve contents of each vial in 2 ml of bidist. water, store aliquotes at -20 �C. For concentration see quality control certificate. 1.8. Enzyme Conjugate, Concentrate 1 vial 250 �l, anti-biotin-alkaline phosphatase in TRIS buffer with stabilizers, dilute 1:81 with Assay Buffer. 1.9. PNPP Substrate Tablets 9 tablets p-nitrophenyl phosphate (pNPP) 1.10.PNPP Substrate Buffer 1 bottle 30 ml, ready for use, contains diethanolamine. 1.11.PNPP Stop Solution 1 bottle 10 ml, ready for use, contains 1 N NaOH with 0.25 M EDTA. 1.12.Adhesive foil 3 pieces

Principle of the Test

Back to Contents The Melatonin ELISA is a competitive enzyme immunoassay for the quantitative determination of melatonin in serum and plasma with extraction. The assay procedure follows the basic principle for competitive immunoassays whereby there is competition between a biotinylated and a non-biotinylated antigen for a fixed number of antibody binding sites. The amount of biotinylated antigen bound to the antibody is inversely proportional to the analyte concentration of the sample. When the system is in equilibrium, the free biotinylated antigen is removed by a washing step and the antibody bound biotinylated antigen is determined by use of anti-biotin alkaline phosphatase as marker and p-nitrophenyl- phosphate 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

Back to Contents A Summary ========= 1. Pipet 50 �l of each extracted standard, controls and patient samples. 2. Pipet 50 �l melatonin-biotin and subsequently 50 �l of antiserum. 3. Seal the plate with adhesive foil and incubate overnight (14-20h) at 2-8�C. 4. Wash three times with assay buffer. 5. Pipet 250 �l of enzyme conjugate. Incubate for 120 min. at room temperature. 6. Wash three times with assay buffer. 7. Pipet 200 ml of PNPP substrate solution. Incubate at room temperature for 20-40 min. 8. Stop the substrate reaction by adding 50 �l of stop solution. 9. Read the optical density at 405 nm (reference wave length 600-650 nm). B. Detailed Instructions ======================== Materials required but not provided - Pipet 50, 500 �l Multipette Eppendorf or similar products 50 - 250 �l and 250 - 1000�l - Centrifuge (alternative: vacuum Manifold, e.g. Mallinekrodt-Baker or Waters) - Evaporator centrifuge (speed-vac, alternative: Sample Concentrator by use of nitrogen, e.g. Techne) - Rotary mixer (Mutli-Tube) - Microtiter plate mixer - ELISA reader capable of reading absorbance at 405 nm - Polystyrene tubes (12 x 75 mm) - Bidist. water - Methanol (p.A.) Preparation of Reagents The contents of the kit can be divided into three seperate runs. The volumes stated below are for one test with 4 strips (32 determinations). If a larger number of strips is to be used, the volumes have to be changed accordingly. 1. Standards Dissolve contents of each vial in 2 ml of bidist. water and wait for 15 min. Mix gently. Aliquots should be stored at - 20 �C. 2. Control Sera Dissolve contents of each vial in 2 ml of bidist. water and wait for 15 min. Mix gently. Aliquots should be stored at - 20 �C. 3. Assay buffer (ready for use) 15 ml of asssay buffer concentrate have to be diluted 1:10 with bidist. water up to 150 ml. This gives the ready to use assay buffer. Store at 2 - 8 �C. 4. Melatonin-biotin Add 2 ml of the ready to use assay buffer to one vial of lyophilysed melatonin-biotin and wait for 15 min. Mix gently and use only once. If a larger volume of melatonin-biotin is required, vials can be pooled. Do not store reconstituted, not used melatonin-biotin. 5. Enzyme conjugate Dilute 70 �l of enzyme conjugate concentrate in 5.6 ml of the ready to use assay buffer. Prepare freshly for each test run. Use only once. Do not store diluted, not used enzyme conjugate. 6. Antiserum Dissolve contents of each vial in 2 ml of bidist. water and wait 15 min. Mix gently. If a larger volume of antiserum is required, vials can be pooled. Do not store reconstituted, not used antiserum. 7. PNPP substrate solution Dissolve 3 PNPP substrate tablets in 8 ml of PNPPsubstrate buffer. IMPORTANT: Prepare the PNPP substrate solution 10 min. prior to its use and use only once. 1) Extraction Procedure Any sample reading greater than the highest standard should be diluted appropriately with ready for use Assay Buffer prior to extraction. Note: Filter or centrifuge the samples prior to extraction in order to avoid clogging of the columns. Each sample including standards and controls have to be extracted! A. Standard version: Procedure for centrifuge and evaporator centrifuge Extraction may be performed in advance. The dried extracts (after evaporation of methanol, see point 8.5.) may be stored at 2 - 8�C or -20�C for up to 24 hours. This procedure yields approximately 90 - 100 % recovery of added Melatonin in samples. 2) Column preparation and conditioning Place one extraction column for each sample to be extracted into polystyrene or glass tubes (> 12 x 75 mm). Add 2 x 1 ml of methanol (undiluted) to columns, centrifuge for 1 minute at 200 x g*. Add 2 x 1 ml of bidist. water to columns, centrifuge for 1 minute at 200 x g. Proceed with sample application without delay in order to avoid the columns getting dry. * Note: g (relative centrifugal force) is not equivalent to rounds per minute (rpm). The rpm value has to be calculated depending on the radius of the centrifuge. Example: At a radius of 142 mm from the rotation centre of the centrifuge, 200 x g are equivalent to approximately 1100 rpm. 3) Sample application Place each column into correspondingly marked tubes. Add 0.5 ml of standards, controls and samples to columns, centrifuge for 1 minute at 200 x g. 4) Washing Add 2 x 1 ml of 10% methanol in bidist. water (v/v) to columns, centrifuge for 1 minute at 500 x g. 5) Elution of extract Place the columns into clean correspondingly marked polystyrene tubes. Add 1 ml of methanol (undiluted) to columns, centrifuge for 1 minute at 200 x g. Remove columns from tubes, avoiding drops to be left at the columns. Use columns for extracting next samples (re-use up to 4 times) or store columns at 4 - 8�C protected from dust. 6) Evaporation and reconstitution of extract Evaporate the methanol to dryness and reconstitute the samples with 0.15 ml of bidistilled water. Vortex for at least 1 minute and assay immediately. B. Alternative extraction procedure: Using a vacuum manifold instead of a centrifuge For the extraction scheme, please follow points 8.1. to 8.5. accordingly. The volumes remain unchanged. Let the solvent pass through the column using vacuum and a flow rate of not more than 5ml/min. For the samples and extracts use a flow rate of not more than 2ml/min. The evaporation of the solvent may be performed by using an evaporator centrifuge or by nitrogen. 7) Assay Procedure 7.1. Pipet 50 �l of each extracted Standards, Controls and patient samples into the appropriate wells. 7.2. Pipet 50 l Melatonin-Biotin and subsequently 50 �l of Antiserum with a multipette into each well and shake the plate carefully. 7.3. Seal the plate with the adhesive foil and incubate overnight (14-20h) at 2 - 8�C. 7.4. Wash each well three times with Assay Buffer (the use of a washer is recommended). Remove the Assay Buffer carefully. Invert plate and remove any remaining liquid by tapping on clean blotting paper. Note: Sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure. 7.5. Pipet 150 l of Enzyme Conjugate into the wells. 7.6. Seal with the adhesive foil and incubate for 120 min. at room temperature on an orbital shaker (500 U/min). Prepare the PNPP Substrate Solution 10 min. before end of incubation period! 7.7. Wash each well 3 x with Assay Buffer (see above). 7.8. Pipet 200 l PNPP Substrate Solution into each well. 7.9. Incubate at room temperature for 20-40 min. on an orbital shaker (500 U/min). 7.10. Stop the substrate reaction by adding 50 l PNPP Stop Solution into each well, briefly mix contents by gently shaking the plate. 7.11. Read the optical density at 405 nm (reference wave length 600-650 nm) with a microtiter plate reader within 60 min. after stopping. 8) Calculation of Results The concentrations of the standards (abscissa, logarithmic) are plotted against their corresponding optical density (ordinate, linear) on a semilogarithmic graph paper. The concentration of the samples can be read directly from this standard curve by using their average optical density. Any sample reading greater than the highest standard should be diluted appropriately with ready for use Assay Buffer prior to extraction and reassayed. 9) Storage and Stability The Melatonin-ELISA kit is shipped at ambient temperature and should be stored in the dark at 2-8�C. After removing assay reagents from the refrigerator, allow them to reach reach room temperature before use. Mix reagents thoroughly by gentle inversion. Do not use components byond the expirationdate shown on the kit labels. Do not mix various lots of any kit component within an individual assay. 10) Specimen Collection and Storage The procedure should be performed with serum or plasma. Do not use hemolytic or lipemic samples. The samples can be stored at 2-8�C for 24 hours. For longer period of storage freeze the samples at -20�C. Repeated freezing and thawing should be avoided. For the determination of daytime values blood collection should be performed after 8.00 a.m. due to the circardian rhythmicity.

Performance Characteristics

Back to Contents 1. Specifity The cross-reactivity of the antiserum has been measured against various compounds, which were processed like samples. Compound Cross-Reactivity (%) Melatonin 100.0 5-Methoxytryptophole 0.5 N-Acetyl-Serotonin 0.4 5-Methoxytryptamine 0.1 5-Hydroxy-L-Tryptophane 0.01 6-Methoxytryptamine <0.01 5-Methoxyindole-3-Acetic <0.01 Acid Serotonin <0.01 DL-Tryptophane <0.01 DL-5-Methoxytryptophane <0.01 2. Sensitivity The lowest detectable level that can be distinguished from zero standard is 3.0 pg/ml. 3. Precision Intra-Assay Variation (Enzyme Immunoassay) (all concentrations in pg/ml) Mean Standard CV n Deviation (%) 6.8 0.59 8.8 12 19.2 2.0 10.5 12 48.9 1.6 3.3 12 Inter-Assay Variation (Enzyme Immunoassay) (all concentrations in pg/ml) Mean Standard CV n Deviation (%) 9.2 1.5 15.7 12 74 5.0 6.9 12 Inter-Assay Variation (Extraction and Immunoassay) (all concentrations in pg/ml) Mean Standard CV n Deviation (%) 5.2 0.99 19.0 10 34 3.0 9.0 10 4. Linearity Unknown patient samples were diluted with ready for use Assay Buffer before sample preparation and assayed. The calculated results are shown in pg/ml. Dilution undiluted 1/2 1/4 1/8 1/16 Sample 1 300 312 324 318 328 (100%) (104%) (108%) (106%) (109%) Sample 2 233 225 233 290 224 (100%) (97%) (100%) (124%) (96%) Sample 3 114 129 133 115 99 (100%) (113%) (116%) (101%) (87%) Sample 4 173 174 202 163 139 (100%) (100%) (117%) (94%) (81%) 5. Recovery Patient samples were enriched with increasing amounts of melatonin (concentrations in pg/ml). Actual Amount Amount Amount Recovery conc. added recovered expected % Sample 1 7.7 5 13.3 12.7 105 7.7 10 17.2 17.7 97 7.7 20 26.2 27.7 95 7.7 40 51.3 47.7 108 7.7 80 85.4 87.7 97 7.7 160 158.4 167.7 94 Sample 2 7.5 5 12.9 12.5 103 7.5 10 18.5 17.5 106 7.5 20 28.0 27.5 102 7.5 40 47.8 47.5 101 7.5 80 87.6 87.5 100 7.5 160 138.0 167.5 82 Sample 3 6.6 5 11.1 11.6 96 6.6 10 16.5 16.6 99 6.6 20 25.4 26.6 95 6.6 40 49.8 46.6 107 6.6 80 82.0 86.6 95 6.6 160 161.6 166.6 97 Sample 4 8.3 5 12.1 12.3 98 8.3 10 17.1 18.3 93 8.3 20 27.8 28.3 98 8.3 40 48.6 48.3 101 8.3 80 88.0 88.3 100 8.3 160 163.2 168.3 97

Expected Values

Back to Contents It is recommended that each laboratory establishes its own range of normal melatonin values. The melatonin levels in humans show a marked circadian rhythmicity characterised by very low levels during day-time and high levels during nighttime, and show a considerable interindividual variation. Furthermore, the melatonin concentration is age dependent. The figure below shows the circadian rhythm of melatonin in serum in a group of 6 healthy persons. The mean value reaches a minimum of about 4.6 pg/ml during daytime at 4 p. m. and a maximum of about 77.5 pg/ml during nighttime at 4 a.m.

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 � 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 pul�ses 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

Back to Contents Potential customers and indications: The clinical indications are 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: - Assay with simple extraction of serum, use of C18 extraction columns from WATERS, 20 columns in the kit included, columns are reusable - NOTICE: The lowest standard of the ELISA is about 3 pg/ml. Values below this standard should be stated as below 3 pg/ml. If a costumer wants to measure volumes in the range of 1-3 pg/&ml he should use the IBL RIA. - Wide standard range, applicable to therapeutic drug monitoring, sample diluent included in the kit. - Excellent correlation to RIA - Convenient for large test series, e.g. circardian rhythmicity studies - Delivery of ready to use microtiter plates. Competitors: - HPLC: very time-consuming, restricted to highly specialized laboratories 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

Back to Contents 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. References 1.Wehrenberg, O et al. (1985); Bedeutung des Epiphysenhormons Melatonin bei Mensch und Tier; Zentralorgan Chirurgie, 231 (7),pp. 381-386. 2.Bartsch, C; et al. (1989); Stage-Dependent Depression of Melatonin in Patients with Primary Breast Cancer; In: CANCER, Vol. 64, No.2, pp.426-433. 3.Brown et al. (1994); Light, melatonin and the sleep wake cycle; J Psychiatr Neurosci, Vol. 19, No 5, 345-353. 4.Czeisler, C A; et al. (1995); Suppression of Melatonin Secretion in some Blind Patients by Exposure to bright light; In: New England Journal of Medicine, 332, 6-11, pp. 7-12. 5.Webb et al. (1995); Role of melatonin in health and disease; Clinical Endocrinology, 42, 221-234. 6.Reiter, RJ (1996); Functional diversity of the pineal hormone melatonin: Its role as an antioxidant; Exp Clin Endocrinol, 104, pp.10-16.


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