We have purified the major metabolite of melatonin, 6-sulphatoxymelatonin, from urine and compared it to its synthetic counterpart. For preparation of the biological material, oral melatonin was administered to human volunteers and their urine extracted onto Amberlite XAD-2 resin to remove urea; the glucuronide metabolites of melatonin were removed by silica chromatography; and 6-sulphatoxymelatonin was separated from N-acetyl serotonin sulphate, the other sulphate metabolite of melatonin, by preparative thin-layer chromatography. Synthetic 6-sulphatoxymelatonin was produced by reacting 6-hydroxymelatonin with chlorosulphonic acid in dimethylformamide; the reaction mixture was purified on Florisil and preparative thin-layer chromatography was used to remove indolic by-products of the reaction. Elemental and X-ray microanalysis of the biological and synthetic products showed that classical methods used for their purification introduced inorganic impurities, such as silicon- and chlorine- containing compounds, which were not detectable by thin-layer chromatography, infrared spectroscopy, nuclear magnetic resonance spectroscopy, or gas chromatography-mass spectrometry. We introduced further purification steps to remove these inorganic impurities, monitoring the process using elemental and X-ray microanalysis. Extensive characterization of the resulting purified products showed that the biological and synthetic compounds were identical.

Abstract: We describe a newly developed enzyme immunoassay (EIA) for the determination of 6‐sulphatoxy‐melatonin (aMT6s) in human urine, using a aMT6s‐bovine serum albumin‐horseradish peroxidase (aMT6s‐BSA‐HRP) conjugate as the enzyme label. The assay incorporates a highly specific antibody raised in rabbits. The EIA has a sensitivity of 2 pg/well (40 pg/ml) with intraassay coefficients of variation of 2.3–6.1% in the range of the assay. The material with the highest level of cross‐reactivity was N‐acetyl serotonin sulphate, with a relative potency of 0.000078%. One hundred thirty‐four urine samples from children and adults at different time points were assayed and the results compared with those from an established radioimmunoassay (RIA) and with a newly developed RIA using the same antibody as the EIA. The correlation coefficient, r, comparing the two RIA's was 0.9869, and the regression equation was log (kit) = 0.9340 log (new) + 0.1213. The correlation coefficient, r, comparing the EIA with the newly developed RIA, was 0.9686, and regression equation log (new) = 0.9674 log (EIA) + 0.0600. The EIA for the measurement of aMT6s in urine represents a new approach in the investigation of pineal function.

The hypothalamic GnRH pulse generator activates the pituitary-gonadal reproductive axis, and contraceptive techniques have advanced to the point where GnRH analogues can block this effect. However, nature has an even finer form of contraception, whereby the GnRH pulse generator is activated or inactivated at different seasons of the year. Darkness affects the retino-pineal nervous pathway to cause the synthesis and release of melatonin from the pineal gland at night. The duration of the night time release of melatonin is longer in winter than in summer; and it is the prolongation in the duration of the night time release of melatonin, with the change of season from summer to winter, which acts as the endocrine signal for inactivating the hypothalamic GnRH pulse generator. Humans are not seasonal breeders, and evidence is presented to indicate that this is due to an impairment of the retino-pineal pathway rather than an impairment of melatonin hypothalamic function. Thus the way is open for utilising melatonin as a human contraceptive, and a melatonin-based contraceptive is at present undergoing phase III clinical trials. The challenge is to develop more refined methods for administering (or releasing) melatonin, so that it has a night time amplitude and duration which mimics that seen in long day breeders.

We report an HPLC assay for melatonin that incorporates automated injection, methanol/water mobile phase, and fluorescence detection. Plasma samples were extracted by solid and liquid phases. Recovery was > 70% for 1-10 mL of plasma extracted, approximately 40 pg-250 ng of melatonin. Samples were dried and reconstituted in 100 mL/L methanol. Injections were 25 microL or 150 microL, depending on sample concentration, and the melatonin peak was eluted in 380 mL/L methanol. The detection limit of the assay was 6 pg on the column, allowing a practical sensitivity in plasma of 11 pmol/L for 8-mL samples and 34 pmol/L for 2-mL samples. More than 100 plasma samples from volunteers and patients were assayed and the results compared with an established RIA. The mean daytime concentration of melatonin was 20.7 pmol/L (SEM = 1.2) and 18.5 pmol/L (SEM = 1.6) for HPLC and RIA, respectively, and the mean nighttime concentration was 82.4 pmol/L (SEM = 6.5) and 82.2 (SEM = 7.3), respectively.

Thirty healthy volunteers were treated with beta-adrenoceptor blocking doses of long-acting propranolol for at least 28 days before being randomized to continue propranolol treatment, receive identical placebo under double-blind conditions, or discontinue all treatment. No evidence of a central nervous withdrawal syndrome occurred during the next 28 days as assessed by changes in psychomotor tests, rating scales, visual analogue scales, tremor recordings and melatonin excretion. Three subjects in the placebo withdrawal group but none in the propranolol group complained of insomnia for up to 14 days of the withdrawal period.

A method for measuring activity of hamsters using a stabilimeter at a 1 second sampling rate with data computer recorded as 5 minutes integrated values was developed. In a single cage without a running wheel a consistent pattern for activity was observed, consisting of (a) low levels of daytime activity until one or two hours before lights off when activity increased significantly; and (b) a peak of nocturnal activity in the first hour of the dark cycle. The inclusion of a running wheel increased and altered significantly the pattern of nocturnal activity. In further experiments animals were housed in two linked cages, one acting as light-proof burrow and the other exposed to light. Measurements were recorded from each cage independently and from two position detectors in the interconnecting tunnel. The results showed: (a) total activity, i.e., the summation of activity in both cages, was not different from activity in a single cage system; (b) low daytime activity was composed of prolonged periods of rest in the burrow plus short periods of activity in the exposed cage; the increased activity one hour before lights off was localised to the light-proof burrow; and (c) after lights off, the animals began to spend increasing periods of time in the exposed cage reaching a maximum after one hour. Replacing artificial with natural light did not change the principal features of behaviour.

Twenty-four-h urine samples, divided into two fractions representing night- and daytime melatonin production, were collected from 115 healthy individuals between the ages of 3 and 80, of known height and weight, and assayed for 6-hydroxy melatonin sulphate (SaMT), a major urinary metabolite of melatonin, by gas chromatography mass spectrometry. The population was divided for analytical purposes into children (boys aged 3-10.99, girls aged 3-9.59), adolescents (males aged 11-17.99, females aged 9.60-17.99), and adults (men and women over 18). The results showed approximately the same excretion over 24 h in all 3 groups but that the night/day ratio was considerably greater in children and adolescents compared to adults (P less than 0.001). However, when the results were expressed as a function of body weight (BW), body surface area (BSA), or creatinine excretion (CE), nocturnal SaMT was higher in children than in adults (P less than 0.001 for all 3 parameters) or adolescents (BW, P less than 0.001; BSA, P less than 0.002; CE, P less than 0.001) and was higher in adolescents than in adults (BW and BSA, P less than 0.001). Children also excreted more during the day than adults (BW, P less than 0.01; CE, P less than 0.001) or adolescents (BW alpha CE, P less than 0.02). Our results show that pineal output barely changes during childhood and adolescence. However, there is an age related decrease in SaMT excretion/unit body mass which correlates with an age-related increase in body mass. We therefore conclude that the decrease in circulating levels of melatonin during growth and sexual maturation is brought about by an increase in body mass.

Melatonin is metabolised by hydroxylation to form 6‐hydroxy‐melatonin by demethylation to form N‐acetyl‐serotonin, which are excreted as sulphate glucuronide conjugates. We required these metabolites as pure powders therefore undertook their isolation characterisation. Three volunteers ingested 1 g each of melatonin, their urine was collected pooled. For the sulphate conjugates, a Lichoprep column was used to concentrate the metabolites to remove most of the urea. The sulphate conjugates were separated from the glucuronides on a Florisil column further purified on a fractogel column. They were separated by high‐performance liquid chromatography (HPLC) resulting in white powders of 6‐hydroxy‐melatonin sulphate (SaMT) N‐acetyl‐serotonin sulphate (SNAS). For the glucuronide conjugates, an aliquot of the pooled urine was taken to dryness, the residue was dissolved in methanol, the solution was filtered. The methanol filtrate was taken to dryness, the residue was applied to a Florisil column. The isolated glucuronide conjugates were recrystallized prior to separation by HPLC, which gave pure white powders of N‐acetyl‐serotonin glucuronide (GNAS) 6‐hydroxy‐melatonin glucuronide (GaMT). Characterisation was achieved by using infrared ultraviolet spectroscopy, thin‐layer chromatography (TLC), gas chromatography‐mass spectrometry (GCMS). These techniques unambiguously confirmed the assigned structures for SaMT SNAS fully supported the assigned structures for GNAS GaMT. Three TLC solvent systems were used, in each case the individual conjugated metabolite appeared as a discreet spot. Purity, as assessed by GCMS, was shown to be greater than 95% for SNAS, SaMT, GaMT to be 88% for GNAS.

Circulating melatonin is hydroxylated to 6-hydroxymelatonin and excretedin urineas the sulfateandglucuronide conjugates.We extractedthesetwo compounds fromunneby usingoctadecylsilane-bonded silicacartridges to eliminate mostof the urea and electrolytes, and silica cartridgesto separate thesulfateandglucuronide conjugates. Afterhydrolyzingtheseparated conjugates enzymlcally, we determined the free hydroxymelatonin by gas chromatography-mass spectrometry. Thoughrecoveries werelowandvariable,we Wereable to quantify the analyteIn the originalsampleby addingdeuterated sulfateand glucuronide conjugates tothe unnesbeforeextraction.

To investigate the motor rhythm of the Syrian hamster under natural photoperiod and to relate these findings to pineal rhythm, animals were left undisturbed in metabolic cages in which stabilimeter systems registered motor activity, photovoltaic cells registered light intensity, and in which urinary output was used for the assay of the major melatonin metabolite 6-sulphatoxymelatonin. The motor activity of two animals was monitored during 1 month and showed a striking similarity. Melatonin secretion was measured during 3 consecutive days and showed a night/day ratio in both animals that was relatively constant and that was coupled to the diurnal rhythm of the animals' activity.

Circulating melatonin is hydroxylated to 6-hydroxymelatonin and excreted in urine as the sulfate and glucuronide conjugates. We extracted these two compounds from urine by using octadecylsilane-bonded silica cartridges to eliminate most of the urea and electrolytes, and silica cartridges to separate the sulfate and glucuronide conjugates. After hydrolyzing the separated conjugates enzymically, we determined the free hydroxymelatonin by gas chromatography-mass spectrometry. Though recoveries were low and variable, we were able to quantify the analyte in the original sample by adding deuterated sulfate and glucuronide conjugates to the urines before extraction.

To investigate whether melatonin (aMT) can be metabolized to N-acetyl serotonin (NAS), a low dose of deuterated aMT was administered to four normal subjects, and their urine samples were analyzed for the presence of deuterated NAS and deuterated 6-hydroxymelatonin (6-HaMT). In one set of experiments, the urine samples were subjected to column chromatography to separate the glucuronide and sulfate conjugates for independent analysis. In another, an internal standard (NAS-sulfate) was used for quantification and total conjugate analysis. Measurement was by gas chromatography-mass spectrometry, and the molecular ions of deuterated and nondeuterated NAS and 6-HaMT were monitored. Deuterated aMT was metabolized to deuterated NAS and deuterated 6-HaMT. The proportion of NAS was less in the sulfate than in the glucuronide conjugates and, overall, represented 15% of the total. Since demethylation is not a pathway that occurs with other pineal methoxyindoles, even at a much larger dose, it seems to be a significant finding with regard to aMT. Thus, it may be important to elucidate the differential metabolism of aMT at different time points and in different age groups.

Though melatonin is primarily metabolised to 6‐hydroxy‐melatonin, we have recently shown that it can also be demethylated to form N‐acetyl‐serotonin. The question therefore arises as to whether demethylation is a general metabolic pathway that can apply to other pineal methoxyindoles. To investigate this possibility we administered deuterated methoxy‐tryptophol (dML) and deuterated methoxy‐tryptamine (dMT) to rats and analysed the urine for the presence of deuterated methoxyindole acetic acid (dMIAA) and deuterated hydroxyindole acetic acid (dHIAA). The method of analysis was gas chromatography mass spectrometry (GCMS), where the relevant molecular ion and fragment ions were monitored. The results showed that the major metabolite in all cases was dMIAA. There was no evidence to suggest that the compounds had been demethylated to form dHIAA. The study therefore indicates that the demethylation of melatonin is a specific metabolic pathway that does not apply to other methoxyindoles.

In a recent study, we showed that melatonin could be metabolized to N-acetylserotonin and 6-hydroxymelatonin. To confirm this finding rats were administered three different forms of deuterated melatonin intraperitoneally. Their urines were analysed by gas chromatography/mass spectrometry and the results showed, in each case, that the appropriate deuterated (or non-deuterated) metabolite had been formed. From these data it is clear that N-acetylserotonin is a urinary metabolite of melatonin.

It has been generally agreed that the metabolism of the pineal hormone melatonin (aMT) consists of 6-hydroxylation followed by sulfate or glucuronide conjugation. The urinary assay of 6-hydroxy-melatonin (6-HaMT) is valued as a means of providing integrated information on aMT production. However, we show, in this study, that aMT has two principal urinary metabolites, N-acetylserotonin (NAS) as well as 6-HaMT. Rats were administered varying doses of aMT and their urines were collected and analyzed by thin layer chromatography and gas chromatography-mass spectrometry (GCMS). Thin layer chromatography of the urinary metabolites showed the expected pattern, a major spot at Rf 46%, the position of 6-sulfatoxy-melatonin, a less intense spot at Rf 32%, the position of 6-glucuronide-melatonin and a weak spot at Rf 78%, the unconjugated metabolite. However, after deconjugation and derivitization, GCMS analysis of the urines, or of the spot at Rf 46%, showed two products, one of which had the same GC retention time and mass spectrum as 6-HaMT, whereas the other had the GC retention time and mass spectrum of NAS. When deuterated aMT was administered, GCMS analysis showed the presence of deuterated 6-HaMT and deuterated NAS, proving that NAS was metabolized directly from aMT and not produced somewhere else in the body in response to aMT. Finally, GCMS analysis of urines after the administration of 6-HaMT or of NAS showed only one metabolic product in each case, i.e. 6-HaMT and NAS, respectively. This suggested that the conversion of aMT to 6-HaMT and NAS resulted from two independent metabolic pathways. It is understandable that research workers who relied entirely on chromatography should have failed to distinguish NAS and its conjugates from 6-HaMT and its conjugates since the chromatographic and staining properties of the two indoles are almost indistinguishable.

It has been shown that negative ion chemical ionization can increase the sensitivity of the mass spectrometric assay of the pineal hormone melatonin. However, it is an exacting assay requiring extensive sample preparation which precludes its use as a general research tool. We have investigated different derivatizing reagents and reaction conditions to demonstrate that a simple negative ion chemical ionization assay can be developed which will measure low picogram or even femtogram levels of the hormone in samples where the data have been ambiguous.