No abstract

We describe a nonextraction procedure, and two extraction procedures, for RIA of melatonin in human plasma. All procedures showed a diurnal rhythm of melatonin in human subjects, with interindividual differences greater than interprocedure differences. However, further investigations demonstrated considerable variability of recovery in the nonextraction procedure, suggesting a variability of binding proteins between samples. Combining recovery and dialysis experiments in the extraction procedures, we demonstrated that chloroform was unable to extract albumin-bound melatonin from a human serum albumin solution but, paradoxically, was able to extract bound and free melatonin from a plasma sample. The methanol extraction procedure extracted free and bound melatonin from all sources. These results indicate that albumin binding can substantially affect the RIA procedures. We conclude that assays should be validated against free and bound melatonin and that the two forms should be independently investigated when assessing bioactivity.

Three patients with idiopathic hypogonadotrophic hypogonadism and 2 with Kallmann's syndrome had variable degrees of GnRH deficiency. One patient with Kallmann's syndrome had apparently normal GnRH activity, which remained normal 5 months later. This patient appears to have the Bauman variant of Kallmann's syndrome.

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.

Elevated nocturnal melatonin is found in women with idiopathic hypogonadotropic hypogonadism (IHH), but it is not known whether this is implicated in the etiology of their GnRH deficiency. It is unlikely that nocturnal melatonin can be implicated in the etiology of the GnRH deficiency of Kallmann's syndrome (KS), because this condition is caused by defective neuronal migration in embryonic life. We therefore measured nocturnal melatonin in women with IHH and KS to determine whether it was elevated in one or both conditions and thereby to determine whether it was implicated as cause or consequence of GnRH deficiency. Four women with IHH, 3 women with KS, and 7 individually matched (age and body size) controls were recruited. Frequent day- and nighttime samples were taken for LH pulsatility studies. All patients showed absent or diminished LH pulsatility, compared with their respective controls. Samples were also taken over 24 h for melatonin and 6-sulphatoxymelatonin (the principle metabolite of melatonin and an independent marker of its secretion). Melatonin and 6-sulphatoxymelatonin levels were elevated in 6 of 7 patients (compared with their matched controls) and were significantly elevated in the KS group (compared with their controls). The finding of elevated nocturnal melatonin (and its metabolite) in GnRH-deficient women with KS (as well as IHH) suggests that nocturnal melatonin is elevated as a consequence of GnRH deficiency, irrespective of its etiology.