Melatonin Can Be Differentially Metabolized in the Rat to Produce N-Acetylserotonin in Addition to 6-Hydroxy-Melatonin

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Abstract
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. aMT has 2 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 TLC and gas chromatography-mass spectrometry (GCMS). TLC 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 derivatization, GCMS analysis of the urines, or of the spot at Rf 46%, showed 2 products, one of which had the same GC retention time and mass spectrum as 6-HaMT; 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 2 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 2 indoles are almost indistinguishable.