Original contributionMelatonin directly scavenges hydrogen peroxide: a potentially new metabolic pathway of melatonin biotransformation
Introduction
Melatonin (N-acetyl-5-methoxytryptamine) is a highly conserved naturally occurring molecule. It is present in virtually all organisms tested, from bacteria [1], to protists [2], plants [3], and mammals [4]. In mammals, the pineal gland is believed to be the major source of circulating melatonin. The pineal gland is also responsible for the circadian melatonin rhythm, which leads to highest blood levels during the scotophase and baseline values during the photophase [4]. Recently, it was reported that a variety of other tissues including the retina [5], lens [6], Harderian gland [7], ovary [8], testes [9], and bone marrow [10], [11] may also synthesize melatonin, and in some tissues melatonin levels are much higher than would be expected based on its concentrations in the blood [10], [11], [12].
Melatonin plays several important physiological functions in mammals, such as reproductive regulation, immune enhancement, and regulation of dark-light signal transduction [13]. Melatonin is also known to be an endogenous free radical scavenger [14], [15] and a broad-spectrum antioxidant [16]. It detoxifies a variety of free radicals and reactive oxygen intermediates including the hydroxyl radical (HO), peroxynitrite anion, singlet oxygen, and nitric oxide [17]. Additionally, melatonin reportedly stimulates several antioxidative enzymes including glutathione peroxidase, glutathione reductase and superoxide dismutase [17]. Since melatonin is an evolutionarily highly conserved molecule and is ubiquitously present in organisms whose metabolism is based on oxygen, it has been speculated that a primary function of melatonin is to defend organisms from oxygen toxicity, a function acquired when melatonin evolved roughly 2.5 billion years ago [18].
H2O2 is a reactive oxygen intermediate which is formed when oxygen accepts two electrons. H2O2 is positioned at the center of the oxygen radical generating chain (Eqn. 1) and its metabolism determines the formation of highly toxic species. H2O2 per se is not toxic at physiological concentrations but, through the Fenton or Haber-Weiss reactions, H2O2 is converted to the highly cytotoxic HO. Once formed, HO attacks any macromolecule within its diffusion distance [19]. Thus, if organisms develop a strategy to scavenge H2O2 so as to avoid HO formation, it would be a highly effective means of defending themselves against oxidative insults. In this study, the ability of melatonin to scavenge H2O2 was investigated, and the metabolites which were produced during this process were isolated and identified.
Section snippets
Materials and methods
Highly pure melatonin was a gift from the Helsinn Chemical Co. (Biasca, Switzerland). All other reagents used and thin-layer chromatography plates were purchased from Sigma Chemicals (St. Louis, MO, USA).
Results
The H2O2 assay used in these studies yielded a linear response for H2O2 concentrations of 1–60 μM (Fig. 1); this is in agreement with previous observations [20]. In the control samples, neither melatonin nor ethanol reacted with either phenol red or HRP in the H2O2 analysis system. The results indicate that melatonin scavenged H2O2 in a concentration-dependent manner (Fig. 2). In the study of the time course of interaction between melatonin and H2O2, we observed that the incubation of H2O2
Discussion
In this investigation, we observed that melatonin scavenged H2O2 in the aqueous phase at pH 7.0 and formed a major metabolite AFMK. It is well known that a trace amount of free iron, if present, will catalyze H2O2 to generate HO. To rule out this possibility, a high concentration of desferoxamine was added to the reaction solution to chelate any free iron. This ensured that the reaction product, AFMK, was not the result of an interaction of melatonin with HO. In this study, AFMK was extracted
Acknowledgements
D.-X. Tan was supported by NIH training grant T32AG00165-13. J. Limson acknowledges the Foundation for Research and Development (South Africa) for postdoctoral support; the work was supported in part by a grant from Amoon Pharmaceutical Co., S.A.E.
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