Melatonin directly scavenges hydrogen peroxide: a potentially new metabolic pathway of melatonin biotransformation

Abstract

A potential new metabolic pathway of melatonin biotransformation is described in this investigation. Melatonin was found to directly scavenge hydrogen peroxide (H₂O₂) to form N¹-acetyl-N²-formyl-5-methoxykynuramine and, thereafter this compound could be enzymatically converted to N¹-acetyl-5-methoxykynuramine by catalase. The structures of these kynuramines were identified using proton nuclear magnetic resonance, carbon nuclear magnetic resonance, and mass spectrometry. This is the first report to reveal a possible physiological association between melatonin, H₂O₂, catalase, and kynuramines. Melatonin scavenges H₂O₂ in a concentration-dependent manner. This reaction appears to exhibit two distinguishable phases. In the rapid reaction phase, the interaction between melatonin and H₂O₂ reaches equilibrium rapidly (within 5 s). The rate constant for this phase was calculated to be 2.3 × 10⁶ M⁻¹s⁻¹. Thereafter, the relative equilibrium of melatonin and H₂O₂ was sustained for roughly 1 h, at which time the content of H₂O₂ decreased gradually over a several hour period, identified as the slow reaction phase. These observations suggest that melatonin, a ubiquitously distributed small nonenzymatic molecule, might serve to directly detoxify H₂O₂ in living organisms. H₂O₂ and melatonin are present in all subcellular compartments; thus, presumably, one important function of melatonin may be complementary in function to catalase and glutathione peroxidase in keeping intracellular H₂O₂ concentrations at steady-state levels.

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.

$$\text{O}{}_2\text{−}\text{e}\text{O}{}_{\mathrm{<mtext>2</mtext>}}{}^{\mathrm{<mtext>•-</mtext>}}\text{−}\text{e}\text{H}{}_{\mathrm{<mtext>2</mtext>}}\text{O}{}_{\mathrm{<mtext>2</mtext>}}\text{−}\text{e}\text{HO}{}^{\mathrm{<mtext>•</mtext>}}\text{−}\text{e}\text{H}{}_{\mathrm{<mtext>2</mtext>}}\text{O}$$

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.