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Melatonin (N-acetyl-5-hydroxytryptamine) is a pineal hormone widely known for its antioxidant properties, both in vivo and by direct capture of free radicals in vitro. Although some metabolites and oxidation products of melatonin have been identified, the molecular mechanism by which melatonin exerts its antioxidant properties has not been totally unravelled. This study investigated the reaction mechanism of oxidation of melatonin by radio-induced reactive oxygen species, generated by gamma radiolysis of water for aqueous solutions of melatonin (from 20 to 200 μm), in the presence or absence of molecular oxygen. The hydroxyl radical was found to be the unique species able to initiate the oxidation process, leading to three main products, e.g. N(1)-acetyl-N(2)-formyl-5-methoxykynurenin (AFMK), N(1)-acetyl-5-methoxykynurenin (AMK) and hydroxymelatonin (HO-MLT). The generation of AFMK and HO-MLT strongly depended on the presence of molecular oxygen in solution: AFMK was the major product in aerated solutions (84%), whereas HO-MLT was favoured in the absence of oxygen (86%). Concentrations of AMK remained quite low, and AMK was proposed to result from a chemical hydrolysis of AFMK in solution. A K-value of 1.1 × 10(-4) was calculated for this equilibrium. Both hydrogen peroxide and superoxide dismutase had no effect on the radio-induced oxidation of melatonin, in good accordance for the second case with the poor reactivity of the superoxide anion towards melatonin. Finally, a reaction mechanism was proposed for the oxidation of melatonin in vitro.

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The oxidation kinetics of glutathione (GSH) by hydrogen peroxide has been studied at neutral pH for different concentration ratios [GSH]0/[H2O2]0 between 0.2 and 2 (5 × 10−4 M ≤ [H2O2]0 ≤ 2.5 × 10−3M; 4 × 10−4 M ≤ [GSH]0 ≤ 2.5 × 10−3 M). In all cases studied, glutathione disulfide GSSG is the main product formed via two different oxidation ways, each of them contributing respectively to 80–85% and 10–15%.Our kinetic data indicate that an important fraction of hydrogen peroxide disappears without oxidizing the thiol function. This can be attributed to a combination between GSH and H2O2 protecting the sulfide group. Chemical evidences of the existence of a peroxide bond with GSSG are described. In our experimental conditions, the overall oxidation equation is 2mol GSH reacting with 2mol H2O2 giving 1mol GSSG. It is very different from the usually accepted stoichiometric reaction:[Formula: see text]A kinetic scheme is proposed and the corresponding rate constants are determined. Keywords: glutathione, hydrogen peroxide, kinetic mechanism, glutathione disulfide.