From Reduction to Oxidation: pH Controlled Reaction of 1 Hydroxyethyl Radical with Caffeic Acid Analogues.

Aims: The aim is to search for newer and better antioxidants through kinetic spectroscopic studies in combination with product analysis and computation. Background: Antioxidant effect of caffeic acid, its derivative, and analogues have been well reported. The antioxidative efficiencies are related to their molecular structure, and two reaction pathways are well accepted, H-atom transfer (HAT) or single electron transfer. 1-hydroxy ethyl radical (1-HER) being an ethanol-derived free radical might be causing the onset of liver injury detected after alcohol administration. 1-HER has also been reported to react with fatty acids and endogenous antioxidants such as glutathione, ascorbic acid, and alpha-tocopherol Objective: The present study is an attempt to understand the reaction mechanism of 1-HER with caffeic acid, its derivative, and analogues in detail. Method: Pulse radiolysis with kinetic absorption spectroscopy has been employed to follow the reaction pathway and identify the intermediates produced in the reaction. The reaction products have been detected using LCMS/MS. Based on these studies, a consolidated mechanism has been proposed. Cyclic voltammetry measurements and computational calculations have been used in support of the proposed mechanism. Result: In the reaction of 1-hydroxy ethyl radical (1-HER) with caffeic acid and its oligomers, reduction takes place below the pKa1, while oxidation occurs with the deprotonated phenolic moiety. The reduction of caffeic acid generates a carbon-centered radical at the double bond of the side chain with a bimolecular rate constant of 1.5x1010 dm3 mol-1 s-1. Notably, a low concentration of oxygen was able to regenerate a part of the caffeic acid molecules in the reduction process. At pH 10 a phenoxyl radical is formed due to oxidation with a much lower bimolecular rate constant (4.2x108 dm3 mol-1 s-1). In the case of di-hydrocaffeic acid, only phenoxyl radical is formed at pH 10 and, no reaction could be observed below pH 8. Conclusion: Change in reactive pattern from reduction to oxidation with change in pH within the same set of reactants has been evidently established in the present study. The results point towards the importance of  unsaturation in the side chain of caffeic acid oligomers for their reaction with 1-HER at neutral pH. The effect of oxygen concentration on the antioxidative protection offered by this class of molecules might be intriguing for the quest of the effectiveness of antioxidants at low concentrations. Other: It may be inferred that the effect of pH on the reactivity pattern as observed is not 1-HER, but substrate-specific, in the present case, phenolic acids. This study generates further scope for in-depth studies on other polyphenols where unsaturation exists in the side chain.

Myoglobin Is a Nitrite Reductase That Generates NO and Regulates Mitochondrial Respiration.

Previous studies have demonstrated that nitrite is an endocrine store of NO that may play an important role in hypoxic vasodilation. Nitrite can be converted to NO enzymatically by deoxyhemoglobin, in a reaction that is under allosteric control with a maximum reaction rate near the hemoglobin P50. In this study, we characterize the nitrite reductase activity of deoxymyoglobin, which reduces nitrite approximately 50-times faster than deoxyhemoglobin due to its lower redox potential. Spectrophotometric measurements show the deoxymyoglobin-nitrite reaction to be second order and linearly dependent on deoxymyoglobin, nitrite and proton concentration with a bimolecular rate constant of 11.7 M−1s−1 at pH 7.4 at 37 degrees Celsius. Using this bimolecular rate constant, we calculate that at physiological concentrations of nitrite (20uM) and myoglobin (25uM), NO will be generated at a rate of 2.85 nM/sec. Since the IC50 of inhibition of mitochondrial respiration is approximately 100nM at physiological oxygen tensions (5–10 μM), we hypothesized that the myoglobin-dependent reduction of nitrite could regulate mitochondrial respiration. Indeed, the addition of deoxymyoglobin in conjunction with nitrite to isolated rat liver mitochondria resulted in the inhibition of respiration, while myoglobin or nitrite alone had no effect. The addition of nitrite to rat heart homogenate, which contains both myoglobin and mitochondria, resulted in a measurable production of NO (with 1mM nitrite addition) and the inhibition of mitochondrial respiration (with 25μM nitrite addition) that was not significantly changed by the addition of the xanthine oxidase inhibitor allopurinol. These data corroborate previous studies demonstrating that NO generated from nitrite reduction can escape heme autocapture to mediate biological responses and confirm that the regulation of mitochondrial respiration by the nitrite-myoglobin reaction is relevant even in a physiological milieu. These data have implications for the modulation of hypoxic respiration, nitrite-dependent hypoxic signaling in tissue, and the regulation of oxygen gradients in the microcirculation.

Oxyhalogen-sulfur chemistry — Kinetics and mechanism of the oxidation of cysteamine by acidic iodate and iodine

The oxidation of cysteamine by iodate and aqueous iodine has been studied in neutral to mildly acidic conditions. The reaction is relatively slow and is heavily dependent on acid concentration. The reaction dynamics are complex and display clock behavior, transient iodine production, and even oligooscillatory production of iodine, depending upon initial conditions. The oxidation product was the cysteamine dimer (cystamine), with no further oxidation observed past this product. The stoichiometry of the reaction was deduced to be IO3– + 6H2NCH2CH2SH → I– + 3H2NCH2CH2S-SCH2CH2NH2 + 3H2O in excess cysteamine conditions, whereas in excess iodate the stoichiometry of the reaction is 2IO3– + 10H2NCH2CH2SH → I2 + 5H2NCH2CH2S-SCH2CH2NH2 + 6H2O. The stoichiometry of the oxidation of cysteamine by aqueous iodine was deduced to be I2 + 2H2NCH2CH2SH → 2I– + H2NCH2CH2S-SCH2CH2NH2 + 2H+. The bimolecular rate constant for the oxidation of cysteamine by iodine was experimentally evaluated as 2.7 (mol L–1)–1 s–1. The whole reaction scheme was satisfactorily modeled by a network of 14 elementary reactions.Key words: cysteamine, cystamine, Dushman reaction, oligooscillations.