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On the Mechanism of the Ascorbic Acid-Induced Release of Nitric Oxide from N-Nitrosated Tryptophan Derivatives: Scavenging of NO by Ascorbyl Radicals

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Abstract

During the past years, there has been increasing interest in endogenous nitric oxide storage compounds. Recently, we briefly reported on the ascorbate-dependent release of nitric oxide (.NO) from N-nitrosotryptophan derivatives. In the present study, the underlying mechanism of .NO release is studied in more detail, primarily utilizing N-acetyl-N-nitrosotryptophan (NANT) as a model compound. The initial rate of the ascorbate-induced release of nitric oxide has been found to correspond to the rate of NANT decay. In this process, N-acetyltryptophan (NAT) is produced almost quantitatively. The final yield of nitrite amounted to around 90 % with respect to the applied amount of NANT. However, the total release of nitric oxide was only 60 %, as determined by using an FNOCT-4(fluorescent nitric oxide cheletropic trap number 4) assay. Besides nitric oxide, a second volatile product, dinitrogen oxide (N2O), has been identified by using 15N NMR spectrometry, strongly indicating the intermediacy of nitroxyl (HNO). The formation of intermediate ascorbyl radical anions during the NANT–ascorbate reaction has been monitored by using ESR spectrometry. Unexpectedly, it was found that the primary oxidized product of vitamin C, dehydroascorbic acid (DHA), efficiently consumes nitric oxide. Since ESR spectrometry further revealed that ascorbyl radical anions are also generated during the spontaneous decay of DHA, the DHA–nitric oxide reaction is related to recombination of .NO with the thus formed ascorbyl radical anions. A conclusively established mechanism of the NANT–ascorbate reaction is presented, with O-nitrosoascorbate as a key intermediate, as additionally supported by CBS-QB3 calculations. The present study suggests that vitamin C and its oxidation products can chemically counterbalance endogenous nitric oxide levels.

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