GC-MS Studies on Nitric Oxide Autoxidation and S-Nitrosothiol Hydrolysis to Nitrite in pH-Neutral Aqueous Buffers: Definite Results Using (15)N and (18)O Isotopes

Nitrite (O=N-O(−), NO(2)(−)) and nitrate (O=N(O)-O(−), NO(3)(−)) are ubiquitous in nature. In aerated aqueous solutions, nitrite is considered the major autoxidation product of nitric oxide ((●)NO). (●)NO is an environmental gas but is also endogenously produced from the amino acid L-arginine by the catalytic action of (●)NO synthases. It is considered that the autoxidation of (●)NO in aqueous solutions and in O(2)-containing gas phase proceeds via different neutral (e.g., O=N-O-N=O) and radical (e.g., ONOO(●)) intermediates. In aqueous buffers, endogenous S-nitrosothiols (thionitrites, RSNO) from thiols (RSH) such as L-cysteine (i.e., S-nitroso-L-cysteine, CysSNO) and cysteine-containing peptides such as glutathione (GSH) (i.e., S-nitrosoglutathione, GSNO) may be formed during the autoxidation of (●)NO in the presence of thiols and dioxygen (e.g., GSH + O=N-O-N=O → GSNO + O=N-O(−) + H(+); pK(a)(HONO), 3.24). The reaction products of thionitrites in aerated aqueous solutions may be different from those of (●)NO. This work describes in vitro GC-MS studies on the reactions of unlabeled ((14)NO(2)(−)) and labeled nitrite ((15)NO(2)(−)) and RSNO (RS(15)NO, RS(15)N(18)O) performed in pH-neutral aqueous buffers of phosphate or tris(hydroxyethylamine) prepared in unlabeled (H(2)(16)O) or labeled H(2)O (H(2)(18)O). Unlabeled and stable-isotope-labeled nitrite and nitrate species were measured by gas chromatography–mass spectrometry (GC-MS) after derivatization with pentafluorobenzyl bromide and negative-ion chemical ionization. The study provides strong indication for the formation of O=N-O-N=O as an intermediate of (●)NO autoxidation in pH-neutral aqueous buffers. In high molar excess, HgCl(2) accelerates and increases RSNO hydrolysis to nitrite, thereby incorporating (18)O from H(2)(18)O into the SNO group. In aqueous buffers prepared in H(2)(18)O, synthetic peroxynitrite (ONOO(−)) decomposes to nitrite without (18)O incorporation, indicating water-independent decomposition of peroxynitrite to nitrite. Use of RS(15)NO and H(2)(18)O in combination with GC-MS allows generation of definite results and elucidation of reaction mechanisms of oxidation of (●)NO and hydrolysis of RSNO.