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 + ; p K 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 t ris (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.