GC-MS approach revealing hidden nitrite reactions in hemolysate using ¹⁸O-labelled nitrite and cysteine-based thiols

In erythrocytes, nitric oxide (NO) and its autoxidation product nitrite undergo multiple reactions with hemoglobin species to form nitrate, Fe-nitrosyl hemoglobin (HbFe^(II)NO), S-nitrosohemoglobin (HbCys^β93SNO), and other intermediates including nitrito-methemoblobin HbFe^(III)ONO, nitro-methemoblobin HbFe^(III)NO₂ and nitrous anhydride (N₂O₃). Here, we report a stable-isotope dilution GC-MS assay that allows studying reactions of nitrite in hemolysate. The method is based on the use of ¹⁸O-labelled nitrite in combination with l-cysteine or N-acetyl-l-cysteine ethyl ester and GC-MS measurement of unlabelled and labelled nitrite and nitrate species. This approach reveals reactions that are hidden at physiological nitrite concentrations.