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 (HbCysβ93SNO), 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.