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(beta 93)SNO), and other intermediates including nitrito-methemoblobin HbFe((III))ONO, nitro-methemoblobin HbFe((III))NO(2) 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 O-18-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.

Automated summary

A new experimental method using stable-isotope dilution GC-MS assay was reported to study reactions of nitrite in hemolysate, revealing reactions that are hidden at physiological nitrite concentrations. This approach allows for the measurement of unlabelled and labelled nitrite and nitrate species in the reactions.