Characterization of phospholipid nitroxidation by LC-MS in biomimetic models and in H9c2 Myoblast using a lipidomic...

Under nitroxidative stress conditions, lipids are prone to be modified by reaction with reactive nitrogen species (RNS) and different modifications were reported to occur in fatty acids. However, in the case of phospholipids (PL) studied under nitroxidative stress conditions, only nitroalkene derivatives of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), were reported when using both in vitro biomimetic conditions and in vivo model system of type 1 diabetes mellitus. Therefore, in order to further explore other nitroxidative modifications of PL, a biomimetic model of nitroxidation combined with liquid chromatography mass spectrometry (MS) and MS/MS approaches were used to characterize the nitrated and nitroxidized derivatives of PCs and PEs. Single and multiple nitrated derivatives of phospholipids (PLs) such as nitroso and dinitroso, nitro, dinitro, and nitronitroso derivatives, together with nitroxidized derivatives were identified. Further, the specific MS/MS fragmentation pathways of these products were studied. Product ions arising from loss of HNO and HNO₂, from the combined loss of HNO (or HNO₂) and polar head groups, [NO_n-FA+O_n+H]⁺ and [NO_n-FA+O_n-H]^- (n=1-2) product ions corresponding to the modified fatty acyl chains were observed, depending on each modification. The knowledge obtained from the study of the MS/MS fragmentation pattern has allowed us to identify nitrated PCs, including NO₂-PC, (NO₂)₂-PCs, (NO₂)(NO)-PC, NO-PC; nitrated PEs, NO₂-PEs; and nitroxidized PCs, (NO₂)(2O)-PC in H9c2 cells under starvation, but not under ischemia or control conditions. The physiological relevance of this nitrated and nitroxidized PCs and PEs species observed exclusively in cardiomyoblast cells (H9c2) under starvation is still unknown but deserves to be explored.