Red cell membrane and plasma linoleic acid nitration products: Synthesis, clinical identification, and quantitation

Nitric oxide ((NO)-N-.) and its reactive metabolites mediate the oxidation, nitration, and nitrosation of DNA bases, amino acids, and lipids. Here, we report the structural characterization and quantitation of two allylic nitro derivatives of linoleic acid (LNO2), present as both free and esterified species in human red cell membranes and plasma lipids. The LNO2 isomers 10-nitro-9-cis,12-cis-octadecadienoic acid and 12-nitro-9-cis,12-cis-octadecadienoic acid were synthesized and compared with red cell and plasma LNO2 species based on chromatographic elution and mass spectral properties. Collision-induced dissociation fragmentation patterns from synthetic LNO2 isomers were identical to those of the two most prevalent LNO2 positional isomers found in red cells and plasma. By using [C-13]LNO2 as an internal standard, red cell free and esterified LNO2 content was 50 +/- 17 and 249 +/- 104 nM, respectively. The free and esterified LNO2 content of plasma was 79 +/- 35 and 550 +/- 275 nM, respectively. Nitrated fatty acids, thus, represent the single largest pool of bioactive oxides of nitrogen in the vasculature, with a net LNO2 concentration of 477 +/- 128 nM, excluding buffy coat cells. These observations affirm that basal oxidative and nitrating conditions occur in healthy humans to an extent that is sufficient to induce abundant membrane and lipoprotein-fatty acid nitration. Given that LNO2 is capable of mediating cGMP and non-cGMP-dependent signaling reactions, fatty acid nitration products are species representing the convergence of No-. and oxygenated lipid cell-signaling pathways.