Nitrite and nitrate-dependent generation of anti-inflammatory fatty acid nitroalkenes

A gap in our understanding of the beneficial systemic responses to dietary constituents nitrate (NO₃⁻), nitrite (NO₂⁻) and conjugated linoleic acid (cLA) is the identification of the downstream metabolites that mediate their actions. To examine these reactions in a clinical context, investigational drug preparations of ¹⁵N-labeled NO₃⁻ and NO₂⁻ were orally administered to healthy humans with and without cLA. Mass spectrometry analysis of plasma and urine indicated that the nitrating species nitrogen dioxide was formed and reacted with the olefinic carbons of unsaturated fatty acids to yield the electrophilic fatty acid, nitro-cLA (NO₂-cLA). These species mediate the post-translational modification (PTM) of proteins via reversible Michael addition with nucleophilic amino acids. The PTM of critical target proteins by electrophilic lipids has been described as a sensing mechanism that regulates adaptive cellular responses, but little is known about the endogenous generation of fatty acid nitroalkenes and their metabolites. We report that healthy humans consuming ¹⁵N-labeled NO₃⁻ or NO₂⁻, with and without cLA supplementation, produce ¹⁵NO₂-cLA and corresponding metabolites that are detected in plasma and urine. These data support that the dietary constituents NO₃⁻, NO₂^- and cLA promote the further generation of secondary electrophilic lipid products that are absorbed into the circulation at concentrations sufficient to exert systemic effects before being catabolized or excreted.