Mass spectrometry-based study defines the human urine nitrolipidome

Nitrated fatty acids (NO2-FA) are an endogenous class of signaling mediators formed mainly during digestion and inflammation. The signaling actions of NO2-FA have been extensively studied, but their detection and characterization lagged. Several different nitrated fatty acid species have been reported in animals and humans, but their formation remains controversial, and a systemic approach to define the endogenous pool of NO2-FA is needed. Herein, we screened for endogenous NO2-FA in urine from healthy human volunteers as this is the main excretion route for NO2-FA and its metabolites, and it provides an excellent matrix for evaluation. Only isomers of two fatty acids, conjugated linoleic and linolenic acid were found to be nitrated. Several, previously unknown, nitrated species were identified and confirmed using high-resolution mass spectrometry, fragmentation analysis, and compared to synthetic nitrated standards, the main group corresponding to nitrated conjugated linolenic acid (NO2-CLnA). In contrast, we were unable to confirm the presence of previously reported nitrated omega-3's, oleic acid, arachidonic acid and alpha- and gamma-linolenic acid, suggesting that their biological formation and presence in humans should be re-evaluated. Metabolite analysis of NO2-CLnA in human urine identified cysteine adducts and beta-oxidation products, which were compared to the metabolic products of nitrated standards obtained using primary mouse hepatocytes. Importantly, NO2-CLnA isomers belong to two defined groups, are electrophilic, participate in Michael addition reactions and account for 39% of total urinary NO2-FA, highlighting their relative abundance and possible role in cell signaling.

Automated summary

Studies have found that isomers of two fatty acids nitrated were discovered in human urine, with the main group corresponding to nitrated conjugated linolenic acid (NO2-CLnA). Previously reported nitrated omega-3's, oleic acid, arachidonic acid and alpha- and gamma-linolenic acid were unable to be confirmed, suggesting the need for a re-evaluation of their biological formation and presence in humans. Metabolite analysis of NO2-CLnA in human urine revealed their relative abundance and possible role in cell signaling through electrophilic properties and Michael addition reactions.