Digestive interaction between dietary nitrite and dairy products generates novel nitrated linolenic acid products

[Display omitted] •Gastric interaction of milk RA and RLA with nitrite leads to NO2-RA and NO2-RLA.•RA and RLA are the only substrates for nitration present in dairy fat.•NO2-RA, NO2-RLA and its metabolites are strongly correlated in plasma and urine.•Plasma levels of NO2-RLA reached up to 350 nM in plasma and 10 nM in urine.•Nitration of dairy fat in mice provides a mechanism for human urine NO2-RLA species. Nitrated fatty acids are important anti-inflammatory and protective lipids formed in the gastric compartment, with conjugated linoleic acid (rumenic acid, RA, 9Z,11E-18:2) being the primary substrate for lipid nitration. The recently reported identification of nitrated rumelenic acid (NO2-RLA) in human urine has led to hypothesize that rumelenic acid (RLA, 9Z,11E,15Z-18:3) from dairy fat is responsible for the formation of NO2-RLA. To evaluate the source and mechanism of NO2-RLA formation, 15N labeled standards of NO2-RLA were synthesized and characterized. Afterward, milk fat with different RA and RLA levels was administered to mice in the presence of nitrite, and the appearance of nitrated fatty acids in plasma and urine followed. We confirmed the formation of NO2-RLA and defined the main metabolites in plasma, urine, and tissues. In conclusion, RLA obtained from dairy products is the main substrate for forming this novel electrophilic lipid reported to be present in human urine.