Nitro-fatty acids are endogenous adaptive signaling mediators detected in animal and human urine, and plasma under basal conditions. Dietary supplementation with conjugated linoleic acid and nitrite/nitrate leads to gastric nitration and increased systemic and urinary levels. Moreover, in mice and rats, tissue levels are found to be elevated not only by gastric formation, but also by mitochondria driven in situ formation. Nitro fatty acids are pleiotropic signaling modulators that have demonstrated protective effects in a variety of pathological conditions including acute and chronic kidney disease, hypertension, heart and kidney ischemia reperfusion, sepsis, metabolic syndrome, diabetes and pulmonary hypertension amongst others. These modified fatty acids are electrophilic and exert their signaling actions by reacting with target proteins to activate Nrf2 and heat shock protein expression, inhibit NF-κB inflammatory signaling and associated fibrotic events. Despite the recent advances nitro fatty acid pharmacology, highly promising preclinical animal model data and undergoing clinical trials, our understanding of formation, absorption, distribution and protein targets is lagging behind. The main reasons for this slow pace were technical challenges associated with analyzing and characterizing biochemical aspects of this reactive species. Based on new evidence, we hypothesize that free NO2-CLA in plasma and tissues is a minor component of the total endogenous pool, represented by glycerol esterified and protein bound forms. These lipid and protein pools are dynamic, bioavailable and dictate transport, storage, signaling and metabolism. To demonstrate this hypothesis, the following Specific Aims are proposed: Aim #1 Define the gastric formation, intestinal absorption and lipoprotein-dependent systemic distribution of NO2-CLA. Aim #2 Evaluate the role of proteins on NO2-CLA transport, storage, plasma and tissue levels. Aim #3 Establish the cardiac tissue distribution of exogenous and endogenous NO2-CLA and its functional protein targets in a rodent model of heart ischemia reperfusion. Overall, this proposal will provide quantitative information on NO2-CLA formation, mechanism of absorption and transport, spatial distribution and protein targets. The findings will directly impact not only the ongoing clinical programs but also how we understand the pharmacology of dietary electrophilic fatty acids.