Electrophilic fatty acid nitroalkenes are systemically transported and distributed upon esterification to complex...

Electrophilic nitro-fatty acids [NO₂-FAs (fatty acid nitroalkenes)] showed beneficial signaling actions in preclinical studies and safety in phase 1 clinical trials. A detailed description of the pharmacokinetics (PK) of NO₂-FAs is complicated by the capability of electrophilic fatty acids to alkylate thiols reversibly and become esterified in various complex lipids, and the instability of the nitroalkene moiety during enzymatic and base hydrolysis. Herein, we report the mechanism and kinetics of absorption, metabolism, and distribution of the endogenously detectable and prototypical NO₂-FA, 10-nitro-oleic acid (10-NO₂-OA), in dogs after oral administration. Supported by HPLC-high-resolution-MS/MS analysis of synthetic and plasma-derived 10-NO₂-OA-containing triacylglycerides (TAGs), we show that a key mechanism of NO₂-FA distribution is an initial esterification into complex lipids. Quantitative analysis of plasma free and esterified lipid fractions confirmed time-dependent preferential incorporation of 10-NO₂-OA into TAGs when compared with its principal metabolite, 10-nitro-stearic acid. Finally, new isomers of 10-NO₂-OA were identified in vivo, and their electrophilic reactivity and metabolism characterized. Overall, we reveal that NO₂-FAs display unique PK, with the principal mechanism of tissue distribution involving complex lipid esterification, which serves to shield the electrophilic character of this mediator from plasma and hepatic inactivation and thus permits efficient distribution to target organs.