Mitochondrial Nitration of Fatty Acids

GUSTAVO R BONACCI, FRANCISCO J SCHOPFER, JEFFREY KOENITZER, STEVEN WOODCOCK, VOLKER RUDOLPH, TANJA RUDOLPH, MARSHA COLE, AND BRUCE A FREEMAN

SAN FRANCISCO (CA)

Congreso; SFRBM's 16th Annual Meeting; 2009

Society for Free Radical Biology and Medicine

The levels of nitrated fatty acids (NO2-FA) are elevated in mitochondria during ischemia-reperfusion. Despite the multiple signaling pathways involving NO2-FA, the mechanism of formation still remains elusive. Several in vitro reactions using nonphysiological conditions demonstrated the relevance of radical reactions involving nitric oxide (NO) and nitrogen dioxide and acidic nitration. Nitrite (NO2−) is the main metabolic product of NO and an important mediator of S-nitrosylation. Herein, we describe that isolated mitochondria is the main source of nitrated oleic, linoleic and linolenic acids, a process characterized by its dependence on NO2− levels (0.01-10 mM) and pH values (6-7.4). Nitration was dependent on enzymatic activities as demonstrated by the inhibition elicited by azide, cyanide or heat treatments. The mitochondrial nitration resulted in the same NO2-FA found during cardiac ischemia-reperfusion. Incubation with 15NO2− resulted in 15N incorporation into mitochondrial NO2-FA. Exogenously added fatty acids did not impact the formation of nitrated species, suggesting that nitration of complex lipids and phospholipase activities may be involved. NO2-FA were detected as early as 5 min after NO2− addition, and increased over time. These products reacted with nucleophiles (e.g. GSH and β-mercaptoethanol) and were readily reduced to nitroalkanes by sodium borohydride. In addition, the formation of GSH adducts was detected in mitochondria. Moreover, the relative position of the nitroalkene moiety was defined using a modified, highly sensitive, MS-MS lithium adduct-based method. In summary, we demonstrated that mitochondria, via an enzymatic mechanism using NO2− as substrate, is a relevant source of NO2-FA at physiological pH values. These NO2-FA are electrophilic, react with GSH and protein targets, and directly impact and modulate mitochondrial homeostasis and respiration.