Fellowship for Petriello: Novel methodologies to quantify anti-inflammatory nitro-fatty acids

Unsaturated fatty acids and notably certain polyunsaturated fatty acids exhibit beneficial effects in clinical settings and experimental models of cardiovascular and metabolic disease, in part through mechanisms that involve attenuation of oxidative stress and inflammation. It is widely hypothesized that these effects involve enzymatic or non-enzymatic conversion of unsaturated fatty acids to biologically active metabolites. Inconsistencies and/or inter individual variation in formation of these active metabolites may account for variable observations that have plagued this field. Accordingly, definitive identification and quantitation of bioactive fatty acid derivatives is essential for both evaluating the beneficial effects of these nutrients and identifying biological markers to monitor the therapeutic efficacy of fatty acid administration. Nitro-fatty acids are a class of fatty acid metabolites that are formed by chemical nitration of the double bonds of unsaturated fatty acids. Several laboratories have shown that nitro-derivatives of some unsaturated fatty acids can decrease inflammation by down regulation of pro-inflammatory mediators such as NFêB and by activating the antioxidant master controller Nrf2. In mouse models, exogenously supplied nitro-fatty acids decrease systemic oxidative stress and inflammation suggesting that these could be biologically relevant mediators. However, the role of endogenously generated nitro-fatty acids as protective mediators of the actions of dietary unsaturated fatty acids is less clear, in large part due to inherent limitations of currently used analytical approaches for the detection and quantitation of free and esterified nitro-fatty acids. Similarly the enzymes and pathways involved in production of nitro-fatty acids in vivo are not well understood. For example, although it is reasonable to speculate that nitric oxide generating enzymes, such as endothelial nitric oxide synthase (eNOS), play a role in cellular nitro-fatty acid production, this has not yet been demonstrated. To address these issues we have developed sensitive, quantitative and specific methods for analysis of nitro-fatty acids using chemical derivatization and high resolution tandem mass spectrometry. With these methods in hand we are now in a position to investigate nitro-fatty acid metabolism in vivo and evaluate the relationship between eNOS activity, nitro fatty acid formation and vascular inflammation.