Nitro-fatty acid metabolome: saturation, desaturation, β-oxidation, and protein adduction

Abstract

Nitrated derivatives of fatty acids (NO 2-FA) are pluripotent cell-signaling mediators that display anti-inflammatory properties. Current understanding of NO 2-FA signal transduction lacks insight into how or if NO 2-FA are modified or metabolized upon formation or administration in vivo. Here the disposition and metabolism of nitro-9-cis-octadecenoic (18:1-NO 2) acid was investigated in plasma and liver after intravenous injection in mice. High performance liquid chromatography-tandem mass spectrometry analysis showed that no 18:1-NO 2 or metabolites were detected under basal conditions, whereas administered 18:1-NO 2 is rapidly adducted to plasma thiol-containing proteins and glutathione. NO 2-FA are also metabolized via β-oxidation, with high performance liquid chromatography- tandem mass spectrometry analysis of liver lipid extracts of treated mice revealing nitro-7-cis-hexadecenoic acid, nitro-5- cis-tetradecenoic acid, and nitro-3-cis-dodecenoic acid and corresponding coenzyme A derivatives of 18:1-NO 2 as metabolites. Additionally, a significant proportion of 18:1-NO 2 and its metabolites are converted to nitroalkane derivatives by saturation of the double bond, and to a lesser extent are desaturated to diene derivatives. There was no evidence of the formation of nitrohydroxyl or conjugated ketone derivatives in organs of interest, metabolites expected upon 18:1-NO 2 hydration or nitric oxide ('NO) release. Plasma samples from treated mice had significant extents of protein-adducted 18:1-NO 2 detected by exchange to added β-mercaptoethanol. This, coupled with the observation of 18:1-NO 2 release from glutathione-18:1-NO 2 adducts, supports that reversible and exchangeable NO 2-FA- thiol adducts occur under biological conditions. After administration of [ 3H]18:1-NO 2,64% of net radiolabel was recovered 90 min later in plasma (0.2%), liver (18%), kidney (2%), adipose tissue (2%), muscle (31%), urine (6%), and other tissue compartments, and may include metabolites not yet identified. In aggregate, these findings show that electrophilic FA nitroalkene derivatives (a) acquire an extended half-life by undergoing reversible and exchangeable electrophilic reactions with nucleophilic targets and (b) are metabolized predominantly via saturation of the double bond and β-oxidation reactions that terminate at the site of acyl-chain nitration. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.