Nitro-fatty Acid Metabolome: Saturation, Desaturation, β-Oxidation, and Protein Adduction

Volker Rudolph,Francisco J Schopfer,Nicholas K.H Khoo,Tanja K Rudolph,Marsha P Cole,Steven R Woodcock,Gustavo Bonacci,Alison L Groeger,Franca Golin-Bisello,Chen-Shan Chen,Paul R.S Baker,Bruce A Freeman

doi:10.1074/jbc.m802298200

Abstract

Nitrated derivatives of fatty acids (NO2-FA) are pluripotent cell-signaling mediators that display anti-inflammatory properties. Current understanding of NO2-FA signal transduction lacks insight into how or if NO2-FA are modified or metabolized upon formation or administration in vivo. Here the disposition and metabolism of nitro-9-cis-octadecenoic (18:1-NO2) 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-NO2 or metabolites were detected under basal conditions, whereas administered 18:1-NO2 is rapidly adducted to plasma thiol-containing proteins and glutathione. NO2-FA are also metabolized via beta-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-NO2 as metabolites. Additionally, a significant proportion of 18:1-NO2 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-NO2 hydration or nitric oxide (*NO) release. Plasma samples from treated mice had significant extents of protein-adducted 18:1-NO2 detected by exchange to added beta-mercaptoethanol. This, coupled with the observation of 18:1-NO2 release from glutathione-18:1-NO2 adducts, supports that reversible and exchangeable NO2-FA-thiol adducts occur under biological conditions. After administration of [3H]18:1-NO2, 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 beta-oxidation reactions that terminate at the site of acyl-chain nitration.

Highlights

The reaction of unsaturated fatty acids with nitric oxide (1⁄7NO)- and nitrite (NO2Ϫ)-derived species, including nitrogen dioxide (1⁄7NO2), peroxynitrite (ONOOϪ), and nitrous acid (HNO2), yields a complex array of oxidized and nitrated products (1– 4)
More recently nitrated unsaturated fatty acids have been reported as products of acidic reactions of NO2Ϫ, radical chain termination reactions induced by 1⁄7NO (7–10), and the oxidation of NO2Ϫ to 1⁄7NO2 by the leukocyte-derived enzyme myeloperoxidase (1)
Various mechanisms can mediate the formation of nitroalkene derivatives of unsaturated fatty acids (11), including homolytic attack of 1⁄7NO2 (12), reaction of 1⁄7NO2 with a pre-existing fatty acid carbon-centered radical (2, 13), and the protonation of nitrite (NO2Ϫ) under acidic conditions to yield an array of HNO2-derived nitrating species (3, 14)

Summary

EXPERIMENTAL PROCEDURES

Materials—A synthesis producing equal yields of 9- and 10-nitro-9-cis-octadecenoic acid regioisomers (collectively termed 18:1-NO2) and [13C]18:1-NO2 was conducted as previously shown (1, 29). For quantification purposes [13C]18:1-NO2 and [13C]nitro-9-cis-12-cisoctadecadienoic acid ([13C]linoleic acid) were added as internal standards to samples obtained from animals treated with saline and [12C]18:1-NO2 prior to extraction with acetonitrile. Controls were performed using oleic acid (final concentration, 200 nM) or an equal volume of saline to separate plasma samples and incubated for 15 min at 37 °C. To assess the total amount of 18:1-NO2 (free and adducted to any plasma components) [13C]18:1-NO2 was added to serum samples as internal standard, and samples were treated with 500 mM ␤-mercaptoethanol (BME) in phosphate-buffered saline for 1 h at 37 °C. BME-ad- and the theoretical fragments corresponding to the difference ducted 18:1-NO2 was quantified after extraction with acetoni- from the mass of 17:0-CoA, which was determined as m/z trile by HPLC-ESI MS/MS as above.

RESULTS

Findings

DISCUSSION