title: Profiling and relative quantification of multiply nitrated and oxidized fatty acids.
abstract: The levels of nitro fatty acids (NO2-FA), such as nitroarachidonic, nitrolinoleic, nitrooleic, and dinitrooleic acids, are elevated under various inflammatory conditions, and this results in different anti-inflammatory effects. However, other multiply nitrated and nitro-oxidized FAs have not been studied so far. Owing to the low concentrations in vivo, NO2-FA analytics usually relies on targeted gas chromatography-tandem mass spectrometry (MS/MS) or liquid chromatography-MS/MS, and thus require standard compounds for method development. To overcome this limitation and increase the number and diversity of analytes, we performed in-depth mass spectrometry (MS) profiling of nitration products formed in vitro by incubating fatty acids with NO2BF4, and ONOO(-). The modified fatty acids were used to develop a highly specific and sensitive multiple reaction monitoring LC-MS method for relative quantification of 42 different nitrated and oxidized species representing three different groups: singly nitrated, multiply nitrated, and nitro-oxidized fatty acids. The method was validated in in vitro nitration kinetic studies and in a cellular model of nitrosative stress. NO2-FA were quantified in lipid extracts from 3-morpholinosydnonimine-treated rat primary cardiomyocytes after 15, 30, and 70 min from stress onset. The relatively high levels of dinitrooleic, nitroarachidonic, hydroxynitrodocosapenataenoic, nitrodocosahexaenoic, hydroxynitrodocosahexaenoic, and dinitrodocosahexaenoic acids confirm the presence of multiply nitrated and nitro-oxidized fatty acids in biological systems for the first time. Thus, in vitro nitration was successfully used to establish a targeted LC-MS/MS method that was applied to complex biological samples for quantifying diverse NO2-FA. Graphical Abstract Schematic representation of study design which combined in vitro nitration of different fatty acids, MS/MS characterization and optimization of MRM method for relative quantification, which was applied to follow dynamic of fatty acid nitration in cellular model of SIN-1 treated cardiomyoctes.
authors.affiliation: Faculty of Chemistry and Mineralogy, Institute of Bioanalytical Chemistry, Leipzig, Germany.
authors.forename: Ivana
authors.initials: I
authors.lastname: Milic
authors.forename: Eva
authors.initials: E
authors.lastname: Griesser
authors.forename: Venukumar
authors.initials: V
authors.lastname: Vemula
authors.forename: Naoya
authors.initials: N
authors.lastname: Ieda
authors.forename: Hidehiko
authors.initials: H
authors.lastname: Nakagawa
authors.forename: Naoki
authors.initials: N
authors.lastname: Miyata
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Galano
authors.forename: Camille
authors.initials: C
authors.lastname: Oger
authors.forename: Thierry
authors.initials: T
authors.lastname: Durand
authors.forename: Maria
authors.initials: M
authors.lastname: Fedorova
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D005227:Fatty Acids / Q000737:chemistry*; D008401:Gas Chromatography-Mass Spectrometry; D032383:Myocytes, Cardiac / Q000378:metabolism; D009566:Nitrates / Q000737:chemistry*; D015538:Nitrosation; D010084:Oxidation-Reduction; D051381:Rats; D053719:Tandem Mass Spectrometry
chemical_list: D005227:Fatty Acids; D009566:Nitrates