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Profiling and relative quantification of multiply nitrated and oxidized fatty acids


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.

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

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The authors are grateful to Ralf Hoffmann (Institute of Bioanalytical Chemistry, University of Leipzig) for providing access to his laboratories and instruments. Financial support from the European Regional Development Fund (European Union and the Free State of Saxony; 100146238 and 100121468 to M.F.) and a stipend to I.M. provided by Universität Leipzig are gratefully acknowledged.

Authors’ contributions

I.M. performed all experimental work, corresponding data evaluation, and contributed to the writing of the manuscript. E.G. and V.V. designed the cell model of nitrosative stress and performed cell culture experiments. N.I., H.N., and N.M. provided the peroxynitrite donor 2,3,5,6-tetramethyl-4-(methylnitrosoamino)phenol. J.M.G., C.O., and T.D. provided internal standards used for liquid chromatography–tandem mass pectrometry. M.F. conceived and designed all experiments and contributed to the writing of the manuscript.

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Correspondence to Maria Fedorova.

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Milic, I., Griesser, E., Vemula, V. et al. Profiling and relative quantification of multiply nitrated and oxidized fatty acids. Anal Bioanal Chem 407, 5587–5602 (2015).

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  • Cardiomyocytes
  • Lipid nitration
  • Lipid peroxidation
  • Nitrated fatty acids
  • Nitrosative stress