Abstract
The oxidation and nitration of unsaturated fatty acids transforms cell membrane and lipoprotein constituents into mediators that regulate signal transduction. The formation of 9-NO2-octadeca-9,11-dienoic acid and 12-NO2-octadeca-9,11-dienoic acid stems from peroxynitrite- and myeloperoxidase-derived nitrogen dioxide reactions as well as secondary to nitrite disproportionation under the acidic conditions of digestion. Broad anti-inflammatory and tissue-protective responses are mediated by nitro-fatty acids. It is now shown that electrophilic fatty acid nitroalkenes are present in the urine of healthy human volunteers (9.9 ± 4.0 pmol/mg creatinine); along with electrophilic 16- and 14-carbon nitroalkenyl β-oxidation metabolites. High resolution mass determinations and coelution with isotopically-labeled metabolites support renal excretion of cysteine-nitroalkene conjugates. These products of Michael addition are in equilibrium with the free nitroalkene pool in urine and are displaced by thiol reaction with mercury chloride. This reaction increases the level of free nitroalkene fraction >10-fold and displays a K(D) of 7.5 × 10(-6) M. In aggregate, the data indicates that formation of Michael adducts by electrophilic fatty acids is favored under biological conditions and that reversal of these addition reactions is critical for detecting both parent nitroalkenes and their metabolites. The measurement of this class of mediators can constitute a sensitive noninvasive index of metabolic and inflammatory status.
Highlights
The oxidation and nitration of unsaturated fatty acids transforms cell membrane and lipoprotein constituents into mediators that regulate signal transduction
This motivated the analysis of NO2-CLA regioisomers and their metabolites in human urine as a noninvasive approach for determining the endogenous production and reactions of these species
LC-ESI-MS/MS analysis of urine using multiple reaction monitoring (MRM) 324.2/46 (m/z corresponding to NO2-CLA and the formation of the NO2Ϫ anion upon collision-induced dissociation (CID)) presented two well-defined chromatographic peaks that coeluted with synthetic 9-15NO2-CLA and 12-15NO2CLA (Fig. 1A–C)
Summary
The oxidation and nitration of unsaturated fatty acids transforms cell membrane and lipoprotein constituents into mediators that regulate signal transduction. The detectable concentrations of fatty acid nitroalkenes and corresponding cysteine conjugates in human urine were strongly influenced by a chemical equilibrium induced by thiol availability and pH. This motivated the analysis of NO2-CLA regioisomers and their metabolites in human urine as a noninvasive approach for determining the endogenous production and reactions of these species.
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