Mass spectrometric analysis of human plasma and urine revealed abundant nitrated derivatives of all principal unsaturated fatty acids. Nitrated palmitoleic, oleic, linoleic, linolenic, arachidonic and eicosapentaenoic acids were detected in concert with their nitrohydroxy derivatives. Two nitroalkene derivatives of the most prevalent fatty acid, oleic acid, were synthesized (9-and 10-nitro-9-cis-octadecenoic acid; OA-NO 2 ), structurally characterized and determined to be identical to OA-NO 2 found in plasma, red cells, and urine of healthy humans. These regioisomers of OA-NO 2 were quantified in clinical samples using 13 C isotope dilution. Plasma free and esterified OA-NO 2 concentrations were 619 ؎ 52 and 302 ؎ 369 nM, respectively, and packed red blood cell free and esterified OA-NO 2 was 59 ؎ 11 and 155 ؎ 65 nM. The OA-NO 2 concentration of blood is ϳ50% greater than that of nitrated linoleic acid, with the combined free and esterified blood levels of these two fatty acid derivatives exceeding 1 M. OA-NO 2 is a potent ligand for peroxisome proliferator activated receptors at physiological concentrations. CV-1 cells co-transfected with the luciferase gene under peroxisome proliferator-activated receptor (PPAR) response element regulation, in concert with PPAR␥, PPAR␣, or PPAR␦ expression plasmids, showed dose-dependent activation of all PPARs by OA-NO 2 . PPAR␥ showed the greatest response, with significant activation at 100 nM, while PPAR␣ and PPAR␦ were activated at ϳ300 nM OA-NO 2 . OA-NO 2 also induced PPAR␥-dependent adipogenesis and deoxyglucose uptake in 3T3-L1 preadipocytes at a potency exceeding nitrolinoleic acid and rivaling synthetic thiazolidinediones. These data reveal that nitrated fatty acids comprise a class of nitric oxide-derived, receptor-dependent, cell signaling mediators that act within physiological concentration ranges.The oxidation of unsaturated fatty acids converts lipids, otherwise serving as cellular metabolic precursors and structural components, into potent signaling molecules including prostaglandins, leukotrienes, isoprostanes, and hydroxy-and hydroperoxyeicosatetraenoates. These enzymatic and auto-catalytic oxidation reactions yield products that orchestrate immune responses, neurotransmission, and the regulation of cell growth. For example, prostaglandins are cyclooxygenase-derived lipid mediators that induce receptor-dependent regulation of inflammatory responses, vascular function, initiation of parturition, cell survival, and angiogenesis (1). In contrast, the various isoprostane products of arachidonic acid auto-oxidation exert vasoconstrictive and pro-inflammatory signaling actions via receptor-dependent and -independent mechanisms (2). A common element of these diverse lipid signaling reactions is that nitric oxide ( ⅐ NO) 6 and other oxides of nitrogen significantly impact lipid mediator formation and bioactivities.The ability of ⅐ NO and ⅐ NO-derived species to oxidize, nitrosate, and nitrate biomolecules serves as the molecular basis for how ⅐ NO influences the sy...

Nitric oxide ( • NO) and its reactive metabolites mediate the oxidation, nitration, and nitrosation of DNA bases, amino acids, and lipids. Here, we report the structural characterization and quantitation of two allylic nitro derivatives of linoleic acid (LNO 2), present as both free and esterified species in human red cell membranes and plasma lipids. The LNO 2 isomers 10-nitro-9-cis,12-cis-octadecadienoic acid and 12-nitro-9-cis,12-cis-octadecadienoic acid were synthesized and compared with red cell and plasma LNO 2 species based on chromatographic elution and mass spectral properties. Collision-induced dissociation fragmentation patterns from synthetic LNO 2 isomers were identical to those of the two most prevalent LNO 2 positional isomers found in red cells and plasma. By using • NO exerts a particularly broad influence on oxidative inflammatory reactions by reacting at diffusion-limited rates with superoxide ( • O 2 Ϫ , k ϭ 1.9 ϫ 10 10 M Ϫ1 ⅐sec Ϫ1 ) to yield peroxynitrite (ONOO Ϫ ) and its conjugate acid, peroxynitritrous acid (ONOOH), the latter of which undergoes homolytic scission to nitrogen dioxide ( • NO 2 ) and hydroxyl radical ( • OH) (2, 6). Also, biological conditions favor the reaction of ONOO Ϫ with CO 2 , generating nitrosoperoxycarbonate (ONO-OCO 2 Ϫ ; k ϭ 3 ϫ 10 4 M Ϫ1 ⅐sec Ϫ1 ), which yields • NO 2 and carbonate ( • CO 3 Ϫ ) radicals by means of homolysis or rearranges to NO 3 Ϫ and CO 2 (7). During inflammation, neutrophil myeloperoxidase and heme proteins, such as myoglobin and cytochrome c, form H 2 O 2 -dependent Compound I intermediates that are direct oxidants and catalyze the consumption of • NO and the oxidation of nitrite (NO 2 Ϫ ) to • NO 2 (8-11). Although the rate of reaction of • NO with O 2 is slow (k ϭ 2 ϫ 10 6 M Ϫ2 ⅐sec Ϫ1 ), the small molecular radius, uncharged nature, and lipophilicity of • NO and O 2 facilitate their diffusion and concentration in membranes and lipoproteins up to 20-fold (12-14). This ''molecular lens'' effect that is induced by solvation in hydrophobic cell compartments accelerates the reaction of • NO with O 2 to yield N 2 O 3 and N 2 O 4 . As a consequence of this diversity of • NO reactions with partially reduced oxygen species, a rich spectrum of products is formed that orchestrates target molecule oxidation, nitrosation, and nitration reactions.Multiple mechanisms account for the nitration of fatty acids by• NO-derived species (15)(16)(17)(18)(19)(20). During both basal cell-signaling and tissue-inflammatory conditions, • NO 2 that is generated by the aforementioned reactions can react with membrane and lipoprotein lipids. Environmental sources also yield • NO 2 as a product of combustion.• NO 2 initiates autooxidation of polyunsaturated fatty acids by means of hydrogen abstraction from the bis-allylic carbon, to form nitrous acid and a resonance-stabilized allylic radical (21). This lipid radical species predominantly reacts with molecular oxygen to form a peroxyl radical. During the unique oxidationreduction conditions of inflammation or ischemia-...

Nitric oxide ( • NO) and • NO-derived reactive species rapidly react with lipids during both autocatalytic and enzymatic oxidation reactions to yield nitrated derivatives that serve as cell signaling molecules. Herein we report the synthesis, purification, characterization, and bioactivity of nitrolinoleate (LNO 2). Nitroselenylation of linoleic acid yielded LNO 2 that was purified by solvent extraction, silicic acid chromatography, and reverse-phase HPLC.