Summary Here we report that Cyclooxygenase-2 (COX-2) mediates the formation of electrophilic fatty acid oxo-derivatives (EFOXs) from the omega-3 fatty acids (ω-3 FA) docosahexaenoic, docosapentaenoic and docosatetraenoic acid. EFOXs produced by activated macrophages were discovered by a mass spectrometry-based “fishing” method, using the nucleophile β-mercaptoethanol (BME) as bait. Intracellular EFOX concentrations ranged from 65 to 350 nM, with acetylsalicylic acid (ASA, aspirin) increasing both rate of production and intracellular concentrations. Due to an electrophilic nature, EFOXs adducted protein cysteine and histidine residues and the cysteine of glutathione (GSH) in activated macrophages. A role for EFOXs as signalling mediators was confirmed by the observations that 17-EFOX-D6 and 17-EFOX-D5 are PPARγ agonists and activate Nrf2-dependent antioxidant responses. They also inhibited cytokine production and inducible nitric oxide synthase (NOS-2) expression in activated macrophages within a biological concentration range. Thus, EFOXs are signalling mediators that can transduce the clinically beneficial effects of ω-3 FA, COX-2 and ASA.

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...

The peroxisome proliferator-activated receptor-␥ (PPAR␥) binds diverse ligands to transcriptionally regulate metabolism and inflammation. Activators of PPAR␥ include lipids and antihyperglycemic drugs such as thiazolidinediones (TZDsThe rapidly expanding global burden of type II diabetes mellitus (DM) 3 and the concomitant increased risk for cardiovascular disease (1, 2) have motivated better understanding of relevant cell signaling pathways and potential therapeutic strategies. One major characteristic of metabolic syndrome and DM is insulin resistance, leading to hyperglycemia and dyslipidemia. Following initial clinical use of TZDs as anti-hyperglycemic agents to treat DM in the late 1990s, the nuclear receptor PPAR␥ was discovered as their molecular target. This receptor is expressed primarily in adipose tissue, muscle, and macrophages, where it regulates glucose uptake, lipid metabolism/ storage, and cell proliferation/differentiation (3-5). Thus, PPAR␥ ligands and allied downstream signaling events play a pivotal role in both the development and treatment of DM (6, 7). This is underscored by the observation that mutations in the C-terminal helix 12 of the ligand-binding domain (LBD) of PPAR␥ (e.g. P467L or V290M) are linked with severe insulin resistance and the onset of juvenile DM (8).The oxidizing inflammatory milieu contributing to the pathogenesis of obesity, diabetes, and cardiovascular disease also promotes diverse biomolecule oxidation, nitrosation, and nitration reactions by O 2 and ⅐ NO-derived species. Although oxidized fatty acids typically propagate proinflammatory conditions, the recently detected class of NO 2 -FA act as anti-inflammatory mediators. Nitroalkene derivatives of oleic acid (OA-NO 2 ) and linoleic acid (LNO 2 ) have been detected in healthy human blood and murine cardiac tissue. The levels of free/unesterified OA-NO 2 are ϳ1-3 nM in human plasma (9, 10), with OA-NO 2 produced at increased rates and present at higher concentrations during inflammatory and metabolic stress (11-13). The signaling actions of NO 2 -FA are primarily ascribed to the electrophilic olefinic carbon situated ␤ to the electron-withdrawing NO 2 substituent, facilitating kinetically rapid and reversible Michael addition with nucleophilic amino acids (i.e. Cys and His) (14). NO 2 -FA adduction of proteins and GSH occurs in model systems and clinically, with this reaction

Nitrated derivatives of fatty acids (NO 2 -FA) are pluripotent cell-signaling mediators that display anti-inflammatory properties. Current understanding of NO 2 -FA signal transduction lacks insight into how or if NO 2 -FA are modified or metabolized upon formation or administration in vivo. Here the disposition and metabolism of nitro-9-cis-octadecenoic (18:1-NO 2 ) 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-NO 2 or metabolites were detected under basal conditions, whereas administered 18:1-NO 2 is rapidly adducted to plasma thiol-containing proteins and glutathione. NO 2 -FA are also metabolized via ␤-oxidation, with high performance liquid chromatographytandem 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-NO 2 as metabolites. Additionally, a significant proportion of 18:1-NO 2 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-NO 2 hydration or nitric oxide ( ⅐ NO) release. Plasma samples from treated mice had significant extents of protein-adducted 18:1-NO 2 detected by exchange to added ␤-mercaptoethanol. This, coupled with the observation of 18:1-NO 2 release from glutathione-18:1-NO 2 adducts, supports that reversible and exchangeable NO 2 -FAthiol adducts occur under biological conditions. After administration of [ 3 H]18:1-NO 2 , 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 ␤-oxidation reactions that terminate at the site of acyl-chain nitration.

Nitroalkene fatty acid derivatives manifest a strong electrophilic nature, are clinically detectable and induce multiple transcriptionally-regulated anti-inflammatory responses. At present, the characterization and quantification of endogenous electrophilic lipids is compromised by their Michael addition with protein and small molecule nucleophilic targets. Herein, we report a trans-nitroalkylation reaction of nitro-fatty acids with β-mercaptoethanol (BME) and apply this reaction to the unbiased identification and quantification of reaction with nucleophilic targets. Trans-nitroalkylation yields are maximal at pH 7 to 8, and occur with physiological concentrations of target nucleophiles. This reaction also amenable to sensitive mass spectrometry-based quantification of electrophilic fatty acid-protein adducts upon electrophoretic resolution of proteins. In-gel trans-nitroalkylation reactions also permit the identification of protein targets without the bias and lack of sensitivity of current proteomic approaches. Using this approach, it was observed that fatty acid nitroalkenes are rapidly metabolized in vivo by a nitroalkene reductase activity and mitochondrial β-oxidation, yielding a variety of electrophilic and non-electrophilic products that could be structurally characterized upon BME-based transnitroalkylation reaction. This strategy was applied to the detection and quantification of fatty acid nitration in mitochondria in response to oxidative inflammatory conditions induced by myocardial ischemia-reoxygenation.