Nitro-fatty Acid Metabolome: Saturation, Desaturation, β-Oxidation, and Protein Adduction*

Nitrated derivatives of fatty acids (NO2-FA) are pluripotent cell-signaling mediators that display anti-inflammatory properties. Current understanding of NO2-FA signal transduction lacks insight into how or if NO2-FA are modified or metabolized upon formation or administration in vivo. Here the disposition and metabolism of nitro-9-cis-octadecenoic (18:1-NO2) 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-NO2 or metabolites were detected under basal conditions, whereas administered 18:1-NO2 is rapidly adducted to plasma thiol-containing proteins and glutathione. NO2-FA are also metabolized via β-oxidation, with high performance liquid chromatography-tandem 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-NO2 as metabolites. Additionally, a significant proportion of 18:1-NO2 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-NO2 hydration or nitric oxide (•NO) release. Plasma samples from treated mice had significant extents of protein-adducted 18:1-NO2 detected by exchange to added β-mercaptoethanol. This, coupled with the observation of 18:1-NO2 release from glutathione-18:1-NO2 adducts, supports that reversible and exchangeable NO2-FA-thiol adducts occur under biological conditions. After administration of [3H]18:1-NO2, 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 β-oxidation reactions that terminate at the site of acyl-chain nitration.

The reaction of unsaturated fatty acids with nitric oxide ( ⅐ NO)-and nitrite (NO 2 Ϫ )-derived species, including nitrogen dioxide ( ⅐ NO 2 ), peroxynitrite (ONOO Ϫ ), and nitrous acid (HNO 2 ), yields a complex array of oxidized and nitrated products (1)(2)(3)(4). The mechanisms of biological fatty acid nitration, the structural isomer distribution of nitrated fatty acids (NO 2 -FAs) 2 and the signaling actions of specific NO 2 -FA regioisomers remain incompletely characterized. Current data reveal that, during fatty acid oxidation and nitration, vinyl nitro regioisomers represent a component of these products that display distinctive chemical reactivities and receptor-dependent signaling actions. Here, we investigate the metabolic fate of the nitroalkene derivative of oleic acid (1,2).
Unsaturated fatty acid nitration was first described in model studies of air-pollutant-induced lipid oxidation where lipids were exposed to high concentrations of ⅐ NO 2 (5,6). More recently nitrated unsaturated fatty acids have been reported as products of acidic reactions of NO 2 Ϫ , radical chain termination reactions induced by ⅐ NO (7)(8)(9)(10), and the oxidation of NO 2 Ϫ to ⅐ NO 2 by the leukocyte-derived enzyme myeloperoxidase (1). Various mechanisms can mediate the formation of nitroalkene derivatives of unsaturated fatty acids (11), including homolytic attack of ⅐ NO 2 (12), reaction of ⅐ NO 2 with a pre-existing fatty acid carbon-centered radical (2,13), and the protonation of nitrite (NO 2 Ϫ ) under acidic conditions (pH 5.5 and lower) to yield an array of HNO 2 -derived nitrating species (3,14). The conditions promoting fatty acid nitration by ⅐ NO and NO 2 Ϫderived species (low oxygen tension, radical formation, and low pH) are not expected to be broadly distributed systemically (e.g. in plasma or extracellular fluids). Rather, nitration reactions will preferably occur during inflammatory or metabolic stress in microenvironments such as the intermembrane space of mitochondria, the low pH environment of the digestive tract, and activated macrophage and neutrophil-rich compartments. Moreover, the acidic, NO 2 Ϫ replete and low O 2 tension conditions that promote nitration reactions are characteristic of inflammatory loci. Although multiple reactions leading to accelerated formation of nitrating species occur at specific anatomic sites, plasma levels of nitrated fatty acids are expected to be low due to events described herein.
Robust electrophilic reactivity and avid nuclear lipid receptor ligand activity have conferred to the class of fatty acid nitroalkene derivatives potent anti-inflammatory properties that occur predominantly via non-cGMP-dependent mechanisms. Nitro derivatives of oleic and linoleic acid inhibit leukocyte and platelet activation (15), vascular smooth muscle proliferation (16), lipopolysaccharide-stimulated macrophage cytokine secretion (17), activate peroxisome proliferator-activated receptor-␥ (1,18), and induce endothelial heme oxygenase 1 expression (19). NO 2 -FA also potently modulate nuclear factor-erythroid 2-related factor 2/Kelch-like ECH-associating protein 1 (Nrf2/Keap1) (16,17) and nuclear factor B (NFB)regulated inflammatory signaling (17). Previous observations of the ⅐ NO-mediated, cGMP-dependent vessel relaxation induced by NO 2 -FA were made under serum-and lipid-free conditions. More recently, it has been appreciated that micellar and membrane stabilization of NO 2 -FA prevents Nef-like aqueous decay reactions and consequent ⅐ NO release, supporting that the predominant signaling actions mediated by NO 2 -FA are ⅐ NO and cGMP-independent (20,21).
Current data indicate that electrophilic adduction of biological targets primarily accounts for NO 2 -FA signal transduction. The high electronegativity of NO 2 substituents, when bound to an alkenyl carbon of fatty acids, confers an electrophilic nature to the adjacent ␤-carbon and enables Michael addition reaction with nucleophiles such as protein His and Cys residues. This process, termed nitroalkylation (22), results in the clinically detectable and reversible adduction of the nucleophilic thiol of glutathione (GSH) and both cysteine and histidine residues of glyceraldehyde-3-phosphate dehydrogenase (23). Furthermore, inhibition of NFB signaling occurs via nitroalkylation of p65 subunit thiols (17), and recent findings reveal that NO 2 -FA activation of peroxisome proliferator-activated receptor-␥ is uniquely induced by covalent nitroalkylation of the ligand binding domain Cys-285. 3 Multiple reports support the endogenous generation and presence of nitrated fatty acids (1,24), first observed in bovine papillary muscles as a vicinal nitrohydroxyeicosatetraenoic acid (25). Nitrolinoleate has been detected in human blood plasma and cholesteryl nitrolinoleate in human plasma and lipoproteins (4,26), with hyperlipidemic and post-prandial conditions elevating plasma levels of NO 2 -FA. Further support for the inflammatory generation of NO 2 -FA comes from lipopolysaccharide and interferon-␥-activated murine J774.1 macrophages, where increased nitration of the acyl chain of cholesteryl linoleate was paralleled by increased macrophage expression and activity of nitric-oxide synthase 2 (27).
To date, insight into the mechanisms of nitroalkene signaling actions overshadows knowledge of the generation, trafficking, and metabolism of nitroalkenes in vivo. Appreciating that NO 2 -FA derivatives are detectable clinically, and that their levels increase following ⅐ NO-dependent oxidative reactions (4,28), challenges still exist in their routine detection. Because the in vivo administration of NO 2 -FA may exert anti-inflammatory benefit, the disposition and metabolite profiles of these species in vivo is of relevance. Here we report that only 2.4% of nitrooctadecenoic acid (18:1-NO 2 ) is immediately detectable in the vascular compartment as native 18:1-NO 2 upon intravenous injection in mice, with the remaining pool of 18:1-NO 2 (a) reversibly bound to plasma and tissue thiols via Michael addition; (b) metabolized to nitro-octadecanoic acid (18:0-NO 2 ) and nitro-octadecadienoic acid (18:2-NO 2 ); and (c) catabolized by hepatic ␤-oxidation following thioester formation with coenzyme A.
Experimental Preparations-All animal studies were approved by the University of Pittsburgh Institutional Animal Care and Use Committee (Approval 0605735-A3). Male C57BL/6 mice, 8 -10 weeks of age (Jackson Laboratories, Bar Harbor, ME), were used for all described procedures. 18:1-NO 2 or [ 13 C]18:1-NO 2 were solvated in 30 l of 20% ethanol to obtain a final concentration of 10 mM for measurements involving free and plasma constituent-adducted 18:1-NO 2 and to a final concentration of 60 mM for measurement of hepatic NO 2 -FA CoA derivatives. Because of limited amounts of [ 13 C]18:1-NO 2 , this molecule was not utilized for hepatic metabolite studies. Injection solutions were prepared freshly for every animal and administered immediately via the tail vein. Injection of 30 l of vehicle was administered to control mice. Blood samples were collected from the saphenous vein prior to 18:1-NO 2 injection and then at 5, 15, 30, and 60 min post injection. Mice were anesthetized using intraperitoneal injection of Nembutal sodium solution (65 mg/kg, Ovation Pharmaceuticals, Deerfield, IL) after 90 min to obtain liver specimens and final blood samples by right ventricular cardiac puncture. Blood samples were transferred to heparinized tubes and stored on ice for further processing. Samples were then stored at Ϫ80°C until further analysis. Liver specimens were frozen in liquid nitrogen and stored at Ϫ80°C for further analysis.
Analysis of 18:1-NO 2 Metabolites-For lipid extraction, 40 l of cold (Ϫ20°C) acetonitrile were added to 10 l of whole blood. Samples were mixed well and centrifuged at 2500 rpm for 15 min at 4°C, and the supernatant was collected. For quantification purposes [ 13 C]18:1-NO 2 and [ 13 C]nitro-9-cis-12-cisoctadecadienoic acid ([ 13 C]linoleic acid) were added as internal standards to samples obtained from animals treated with saline and [ 12 C]18:1-NO 2 prior to extraction with acetonitrile. Qualitative and quantitative lipid analyses were conducted by using high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI MS/MS) using either a hybrid triple quadrupole mass spectrometer (API 4000) or a triple quadrupole mass spectrometer (API 5000, Applied Biosystems/MDS Sciex, Framingham, MA). NO 2 -FA molecular species were resolved by integrated reversed-phase HPLC (Shimadzu CBM20A, Japan) employing a 150-mm ϫ 2-mm C18 Luna column (particle size, 3 m, Phenomenex, Belmont, CA) at a flow rate of 0.25 ml/min using a gradient elution with 0.1% acetic acid as solvent A and 0.1% acetic acid in 100% acetonitrile as solvent B. Elution was carried out with the following gradient profile: 0 -3 min 3% of B, 3-6 min of 3-50% B, 6 -45 min 50 -99% of B, 45-53 min 99% of B, and 53.1-65 min 3% of B. Electrospray voltage was Ϫ4.5 kV, and the source temperature was set at 550°C. Mass spectrometric detection of NO 2 -FA was first performed using the precursor ion scan mode set to detect molecules that, upon collision-induced dissociation (CID), generate a fragment corresponding to NO 2 Ϫ (m/z 46). The precursor masses of molecules containing a nitro functional group were identified, and multiple reaction monitoring (MRM) transitions were used to detect and quantify NO 2 -FA molecular species using a collision energy of Ϫ32.0 eV. The mass transition of m/z 326/46 was used to detect 18:1-NO 2 with the appearance of 46 atomic mass units being consistent with the formation of NO 2 Ϫ . Mass transitions for ␤-oxidation metabolites of 18:1-NO 2 were calculated according to expected differences in mass, i.e. to account for each loss of an ethyl moiety (-CH 2 -CH 2 -) as to be expected in the course of ␤-oxidation a mass of 28 was subtracted for Q1 (e.g. 326 -28 ϭ 298 for nitro-7-cis-hexadecenoic acid), whereas Q3 remained unaltered (Table 1). Similarly, monitoring for 18:0-NO 2 and 18:2-NO 2 was performed allowing for the respective changes in masses (Table 1). Additionally, expected MRM transitions of nitrohydroxyl and conjugated ketone derivatives were employed. Structural confirmation of observed compounds was carried out by MS/MS analysis using the same HPLC settings described earlier. After confirmation of structure, quantification of biological samples was performed using a 20-ϫ 2-mm reversedphase column (Mercury MS Gemini 3 C18, 110 Å, Phenomenex, Torrance, CA) with a flow rate of 0.75 ml/min and a linear gradient of solvent B (11-99% in 3.5 min). For quantification of 18:1-NO 2 and 18:2-NO 2 , peak areas were assessed using Analyst 1.4.2 quantification software (Applied Biosystems/MDS Sciex, Thornhill, Ontario, Canada), and ratios of analytes to internal standard were calculated for determination of concentration. Peak areas for 18:0-NO 2 were determined as for 18:1-NO 2 . An external standard curve of nitro-octadecanoic acid was used to determine concentration. The same approaches for quantification were used to approximate concentrations of the metabolites of 18:1-NO 2 and 18:0-NO 2 . Because no standards were available for these metabolites standards for 18:1-NO 2 and 18:0-NO 2 , respectively, were used to correct for any losses and values reported as area ratio.
Metabolism of 18:1-NO 2 to 18:0-NO 2 Acid in Vitro-Peripheral human blood was collected by venipuncture into heparinized tubes with Institutional Review Board approval (number 0606145). Blood was centrifuged (2500 rpm, 4°C, 15 min) to obtain plasma. To assess the total amount of 18:1-NO 2 (free and adducted to any plasma components) [ 13 C]18:1-NO 2 was added to serum samples as internal standard, and samples were treated with 500 mM ␤-mercaptoethanol (BME) in phosphate-buffered saline for 1 h at 37°C. Under these conditions, nitroalkylated adducts undergo an exchange reaction where the nitroalkylated moiety transnitroalkylates with BME to form BME adducts (BME-18:1-NO 2 ), and the original protein amino acid moiety is restored to its reduced form. Samples were then analyzed by HPLC-ESI MS/MS using the same chromatographic gradient as for quantification of free NO 2 -FA. Detection of BME-adducted NO 2 -FA was performed in MRM scan mode using mass transitions of m/z x ϩ 78 to m/z x (where x ϭ the mass of the nascent NO 2 -FA and 78 is the atomic mass units of a neutral loss of BME).
For assessment of 18:1-NO 2 adducted to albumin, serum proteins were separated by gel electrophoresis (Criterion XT Precast Gel, Bio-Rad, Hercules, CA). After separation, bands of albumin were detected by Coomassie staining, excised, and cut in 1-mm 3 cubes in 400 l of phosphate buffer (50 mM, pH 7.4) containing [ 13 C]18:1-NO 2 as an internal standard. Subsequently, BME was added to a final concentration of 500 mM, and samples were incubated for 2 h to transnitroalkylate 18:1-NO 2 from albumin nucleophiles to BME. Finally, BME-adducted 18:1-NO 2 was quantified after extraction with acetonitrile by HPLC-ESI MS/MS as above. To estimate the concentration of 18:1-NO2-adducted to albumin a plasma albumin concentration of 30 mg/ml was assumed.
Synthetic GS-18:1-NO 2 was added to 2 ml of phosphate buffer (50 mM, pH 7.4) and incubated for 6 h at 37°C. Release of free, non-GSH-adducted 18:1-NO 2 was assessed after 0 min, 30 min, 1 h, 3 h, and 6 h. For this, 100 l were collected from the phosphate buffer solution, acidified to pH 4 using 10% formic acid and diluted with acetonitrile. GSH-adducted and free 18:1-NO 2 were measured with HPLC-ESI MS/MS as indicated above. To test for the characteristic electrophilic activity of reversibly released 18:1-NO 2 , samples were incubated with 500 mM BME for 30 min at 37°C. Subsequently, BME-adducted 18:1-NO 2 was assessed by HPLC-ESI MS/MS.
Analysis of CoA Derivatives of Nitro-fatty Acids-For measurement of NO 2 -FA metabolites, liver specimens dissected from anesthetized mice 90 min after injection of lipid derivatives were frozen with liquid nitrogen and stored at Ϫ80°C until further analysis. For lipid extraction, specimens were homogenized (sample weight between 620 and 710 mg), 1 ml of water containing 5 nM 17:0-CoA as an internal standard was added. Thereafter NO 2 -FA derivatives were extracted using 4 ml of cold acetonitrile, centrifuged at 2500 rpm for 15 min at 4°C, and supernatants were collected. HPLC-ESI MS analysis was performed as described previously for fatty acyl-CoA derivatives (30). Briefly, NO 2 -FA-CoA derivatives were resolved by HPLC (CBM20A, Shimadzu, Japan) with a 150-ϫ 2-mm C18 Luna column (particle size, 3 m; Phenomenex) at a flow rate of 0.25 ml/min. A linear gradient elution was carried out using 0.1% NH 4 OH (solvent A) and 0.1% NH 4 OH in acetonitrile (solvent B, 0 -48% of B) over 45 min. Mass spectrometric analysis was conducted in the positive ion mode using the MRM scan mode (30). Mass transitions for NO 2 -FA-CoA derivatives were calculated according to the expected masses for the different species and the theoretical fragments corresponding to the difference from the mass of 17:0-CoA, which was determined as m/z 1020.3/513.3 ( Table 1). The description of CoA derivatives was qualitative, because no internal standards were used.
Assessment of Tissue Distribution Using [ 3 H]18:1-NO 2 -30-l aliquots of 10 mM 18:1-NO 2 containing ϳ0.4 Ci of [ 3 H]18:1-NO 2 -18:1 in 20% ethanol were injected intravenously into the tail vein of C57BL/6 mice. After 90 min mice were anesthetized, and blood was taken by cardiac puncture to obtain serum. Specimens of liver, kidney, fat, muscle, spleen, and feces were weighed and homogenized in phosphate buffer (50 mM, pH 7.4). The tissue solubilizer Soluene 350 (PerkinElmer Life Sciences) was added to each homogenate, and the mixture was incubated 6 h at 50°C. After 6 h samples were cooled to room temperature, 200 l of 30% hydrogen peroxide was added in four aliquots, and the mixture was incubated for 30 min at 50°C. Then, 5 ml of scintillation fluid (Hionic-Fluor, PerkinElmer Life Sciences) was added to each vial. Samples were measured after 1 h of dark adaptation. This procedure was repeated three times for each tissue. For calculation of the percentage of recovered specific activity per organ, values were either normalized to the net weight of the organ or, in the case of fat and muscle total weight, were estimated according to expected normal values (1.25 g for fat, 10 g for muscle).

Detection and Identification of Free 18:1-NO 2 and Its
Metabolites-A complete, representative chromatogram showing the precursor ions of NO 2 Ϫ (46 atomic mass units) from a vehicle-treated and 18:1-NO 2 -treated animal is shown in Fig. 1, A and B. Assessment of blood samples in the MRM scan mode allowed identification of 18:1-NO 2 as well as all predicted metabolites of ␤-oxidation, i.e. nitro-7-cis-hexadecenoic acid (16:1-NO 2 ), nitro-5-cis-tetradecenoic acid (14:1-NO 2 ), and nitro-3-cis-dodecenoic acid (12:1-NO 2 ) when monitoring for the calculated mass transitions shown in Table 1. Products of ␤-oxidation with shorter fatty acid chain length were not  (Fig. 2A). Concurrent MRM scanning also revealed metabolites exhibiting a mass that was 2 atomic mass units greater than for each of the observed nitroalkenes, reflecting the reduction of the double bond. These metabolites were confirmed by MS/MS analysis as the nitroalkanes 18:0-NO 2 , nitro-hexadecanoic acid (16:0-NO 2 ), nitro-tetradecanoic acid (14:0-NO 2 ), and nitro-dodecanoic acid (12:0-NO 2 , Figs. 2B and 3). 18:0-NO 2 was further confirmed by comparison with the synthetic derivative. Additional metabolites were detected that displayed a mass 2 atomic mass units less than observed for the nitroalkene derivative, reflecting an additional monounsaturation step, and was confirmed as the corresponding nitroalkadienes by MS/MS analysis and comparison with the synthetic derivative (Figs. 2B and 3). Because desaturation of fatty acids in mammals occurs between the existing double bond and the C terminus, typically at positions 6 and 7 (31)(32)(33)(34), these metabolites were assumed to be nitro-6-cis-9-cisoctadecadienoic acid (18:2-NO 2 ) and the corresponding ␤-oxidation metabolites nitro-4-cis-7-cis-hexadecadienoic acid (16:2-NO 2 ) and nitro-2-cis-5-cis-tetradecadienoic acid   Table 1. Nitrohydroxyl or conjugated ketone derivatives were not detected in tissue compartments of interest. As expected, metabolites with shorter chain lengths displayed shorter retention times. Retention times for nitroalkanes were slightly increased compared with 18:1-NO 2 . As expected, because of the increased degree of unsaturation, nitroalkadienes eluted earlier than corresponding nitroalkenes.
Quantification of free 18:1-NO 2 and Its Metabolites-Concentrations of whole blood 18:1-NO 2 at different times after administration and its metabolite 18:0-NO 2 are shown in Fig. 4. The peak concentration of 18:1-NO 2 was 212 Ϯ 25 nM, occurring 5 min after injection. The peak concentration of 18:0-NO 2 was also observed 5 min after injection of 18:1-NO 2 (85 Ϯ 39 nM) attaining ϳ40% of 18:1-NO 2 concentration at this time point. Convergence of the concentration of both species was observed within the remaining 90 min. ␤-Oxidation metabolites of nitroalkanes displayed higher area ratios than nitroalkene metabolites, however these differences were not statistically significant. In contrast to 18:1-NO 2 and 18:0-NO 2 , which already peaked after 5 min, peak concentrations of ␤-oxidation metabolites occurred 60 min after injection. 18:2-NO 2 and its ␤-oxidation metabolites yielded considerably lower concentrations compared with 18:0 and 18:1 metabolites (peak concentration of 1.8 Ϯ 0.9 nM 5 min after injection, not shown).
Saturation of 18:1-NO 2 to 18:0-NO 2 -No saturation of 18:1-NO 2 to 18:0-NO 2 was induced by human plasma ex vivo (not shown). Incubation of bovine aortic endothelial cell with 18:1-NO 2 , however, yielded increasing levels of 18:0-NO 2 over 90 min, with the metabolite 18:0-NO 2 also accumulating in media within 15 min (Fig. 5, A and B). Lipid extracts of cells treated with 18:1-NO 2 revealed an isotope peak of 18:1-NO 2 in the MS  Venous blood of treated mice was extracted and prepared for mass spectrometric analysis as described under "Experimental Procedures." Concentrations of 18:1-NO 2 were calculated using [ 13 C] 18:1-NO 2 as internal standard, which was added during sample preparation to correct for any losses. 18:0-NO 2 was quantitated using an external standard curve of nitro-octadecanoic acid, which was linear over four orders of magnitude (0.08 -80.00 nM). Top left panel, a two-phase decline of the 18:1-NO 2 concentration with the first phase (5-15 min) predominantly reflecting distribution of the compound into extraplasmatic compartments and the second phase (15-90 min) predominantly reflecting elimination. 18:0-NO 2 concentration already after 5 min reaches 40% of the concentration of 18:1-NO 2 and converges with 18:1-NO 2 concentration after 60 min. Values given for ␤-oxidation metabolites were calculated in relation to the [ 13 C]18:1-NO 2 internal standard for the sake of comparability. Comparisons are based upon the assumption that fragmentation efficiencies are similar between metabolites. Values are therefore given as area ratio. Metabolites of nitro-octadecanoic acid exhibited higher values as the corresponding metabolites of 18:1-NO 2 . However, areas under the curve were not statistically different. transition for 18:0-NO 2 , which co-eluted with 18:1-NO 2 at 3.60 min, whereas the actual peak of 18:0-NO 2 eluted at 3.72 min (Fig. 5C). In control cells treated with HBSS or oleic acid no detectable 18:1-NO 2 or 18:0-NO 2 was observed. 18:0-NO 2 was not detected in media incubated for 90 min with 18:1-NO 2 .
The observation that 18:1-NO 2 is either reduced to 18:0-NO 2 or further desaturated to 18:2-NO 2 motivated the experiment illustrated in Fig. 6, which was performed to characterize the peaks typically eluting before and after 18:1-NO 2 when monitoring for the mass transition m/z 326/46. Incubation of a blood sample from an 18:1-NO 2 -treated animal with BME after lipid extraction revealed a lack of electrophilic reactivity of the compounds eluting before and after 18:1-NO 2 . Although the earlier eluting peak (peak 1 in Scheme 1) is most likely an undefined non-covalent adduct of 12:0-NO 2 , a possible explanation for the peak eluting later could be the presence of a nitroalkane configuration of an 18-carbon alkenyl derivative, which would result from desaturation and subsequent saturation of 18:1-NO 2 (peak 3 in Scheme 1). To test for electrophilic reactivity the extracted blood sample, which gave the HPLC elution profile illustrated in panel A, was treated with BME as demonstrated in B. As expected, the characteristic peak of 18:1-NO 2 disappeared in the mass transition m/z 326/46 (arrow) and a new peak eluted shortly before (*), whereas peaks 1 and 3 remain unaltered suggesting a lack of electrophilic reactivity. As demonstrated in C this new peak co-eluted with the BME-adducted 18:1-NO 2 (*), which can be explained by partial in-source fragmentation of the BME-adducts resulting in the release of the free fatty acid ion, which then is detectable in its actual mass transition m/z 326/46. D and E illustrate HPLC elution profiles of free and BME-adducted [ 13 C]18:1-NO 2 . Although peak 1 is most likely explained by an undefined noncovalent adduct of 12:0-NO 2 , we propose the presence of a "nitroalkane-alkene" as a result of saturation of the 9-cis-double bond with concomitant desaturation of the bond between carbons 6 and 7 (see Scheme 1) as explanation for peak 3. A, serum obtained 90 min after injection was used to assess adduction of nitro-9-cis-octadecenoic acid to plasma components. Samples were either treated directly with BME to acquire total 18:1-NO 2 , or only albumin was incubated with BME after protein separation by gel electrophoresis to obtain albumin-adducted 18:1-NO 2 , or analyzed without BME treatment to assess free 18:1-NO 2 . The bar graph demonstrates that only 5.7% of 18:1-NO 2 is present in its free form. B, the left-hand panel shows the elution profile of BME-18:1-NO 2 as assessed in MRM scan mode. The product ion scan of this moiety is displayed with the major fragments representing the parent ion  No detectable concentrations of BME-adducted 18:1-NO 2 could be obtained. B, after treatment of samples with BME, GSH-18:1-NO 2 and free 18:1-NO 2 were no longer detectable. Equal levels of BME-adducted 18:1-NO 2 for all time points suggest complete transfer of 18:1-NO 2 to BME. C, synthesized GSH-18:1-NO 2 spontaneously decomposes to GSH and 18:1-NO 2 demonstrating the reversibility of the electrophilic adduction of 18:1-NO 2 . In the presence of BME, free and GSH-adducted 18:1-NO 2 were adducted to this stronger nucleophile. SCHEME 1. Proposed mechanism for the generation of a "nitro-alkanealkene" from 18:1-NO 2 . The nitro-alkane-alkene can be formed either via the oxidation of 18:1-NO 2 to 18:2-NO 2 and the subsequent desaturation of the 9,10-double bond or via reduction of the 9,10-double bond of 18:1-NO 2 and subsequent oxidation of 18:0-NO 2 in the 6,7-position.

Determination of Electrophilic NO 2 -FA Adduction-
The total concentration of 18:1-NO 2 in serum 90 min after injection as assessed with BME pretreatment was 541.0 nM, whereas free 18:1-NO 2 had a concentration of 30.9 nM, which was consistent with the concentration of free 18:1-NO 2 measured in whole blood (Fig. 7, A and B). ␤-Oxidation metabolites of 18:1-NO 2 and 18:2-NO 2 were also found to be adducted to BME (data not shown). After separation of plasma proteins by gel electrophoresis, it was possible to quantify adduction of 18:1-NO 2 to albumin. The concentration of 18:1-NO 2 adducted to albumin was estimated to be 287.5 nM (Fig. 7A). Furthermore HPLC-ESI MS/MS allowed qualitative assessment of GSH-adducted 18:1-NO 2 (Fig. 7C). Incubation of previously synthesized GS-18:1-NO 2 in phosphate buffer revealed the reversibility of the covalent adduction of 18:1-NO 2 to GSH (Fig. 8, A and B).
Assessment of Tissue Distribution of [ 3 H]18:1-NO 2 -Ninety minutes after intravenous injection of 3 H-labeled 18:1-NO 2 , the greatest proportion of specific activity was recovered in muscle (30.6%) and liver (17.9%). In contrast, all other organs contained Ͻ5% of net administered 3 H-label. Plasma accounted for 0.5% of administered 3 H-labeled 18:1-NO 2 . Around 9% of specific activity was excreted within 90 min (5.6% in urine, 3.5% in feces, Fig. 11). Because only liver and plasma were investigated in detail, we do not exclude the potential formation of alternative metabolites in other tissue compartments. The use of [ 3 H]18: 1-NO 2 to assess extents of protein adduction was complicated by protein aggregation and quenching by reagents upon liquid scintillation counting.

DISCUSSION
Nitro-9-cis-octadecenoic acid undergoes multiple metabolic modifications and biochemical reactions after intravenous injection: (i) A significant amount of 18:1-NO 2 is saturated to FIGURE 9. CoA derivatives of 18:1-NO 2 and its metabolites in liver samples 90 min after intravenous injection. Liver samples of animals treated with vehicle or 18:1-NO 2 were frozen with liquid nitrogen and homogenized. CoA derivatives were extracted using acetonitrile. Analysis was performed by HPLC-ESI MS in the MRM scan mode using mass transitions according to the expected differences of compounds to heptadecanoic acid-CoA (Table 1). Monitoring was performed for 18:1-NO 2 -CoA, 18:0-NO 2 -CoA, 18:2-NO 2 -CoA, and their respective metabolites. CoA derivatives of all observed ␤-oxidation metabolites could be detected in all treated animals. No CoA derivatives of nitrated fatty acids were detected in control animals. Each HPLC elution profile is presented with base peak intensity and does not reflect quantity relative to the other profiles. Multiple peaks were recorded for mass transitions of some metabolites. Identification of the peak reflecting the CoA derivative was carried out using elution times and EPI analysis (see Fig. 10).
18:0-NO 2 5 min after injection. Modest extents of desaturation to 18:2-NO 2 were also observed, with plasma levels only 1% of 18:1-NO 2 . (ii) ␤-Oxidation metabolites of 18:1-NO 2 , 18:0-NO 2 , and 18:2-NO 2 along with respective CoA derivatives are formed, with metabolite ion intensities 80-to 130-fold lower than their respective 18-carbon parent molecule for the different time points. (iii) Over 90% of nitro-9-cis-octadecenoic acid in the circulation is not present in the free form, but rather is rapidly adducted to plasma macromolecules via Michael addition (see Scheme 2). This reaction is reversible, indicating these adducts serve as a reservoir of NO 2 -FA.
Blood levels of "free" 18:1-NO 2 decrease after intravenous injection via biphasic kinetics. The first phase, between 5 and 15 min, reflects the rapid distribution into extravascular compartments that is typical of lipophilic compounds. The second phase involves elimination of these compartments after saturation of extravascular compartment levels. A peak concentration of 18:1-NO 2 , 212 nM, was measured 5 min after injection and displayed a half-life of ϳ8 min.
Capture of plasma 18:1-NO 2 with BME permitted differentiation of free and adducted species, and revealed that only 6% of 18:1-NO 2 was in free form; with the majority adducted to plasma components. Accordingly, 18:1-NO 2 was adducted to albumin at an estimated concentration of 287.5 nM, corresponding to 53% of total 18:1-NO 2 . Of note, the electrophilic adduction of 18:1-NO 2 to protein thiols is reversible. In support of this, spontaneous release of free 18:1-NO 2 from previously synthesized GSH-adducted 18:1-NO 2 was observed, affirming the reversibility of nitroalkylation reactions. The transnitroalkylation of 18:1-NO 2 from albumin to BME also supports the reversibility of 18:1-NO 2 adduction to plasma proteins. Plasma protein-adducted 18:1-NO 2 thus represents a reservoir of NO 2 -FA that temporarily restrains electrophilic reactivity that can subsequently release NO 2 -FA when equilibria are shifted. More generally, because reversibility of signaling reactions is a prerequisite for signal transduction, this finding further corroborates the evolving role of electrophilic NO 2 -FA as signaling mediators and affirms previous reports regarding electrophilic adduction as a signaling mechanism (22,35,36). Robust evidence supports protein conjugation of NO 2 -FA in the cell, an event in part regulated by multidrug resistance protein-1-mediated efflux of penultimate GSH-NO 2 -FA adducts (23,37).
Conversion of 18:1-NO 2 to 18:0-NO 2 was detectable 5 min after intravenous injection of 18:1-NO 2 , reaching 40% of the concentration of the injected 18:1-NO 2 at this time. The extent of 18:1-NO 2 saturation in vivo and the observation that this reaction is mediated by bovine aortic endothelial cells in vitro,  Table 1. In the right column identifying EPI fragmentation patterns, which were used for characterization of the different metabolites, are illustrated. Relative intensities are displayed, which do not allow for quantity relative to the other profiles. but not human plasma, indicates an enzymatically catalyzed rather than spontaneous reaction. Because saturation of 18:1-NO 2 to 18:0-NO 2 leads to loss of electrophilic reactivity, this represents a mechanism for cellular inactivation of reactive electrophiles. Although enzymes competent to catalyze the saturation of nitroalkenes have been reported, including the flavin mononucleotide-containing NADPH oxido-reductase "old yellow enzyme" (38), these enzymes are only reported for yeast, plants, and bacteria (39). The identity of enzymes responsible for nitroalkene reduction in mammalian cells remains to be defined.
In comparison to nitroalkane formation, nitroalkadienes were generated to much lower extents after intravenous injection of 18:1-NO 2 . Because mammals typically desaturate fatty acids between the carboxyl group and an already existing olefin the additional desaturation of 18:1-NO 2 , is most likely inserted between carbons 6 and 7 (31)(32)(33)(34). Conversion of nitroalkenes to nitroalkadienes suggests the inclusion of nitroalkenes into synthetic pathways for polyunsaturated fatty acids. The presence of nitrated linolenic, arachidonic, and eicosapentaenoic acids, all of which are kinetically more likely to become nitrated than oleic acid, has been reported in vivo (1). The observation that 18:1-NO 2 acid undergoes saturation and desaturation also provides a possible explanation for the origin of the nonelectrophilic isobaric species that elutes after the 18:1-NO 2 peak when monitoring for the characteristic mass transition m/z 326/46 both in Fig. 6 and biological samples (not shown). This peak is commonly observed when treating rodents and cells with 18:1-NO 2 . Saturation of the double bond between carbons 9 and 10, along with desaturation at another location, e.g. between carbons 6 and 7, could result in a nitroalkene-alkane, (e.g. 9-nitro-6-cis-octadecenoic acid), that exhibits the same mass transition as 9-nitro-9-cis-octadecenoic acid but displays a different HPLC retention time and no electrophilic reactivity (Scheme 1).
Nitroalkenes, as well as nitroalkanes and nitroalkadienes, undergo ␤-oxidation. Thus, ␤-oxidation metabolites for all three species were detectable that displayed expected mass transitions and the decreasing retention times characteristic of smaller molecules. In the tissue compartments of interest, concentrations for these metabolites were 80-to 130-fold less than concentrations of the parent NO 2 -FA, based on the assumption that fragmentation efficiencies between metabolites are comparable. Collision-induced product fragmentation via MS/MS confirmed these metabolites. The detection and characterization of CoA derivatives of metabolites detected in liver samples of treated mice further support these findings. No metabolites with chain lengths shorter than 12 carbons were detected for free NO 2 -FA and their CoA derivatives. The metabolite of the two unsaturated species at this stage would be nitro-3-cis-dodecenoic acid. For this acid to be further oxidized by ␤-oxidation, a ⌬ 3 -cis-⌬ 2 -trans-enoyl-CoAisomerase must convert the 3-cis-double bond to a 2-transdouble bond. The presence of the nitro group that is located either on carbon 3 or 4, depending on whether it is a metabolite of the 9-or 10-NO 2 regioisomer of 18:1-NO 2 , prevents this enzymatic step and therefore any further ␤-oxidation. In the case of nitroalkanes, further oxidation of 12:0-NO 2 to nitrodecanoic acid was expected. As previously noted, nitroselenation-catalyzed synthesis of 18:1-NO 2 yields two regioisomers, 9-nitro-9-cis-octadecenoic acid and 10-nitro-9-cis-octadecenoic acid, which at this stage of metabolism would result in either 3-or 4-nitro-dodecanoic acid. The former metabolite is unlikely to be further ␤-oxidized, because the nitro-bonded carbon would be destined as the carboxylate carbon of the product. Because no nitrated fatty acid metabolite of chain length Ͻ12 carbons can be formed by ␤-oxidation, relatively greater levels of 12:1-NO 2 and 12:0-NO 2 are expected and were observed (Fig. 4).
The finding that NO 2 -FA undergo ␤-oxidation has multiple implications. First, as a consequence of shorter chain length, ␤-oxidation metabolites will be less hydrophobic. This will not only influence partitioning between hydrophobic and hydrophilic compartments and consequent anatomic distribution, but can also affect chemical reactivity and pharmacological profiles by altering accessibility to reaction targets. This concept is reminiscent of the differential regulation of myocyte and pancreatic ␤-cell ATP-sensitive K ϩ -channels by acyl-CoA esters depending on respective chain length (40 -42). Second, SCHEME 2. Overview of the possible metabolic modifications of 18:1-NO 2 and its disposition after intravenous injection in vivo. The assumed extracellular (large box), intracellular (large oval), and intramitochondrial (small oval) locations of distributional and metabolic steps of 18:1-NO 2 are illustrated. modified fatty acids undergo ␤-oxidation with kinetics that differ from the parent native fatty acid. For example, 5-hydroxydecanoate-CoA exhibits a 5-fold lower V max at the penultimate step of ␤-oxidation compared with the corresponding non-hydroxylated fatty acid, eventually resulting in inhibition of the ␤-oxidation of decanoyl-CoA (43). Whether NO 2 -FA acts in a similar fashion is of relevance to the response of tissues to ischemic insult and warrants further investigation. Finally, the fact that NO 2 -FA undergo ␤-oxidation upon formation of CoA thioester derivatives affirms that these species gains intramitochondrial access. Studies of HEK-293 cell mitochondrial fractions reveal that mitochondria contain a myriad of protein targets that selectively interact with thiol-reactive electrophiles (44). Esterification of 18:1-NO 2 to membrane and lipoprotein phospholipids is also a candidate metabolic disposition of 18:1-NO 2 in vivo, but was not addressed in herein.
The tissue distribution of specific activity after injection of [ 3 H]18:1-NO 2 showed liver having the greatest organ specific radioactivity, and the greatest percentage of administered radioactivity per whole organ was in muscle and liver. This indicates that 18:1-NO 2 traffics much like a native fatty acid in vivo. The observation of only 0.5% of administered radioactivity in the plasma compartment 90 min after administration agrees with independent mass spectrometry-based quantitation. Thus, intravenous administration of 300 nmol of [ 3 H]18:1-NO 2 gave a net concentration of 18:1-NO 2 , after BME "capture" of adducted species, of 541 nM 18:1-NO 2 in blood. Assuming a blood volume of 4 ml in mice, this is ϳ2.2 nmol or 0.7% of the administered amount. Quantitative limitations apply to the interpretation of these data, because the measured radioactivity reflects not only 18:1-NO 2 but also its metabolites.
No basal 18:1-NO 2 was detected in plasma and liver of the C57BL/6 cohort of mice used for the present metabolism study. We and others have detected nitro-oleate in rodents and humans in other instances, as well as metabolites reported herein. There are a number of mitigating factors in the detection and levels of fatty acid nitration products. For example, gastric acidification results in nitration of dietary fatty acids present in rodent chow, an event subject to dietary NO 2 Ϫ and unsaturated fatty acid levels. Also, plasma and organ levels of oleate and linoleate nitration products are affected by underlying inflammatory conditions (e.g. lipopolysaccharide treatment (27) and ischemic preconditioning). 3 Finally, the present study reveals that Michael addition reactions and metabolism (saturation, desaturation, and ␤-oxidation) affect detectable levels of "free" fatty acid nitration products. The initial report of ϳ500 nM nitro-oleate in human plasma (1) is now viewed to be higher than current measurements, with the original value complicated by non-covalent complexes of nitrite and oleate.
The metabolism of exogenously administered 18:1-NO 2 was evaluated to reveal the spectrum of reactions that endogenously produced nitro-fatty acid derivatives can undergo. Due to these rapid and diverse reactions, it is expected that specific organs, cells, and subcellular compartments responsible for fatty acid nitration will display levels higher than those detected in plasma. Thus, the reactions described herein are reflective of the trafficking and metabolic events expected for endogenous fatty acid nitration products. In summary, 18:1-NO 2 undergoes a rapid and substantial modification that affects subsequent chemical reactivity and signaling actions. Specifically, the reversible adduction of 18:1-NO 2 to biological nucleophiles and conversion to 18:0-NO 2 induces a rapid and transient neutralization of electrophilic reactivity. This adduction of nitroalkenes to nucleophilic targets thus both modifies the timing and sites of NO 2 -FA signaling and accounts for many of the anti-inflammatory and adaptive signaling actions of these species.