Nitro-fatty Acid Metabolome: Saturation, Desaturation, β-Oxidation, and Protein Adduction
2008; Elsevier BV; Volume: 284; Issue: 3 Linguagem: Inglês
10.1074/jbc.m802298200
ISSN1083-351X
AutoresVolker Rudolph, Francisco J. Schöpfer, Nicholas K.H. Khoo, Tanja K. Rudolph, Marsha P. Cole, Steven R. Woodcock, Gustavo Bonacci, Alison L. Groeger, Franca Golin-Bisello, Chen-Shan Chen, Paul R.S. Baker, Bruce Α. Freeman,
Tópico(s)Nitric Oxide and Endothelin Effects
ResumoNitrated 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. 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 (•NO)- and nitrite (NO2−) species, including nitrogen dioxide (•NO2), peroxynitrite (ONOO–), and nitrous acid (HNO2), yields a complex array of oxidized and nitrated products (1Baker P.R. Lin Y. Schopfer F.J. Woodcock S.R. Groeger A.L. Batthyany C. Sweeney S. Long M.H. Iles K.E. Baker L.M. Branchaud B.P. Chen Y.E. Freeman B.A. J. Biol. Chem. 2005; 280: 42464-42475Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 2O'Donnell V.B. Eiserich J.P. Chumley P.H. Jablonsky M.J. Krishna N.R. Kirk M. Barnes S. rley-Usmar V.M. Freeman B.A. Chem. Res. Toxicol. 1999; 12: 83-92Crossref PubMed Scopus (245) Google Scholar, 3Napolitano A. Camera E. Picardo M. d'Ischia M. J. Org. Chem. 2000; 65: 4853-4860Crossref PubMed Scopus (57) Google Scholar, 4Lima E.S. Di M.P. Abdalla D.S. J. Lipid Res. 2003; 44: 1660-1666Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The mechanisms of biological fatty acid nitration, the structural isomer distribution of nitrated fatty acids (NO2-FAs) 2The abbreviations used are: NO2-FA, nitrated fatty acid; HPLC, high-performance liquid chromatography; ESI-MS, electrospray ionization-mass spectrometry; MS/MS, tandem MS; CID, collision-induced dissociation; MRM, multiple reaction monitoring; HBSS, Hanks' balanced salt solution; BME, β-mercaptoethanol; EPI, enhanced product ion.2The abbreviations used are: NO2-FA, nitrated fatty acid; HPLC, high-performance liquid chromatography; ESI-MS, electrospray ionization-mass spectrometry; MS/MS, tandem MS; CID, collision-induced dissociation; MRM, multiple reaction monitoring; HBSS, Hanks' balanced salt solution; BME, β-mercaptoethanol; EPI, enhanced product ion. and the signaling actions of specific NO2-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 (1Baker P.R. Lin Y. Schopfer F.J. Woodcock S.R. Groeger A.L. Batthyany C. Sweeney S. Long M.H. Iles K.E. Baker L.M. Branchaud B.P. Chen Y.E. Freeman B.A. J. Biol. Chem. 2005; 280: 42464-42475Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 2O'Donnell V.B. Eiserich J.P. Chumley P.H. Jablonsky M.J. Krishna N.R. Kirk M. Barnes S. rley-Usmar V.M. Freeman B.A. Chem. Res. Toxicol. 1999; 12: 83-92Crossref PubMed Scopus (245) Google Scholar). Unsaturated fatty acid nitration was first described in model studies of air-pollutant-induced lipid oxidation where lipids were exposed to high concentrations of •NO2 (5Finlayson-Pitts B.J. Sweetman L.L. Weissbart B. Toxicol. Appl. 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Freeman B.A. Biochemistry. 1997; 36: 15216-15223Crossref PubMed Scopus (232) Google Scholar), and the oxidation of NO2− to •NO2 by the leukocyte-derived enzyme myeloperoxidase (1Baker P.R. Lin Y. Schopfer F.J. Woodcock S.R. Groeger A.L. Batthyany C. Sweeney S. Long M.H. Iles K.E. Baker L.M. Branchaud B.P. Chen Y.E. Freeman B.A. J. Biol. Chem. 2005; 280: 42464-42475Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Various mechanisms can mediate the formation of nitroalkene derivatives of unsaturated fatty acids (11Trostchansky A. Rubbo H. Free Radic. Biol. Med. 2008; 44: 1887-1896Crossref PubMed Scopus (102) Google Scholar), including homolytic attack of •NO2 (12Gallon A.A. Pryor W.A. Lipids. 1994; 29: 171-176Crossref PubMed Scopus (54) Google Scholar), reaction of •NO2 with a pre-existing fatty acid carbon-centered radical (2O'Donnell V.B. Eiserich J.P. Chumley P.H. Jablonsky M.J. Krishna N.R. Kirk M. Barnes S. rley-Usmar V.M. Freeman B.A. Chem. Res. Toxicol. 1999; 12: 83-92Crossref PubMed Scopus (245) Google Scholar, 13O'Donnell V.B. Freeman B.A. Circ. Res. 2001; 88: 12-21Crossref PubMed Scopus (229) Google Scholar), and the protonation of nitrite (NO2−) under acidic conditions (pH 5.5 and lower) to yield an array of HNO2-derived nitrating species (3Napolitano A. Camera E. Picardo M. d'Ischia M. J. Org. Chem. 2000; 65: 4853-4860Crossref PubMed Scopus (57) Google Scholar, 14Napolitano A. Camera E. Picardo M. d'Ishida M. J. Org. Chem. 2002; 67: 1125-1132Crossref PubMed Scopus (35) Google Scholar). The conditions promoting fatty acid nitration by •NO and NO2− 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, NO2− replete and low O2 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 (15Coles B. Bloodsworth A. Clark S.R. Lewis M.J. Cross A.R. Freeman B.A. O'Donnell V.B. Circ. Res. 2002; 91: 375-381Crossref PubMed Scopus (143) Google Scholar), vascular smooth muscle proliferation (16Villacorta L. Zhang J. Garcia-Barrio M.T. Chen X.L. Freeman B.A. Chen Y.E. Cui T. Am. J. Physiol. 2007; 293: H770-H776Crossref PubMed Scopus (123) Google Scholar), lipopolysaccharide-stimulated macrophage cytokine secretion (17Cui T. Schopfer F.J. Zhang J. Chen K. Ichikawa T. Baker P.R. Batthyany C. Chacko B.K. Feng X. Patel R.P. Agarwal A. Freeman B.A. Chen Y.E. J. Biol. Chem. 2006; 281: 35686-35698Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar), activate peroxisome proliferator-activated receptor-γ (1Baker P.R. Lin Y. Schopfer F.J. Woodcock S.R. Groeger A.L. Batthyany C. Sweeney S. Long M.H. Iles K.E. Baker L.M. Branchaud B.P. Chen Y.E. Freeman B.A. J. Biol. Chem. 2005; 280: 42464-42475Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 18Schopfer F.J. Lin Y. Baker P.R. Cui T. Garcia-Barrio M. Zhang J. Chen K. Chen Y.E. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2340-2345Crossref PubMed Scopus (365) Google Scholar), and induce endothelial heme oxygenase 1 expression (19Wright M.M. Schopfer F.J. Baker P.R. Vidyasagar V. Powell P. Chumley P. Iles K.E. Freeman B.A. Agarwal A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4299-4304Crossref PubMed Scopus (109) Google Scholar). NO2-FA also potently modulate nuclear factor-erythroid 2-related factor 2/Kelch-like ECH-associating protein 1 (Nrf2/Keap1) (16Villacorta L. Zhang J. Garcia-Barrio M.T. Chen X.L. Freeman B.A. Chen Y.E. Cui T. Am. J. Physiol. 2007; 293: H770-H776Crossref PubMed Scopus (123) Google Scholar, 17Cui T. Schopfer F.J. Zhang J. Chen K. Ichikawa T. Baker P.R. Batthyany C. Chacko B.K. Feng X. Patel R.P. Agarwal A. Freeman B.A. Chen Y.E. J. Biol. Chem. 2006; 281: 35686-35698Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar) and nuclear factor κB (NFκB)-regulated inflammatory signaling (17Cui T. Schopfer F.J. Zhang J. Chen K. Ichikawa T. Baker P.R. Batthyany C. Chacko B.K. Feng X. Patel R.P. Agarwal A. Freeman B.A. Chen Y.E. J. Biol. Chem. 2006; 281: 35686-35698Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Previous observations of the •NO-mediated, cGMP-dependent vessel relaxation induced by NO2-FA were made under serum- and lipid-free conditions. More recently, it has been appreciated that micellar and membrane stabilization of NO2-FA prevents Nef-like aqueous decay reactions and consequent •NO release, supporting that the predominant signaling actions mediated by NO2-FA are •NO and cGMP-independent (20Schopfer F.J. Baker P.R. Giles G. Chumley P. Batthyany C. Crawford J. Patel R.P. Hogg N. Branchaud B.P. Lancaster Jr., J.R. Freeman B.A. J. Biol. Chem. 2005; 280: 19289-19297Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 21Lima E.S. Bonini M.G. Augusto O. Barbeiro H.V. Souza H.P. Abdalla D.S. Free Radic. Biol. Med. 2005; 39: 532-539Crossref PubMed Scopus (113) Google Scholar). Current data indicate that electrophilic adduction of biological targets primarily accounts for NO2-FA signal transduction. The high electronegativity of NO2 substituents, when bound to an alkenyl carbon of fatty acids, confers an electrophilic nature to the adjacent β-carbon and enables Michael addition reaction with nucleophiles such as protein His and Cys residues. This process, termed nitroalkylation (22Baker L.M. Baker P.R. Golin-Bisello F. Schopfer F.J. Fink M. Woodcock S.R. Branchaud B.P. Radi R. Freeman B.A. J. Biol. Chem. 2007; 282: 31085-31093Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), 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 (23Batthyany C. Schopfer F.J. Baker P.R. Duran R. Baker L.M. Huang Y. Cervenansky C. Branchaud B.P. Freeman B.A. J. Biol. Chem. 2006; 281: 20450-20463Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Furthermore, inhibition of NFκB signaling occurs via nitroalkylation of p65 subunit thiols (17Cui T. Schopfer F.J. Zhang J. Chen K. Ichikawa T. Baker P.R. Batthyany C. Chacko B.K. Feng X. Patel R.P. Agarwal A. Freeman B.A. Chen Y.E. J. Biol. Chem. 2006; 281: 35686-35698Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar), and recent findings reveal that NO2-FA activation of peroxisome proliferator-activated receptor-γ is uniquely induced by covalent nitroalkylation of the ligand binding domain Cys-285. 3V. Rudolph, F. J. Schopfer, N. K. H. Khoo, T. K. Rudolph, M. P. Cole, S. Woodcock, G. Bonacci, A. Groeger, F. Golin-Bisello, C. S. Chen, P. R. S. Baker, and B. A. Freeman, unpublished observation.3V. Rudolph, F. J. Schopfer, N. K. H. Khoo, T. K. Rudolph, M. P. Cole, S. Woodcock, G. Bonacci, A. Groeger, F. Golin-Bisello, C. S. Chen, P. R. S. Baker, and B. A. Freeman, unpublished observation. Multiple reports support the endogenous generation and presence of nitrated fatty acids (1Baker P.R. Lin Y. Schopfer F.J. Woodcock S.R. Groeger A.L. Batthyany C. Sweeney S. Long M.H. Iles K.E. Baker L.M. Branchaud B.P. Chen Y.E. Freeman B.A. J. Biol. Chem. 2005; 280: 42464-42475Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 24Baker P.R. Schopfer F.J. Sweeney S. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11577-11582Crossref PubMed Scopus (178) Google Scholar), first observed in bovine papillary muscles as a vicinal nitrohydroxyeicosatetraenoic acid (25Balazy M. Iesaki T. Park J.L. Jiang H. Kaminski P.M. Wolin M.S. J. Pharmacol. Exp. Ther. 2001; 299: 611-619PubMed Google Scholar). Nitrolinoleate has been detected in human blood plasma and cholesteryl nitrolinoleate in human plasma and lipoproteins (4Lima E.S. Di M.P. Abdalla D.S. J. Lipid Res. 2003; 44: 1660-1666Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 26Lima E.S. Di M.P. Rubbo H. Abdalla D.S. Biochemistry. 2002; 41: 10717-10722Crossref PubMed Scopus (91) Google Scholar), with hyperlipidemic and post-prandial conditions elevating plasma levels of NO2-FA. Further support for the inflammatory generation of NO2-FA comes from lipopolysaccharide and interferon-γ-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 (27Ferreira A.M. Ferrari M. Trostchansky A. Batthyany C. Souza J.M. Alvarez M.N. Lopez G.V. Baker P.R. Schopfer F.J. O'Donnell V. Freeman B.A. Rubbo H. Biochem. J. 2008; 10.1042/BJ20080701Google Scholar). To date, insight into the mechanisms of nitroalkene signaling actions overshadows knowledge of the generation, trafficking, and metabolism of nitroalkenes in vivo. Appreciating that NO2-FA derivatives are detectable clinically, and that their levels increase following •NO-dependent oxidative reactions (4Lima E.S. Di M.P. Abdalla D.S. J. Lipid Res. 2003; 44: 1660-1666Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 28Freeman B.A. Baker P.R. Schopfer F.J. Woodcock S.R. Napolitano A. d'Ischia M. J. Biol. Chem. 2008; 283: 15515-15519Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar), challenges still exist in their routine detection. Because the in vivo administration of NO2-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-NO2) is immediately detectable in the vascular compartment as native 18:1-NO2 upon intravenous injection in mice, with the remaining pool of 18:1-NO2 (a) reversibly bound to plasma and tissue thiols via Michael addition; (b) metabolized to nitro-octadecanoic acid (18:0-NO2) and nitro-octadecadienoic acid (18:2-NO2); and (c) catabolized by hepatic β-oxidation following thioester formation with coenzyme A. Materials—A synthesis producing equal yields of 9- and 10-nitro-9-cis-octadecenoic acid regioisomers (collectively termed 18:1-NO2) and [13C]18:1-NO2 was conducted as previously shown (1Baker P.R. Lin Y. Schopfer F.J. Woodcock S.R. Groeger A.L. Batthyany C. Sweeney S. Long M.H. Iles K.E. Baker L.M. Branchaud B.P. Chen Y.E. Freeman B.A. J. Biol. Chem. 2005; 280: 42464-42475Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 29Lim D.G. Sweeney S. Bloodsworth A. White C.R. Chumley P.H. Krishna N.R. Schopfer F. O'Donnell V.B. Eiserich J.P. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15941-15946Crossref PubMed Scopus (104) Google Scholar). In some experiments, [3H]18:1-NO2 was utilized, prepared by a similar synthetic and purification strategy using 9,10-[3H]-cis-octadecenoic acid as the starting material. The 9- and 10-nitro regioisomers of octadecenoic acid were not differentiated for the present study and for shorter acyl chain length β-oxidation products, the NO2 position was assumed to remain on these carbons (e.g. becoming 3-nitro-3-cis-dodecenoic acid and 4-nitro-3-cis-dodecenoic acid). CoA-heptadecanoic acid (17:0-CoA) was from Sigma. Solvents used for extractions and mass spectrometric (MS) analysis were from Burdick and Jackson (Muskegon, MI). C57/Bl6 mice were from Jackson Laboratory (Bar Harbor, ME). Insulin syringes for tail vein injections were from BD Biosciences. 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-NO2 or [13C]18:1-NO2 were solvated in 30 μl of 20% ethanol to obtain a final concentration of 10 mm for measurements involving free and plasma constituent-adducted 18:1-NO2 and to a final concentration of 60 mm for measurement of hepatic NO2-FA CoA derivatives. Because of limited amounts of [13C]18:1-NO2, 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 μl of vehicle was administered to control mice. Blood samples were collected from the saphenous vein prior to 18:1-NO2 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 –80 °C until further analysis. Liver specimens were frozen in liquid nitrogen and stored at –80 °C for further analysis. Analysis of 18:1-NO2 Metabolites—For lipid extraction, 40 μl of cold (–20 °C) acetonitrile were added to 10 μl 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 [13C]18:1-NO2 and [13C]nitro-9-cis-12-cis-octadecadienoic acid ([13C]linoleic acid) were added as internal standards to samples obtained from animals treated with saline and [12C]18:1-NO2 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). NO2-FA molecular species were resolved by integrated reversed-phase HPLC (Shimadzu CBM20A, Japan) employing a 150-mm × 2-mm C18 Luna column (particle size, 3 μm, 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 –4.5 kV, and the source temperature was set at 550 °C. Mass spectrometric detection of NO2-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 NO2− (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 NO2-FA molecular species using a collision energy of –32.0 eV. The mass transition of m/z 326/46 was used to detect 18:1-NO2 with the appearance of 46 atomic mass units being consistent with the formation of NO2−. Mass transitions for β-oxidation metabolites of 18:1-NO2 were calculated according to expected differences in mass, i.e. to account for each loss of an ethyl moiety (-CH2-CH2-) as to be expected in the course of β-oxidation a mass of 28 was subtracted for Q1 (e.g. 326–28 = 298 for nitro-7-cis-hexadecenoic acid), whereas Q3 remained unaltered (Table 1). Similarly, monitoring for 18:0-NO2 and 18:2-NO2 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- × 2-mm reversed-phase column (Mercury MS Gemini 3μ 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-NO2 and 18:2-NO2, 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-NO2 were determined as for 18:1-NO2. 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-NO2 and 18:0-NO2. Because no standards were available for these metabolites standards for 18:1-NO2 and 18:0-NO2, respectively, were used to correct for any losses and values reported as area ratio.TABLE 1MRM transitions for free, adducted, and CoA-activated nitrated fatty acids Open table in a new tab Metabolism of 18:1-NO2 to 18:0-NO2 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. NO2-OA was added to a final concentration of 200 nm and incubated for 15 min at 37 °C. Controls were performed using oleic acid (final concentration, 200 nm) or an equal volume of saline to separate plasma samples and incubated for 15 min at 37 °C. Sample processing for HPLC-ESI MS/MS analysis was then performed as for whole blood. Conversion of 18:1-NO2 to 18:0-NO2 was assessed by scanning for the corresponding transitions using HPLC-ESI MS/MS in the MRM scanning mode. Separately, bovine aortic endothelial cells (passages 7–9) were grown to confluence on 6-well plates and incubated at 37 °C for 90 min with 0.15 m NaCl, oleic acid (5 μm, as control), 18:1-NO2 (5 μm), or [13C]18:1-NO2 (5 μm) in 3 ml of Hanks' buffered salt solution (HBSS). Cell medium (200 μl) was collected at baseline and after 5, 15, 30, and 90 min. After 90 min, cells were washed twice with HBSS and collected by scraping in 200 μl of HBSS. Media samples, cells, and a parallel HBSS solution, which was also incubated with lipids at 37 °C for 90 min, were all treated with acetonitrile as above to deproteinize and extract lipids. Further analysis was performed by HPLC-ESI MS/MS. Analysis of NO2-FA Adduction—Serum samples obtained 90 min after injection of 18:1-NO2 were used to investigate nitroalkylation of plasma components. Free 18:1-NO2 was measured as above using HPLC-ESI MS/MS after lipid extraction with acetonitrile. For evaluation of the presence of glutathione (GSH)-adducted 18:1-NO2 (GS-18:1-NO2) the same HPLC-ESI MS/MS approach was employed. The positive mass transition of m/z 633.3/306.3 was used in the MRM scan mode, where 306.3 is the mass of glutathione and 633.3 is the mass of the adduct of 18:1-NO2 to glutathione. To assess the total amount of 18:1-NO2 (free and adducted to any plasma components) [13C]18:1-NO2 was added to serum samples as internal standard, and samples were treated with 500 mm β-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-NO2), 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 NO2-FA. Detection of BME-adducted NO2-FA was performed in MRM scan mode using mass transitions of m/zx + 78 to m/zx (where x = the mass of the nascent NO2-FA and 78 is the atomic mass units of a neutral loss of BME). For assessment of 18:1-NO2 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-mm3 cubes in 400 μl of phosphate buffer (50 mm, pH 7.4) containing [13C]18:1-NO2 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-NO2 from albumin nucleophiles to BME. Finally, BME-adducted 18:1-NO2 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. To evaluate the reversibility of 18:1-NO2-nucleophile alkylation reactions further, glutathione-adducted 18:1-NO2 (GS-18: 1-NO2) was synthesized and purified. Glutathione (GSH) (300 mm) was solvated in 500 mm potassium phosphate buffer (final pH 7.4) and treated with 1.5 mm of 18:1-NO2 at 37 °C for 30 min. The reaction was stopped by acidification with formic acid at a final pH of 2.0. GS-18:1-NO2 was purified from residual GSH by reversed-phase chromatography. Samples were loaded onto PrepSep™ C18 columns (Fisher Scientific, Pittsburgh, PA) and e
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