Produção Nacional
Revisado por pares
Cholesteryl nitrolinoleate, a nitrated lipid present in human blood plasma and lipoproteins
2003; Elsevier BV; Volume: 44; Issue: 9 Linguagem: Inglês
10.1194/jlr.m200467-jlr200
ISSN1539-7262
AutoresÉmerson Silva Lima, Paolo Di Mascio, Dulcinéia Saes Parra Abdalla,
Tópico(s)Electron Spin Resonance Studies
ResumoNitric oxide (•NO) and •NO-derived reactive species (e.g., peroxynitrite anion, nitrogen dioxide radical) react with lipids containing unsaturated fatty acids to generate nitrated species. In the present work, we synthesized, characterized, and detected a nitrated derivative of cholesteryl linoleate (Ch18:2) in human blood plasma and lipoproteins using a high-pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry method. It was synthesized by a reaction of Ch18:2 with nitronium tetrafluoroborate, yielding a species with m/z 711, which is characteristic of the cholesteryl nitrolinoleate (Ch18:2NO2) ammonium adduct. The presence of the nitro group was confirmed by using [15N]nitrite, which gave a product with m/z 712, with the same chromatographic and spectrometric characteristics of those of m/z 711. Furthermore, a C-NO2 structure was also demonstrated in Ch18:2NO2 by infrared analysis (Vmax 1549, 1374 cm−1). A stable product with m/z of 711, showing the same chromatographic characteristics and fragmentation pattern as those of synthesized standard, was found in human blood plasma and lipoproteins of normolipidemic subjects.The presence of this novel nitrogen-containing lipid product in human plasma and lipoproteins could represent a potential indicator of the oxidative/nitrative roles that •NO or its metabolites play during in vivo lipid oxidation, generating a compensatory mechanism of protection in vascular disease. Nitric oxide (•NO) and •NO-derived reactive species (e.g., peroxynitrite anion, nitrogen dioxide radical) react with lipids containing unsaturated fatty acids to generate nitrated species. In the present work, we synthesized, characterized, and detected a nitrated derivative of cholesteryl linoleate (Ch18:2) in human blood plasma and lipoproteins using a high-pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry method. It was synthesized by a reaction of Ch18:2 with nitronium tetrafluoroborate, yielding a species with m/z 711, which is characteristic of the cholesteryl nitrolinoleate (Ch18:2NO2) ammonium adduct. The presence of the nitro group was confirmed by using [15N]nitrite, which gave a product with m/z 712, with the same chromatographic and spectrometric characteristics of those of m/z 711. Furthermore, a C-NO2 structure was also demonstrated in Ch18:2NO2 by infrared analysis (Vmax 1549, 1374 cm−1). A stable product with m/z of 711, showing the same chromatographic characteristics and fragmentation pattern as those of synthesized standard, was found in human blood plasma and lipoproteins of normolipidemic subjects. The presence of this novel nitrogen-containing lipid product in human plasma and lipoproteins could represent a potential indicator of the oxidative/nitrative roles that •NO or its metabolites play during in vivo lipid oxidation, generating a compensatory mechanism of protection in vascular disease. Nitric oxide (•NO) is an endogenously produced free radical that can modulate several relevant biological actions. •NO is the primary source of several substances with oxidant and nitrating activities [e.g., nitrogen dioxide radical (•NO2), peroxynitrite anion] (Reaction 1) that can yield nitrated and/or nitrosylated endogenous substances (1Eiserich J.P. Patel R.P. O'Donnell V.B. Pathophysiology of nitric oxide and related species: free radical reactions and modification of biomolecules.Molec. Aspects Med. 1998; 19: 221-357Crossref PubMed Scopus (190) Google Scholar, 2Wink D.A. Mitchell J.B. Chemical biology of nitric oxide: insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide.Free Radic. Biol. Med. 1998; 25: 434-456Crossref PubMed Scopus (1310) Google Scholar). Nitrated lipid formation was initially described in studies that aimed at modeling acute and chronic exposures of pulmonary lipids to NO2 in severely polluted urban atmospheres, providing evidence that at low ppm levels, NO2 reacts with unsaturated fatty acids, leading to nitrolipids formation (3Pryor W.A. Lightsey J.W. Mechanisms of nitrogen dioxide reactions: initiation of lipid peroxidation and the production of nitrous acid.Science. 1981; 214: 435-437Crossref PubMed Scopus (223) Google Scholar, 4Pryor W.A. Lightsey J.W. Church D.F. Reaction of nitrogen dioxide with alkene and polyunsaturated fatty acids: addition and hydrogen abstraction mechanism.J. Am. Chem. Soc. 1982; 104: 6685-6692Crossref Scopus (154) Google Scholar, 5Giamalva D.H. Kenion G.B. Church D.F. Pryor W.A. Rates and mechanisms of reaction of nitrogen dioxide with alkenes in solution.J. Am. Chem. Soc. 1987; 109: 7059-7063Crossref Scopus (87) Google Scholar). Nitrated lipid formation has also been shown in a variety of in vitro model systems, including unsaturated free fatty acids, phosphatidylcholine liposomes, LDL oxidized by copper, endothelial cells, and macrophages (6Rubbo H. Freeman B.A. Nitric oxide regulation of lipid peroxidation reactions: formation and analysis of nitrogen-containing oxidized lipid derivatives.Methods Enzymol. 1996; 269: 385-394Crossref PubMed Google Scholar, 7Rubbo H. Parthasarathy S. Barnes S. Kalyanaraman M.K.B. Freeman B.A. Nitric oxide inhibition of lipoxygenase-dependent liposome and low-density lipoprotein oxidation: termination of radical chain propagation reactions and formation of nitrogen-containing oxidized lipid derivatives.Arch. Biochem. Biophys. 1995; 324: 15-25Crossref PubMed Scopus (237) Google Scholar, 8Hayashi K. Noguchi N. Niki E. Action of nitric oxide as an antioxidant against oxidation of soybean phosphatidyl choline liposomal membranes.FEBS Lett. 1995; 370: 37-40Crossref PubMed Scopus (71) Google Scholar, 9Yates M.T. Lambert L.E. Whitten J.P. McDonald L. Mano M. Ku G. Mao S.J.T. A protective role for nitric oxide in the oxidative modification of low-density lipoproteins by mouse macrophages.FEBS Lett. 1992; 309: 135-138Crossref PubMed Scopus (105) Google Scholar). This formation can be primarily a consequence of •NO or its metabolites reacting with lipid-derived radicals (e.g., L•, LO•, LOO•) via diffusion-limited rates (109 to 1011 mol/l−1/s−1) (10Maricq M.J. Szente J.J. Kinetics of the reaction between ethyl peroxy radicals and nitric oxide.J. Phys. Chem. 1996; 96: 986-992Crossref Scopus (44) Google Scholar, 11Padmaja S. Huie R.E. The reaction of nitric oxide with peroxyl radicals.Biochem. Biophys. Res. Commun. 1993; 15: 539-544Crossref Scopus (275) Google Scholar) leading to the formation of nitrated products with structural characteristics of nitrolinoleate, nitritelinoleate (LONO), nitratelinoleate (LONO2), L(O)NO2, and nitrohydroxylinoleate (12Galon A.A. Pryor W.A. The identification of the allylic nitrite and nitro derivatives of the methyl linoleate and methyl linolenate by negative chemical ionization mass spectrometry.Lipids. 1993; 28: 125-133Crossref PubMed Scopus (43) Google Scholar, 13Galon A.A. Pryor W.A. The reaction of low levels of nitrogen dioxide with methyl linoleate in the presence and absence of oxygen.Lipids. 1994; 29: 171-176Crossref PubMed Scopus (54) Google Scholar, 14Rubbo H. Radi R. Trujillo M. Telleri R. Kalyanaraman B. Barnes S. Kirk M. Freeman B.A. Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation.J. Biol. Chem. 1994; 269: 26066-26075Abstract Full Text PDF PubMed Google Scholar, 15O'Donnell V.B. Eiserich J.P. Bloodsworth A. Chumley P.H. Kirk M. Barnes S. Darley-Usmar V.M. Freeman B.A. Nitration of unsaturated fatty acids by nitric oxide-derived reactive species.Methods Enzymol. 1999; 301: 455-471Google Scholar, 16O'Donnell V.B. Eiserich J.P. Chumley P.H. Jablonsky M.J. Kirk M. Barnes S. Darley-Usmar V.M. Freeman B.A. Nitration of unsatured fatty acids by nitric oxide-derived reactive nitrogen species peroxynitrite, nitrous acid, nitrogen dioxide and nitronium ion.Chem. Res. Toxicol. 1999; 12: 83-92Crossref PubMed Scopus (246) Google Scholar, 17Napolitano A. Camera E. Picardo M. D'Ischia M. Acid-promoted reactions of ethyl linoleate with nitrite ions: formation and structural characterization of isomeric nitroalkene, nitrohydroxy and novel 3-nitro-1,5-hexadiene and 1,5-dinitro-1, 3-pentadiene products.J. Org. Chem. 2000; 65: 4853-4860Crossref PubMed Scopus (57) Google Scholar, 18Napolitano A. Camera E. Picardo M. D'Ischia M. Reaction of hydro(pero)xy derivatives of polyunsaturated fatty acids/esters with nitrite ions under acidic conditions. Unusual nitrosative breakdown of methyl 13-hydro(pero)xyoactadeca-9,1-dienoate to a novel 4-nitro-2-oximinoalk-3-enal product.J. Org. Chem. 2002; 67: 1125-1132Crossref PubMed Scopus (35) Google Scholar). Nitrolipids can also be formed by a direct reaction of •NO2 with nonoxidized lipids at physiological pH (16O'Donnell V.B. Eiserich J.P. Chumley P.H. Jablonsky M.J. Kirk M. Barnes S. Darley-Usmar V.M. Freeman B.A. Nitration of unsatured fatty acids by nitric oxide-derived reactive nitrogen species peroxynitrite, nitrous acid, nitrogen dioxide and nitronium ion.Chem. Res. Toxicol. 1999; 12: 83-92Crossref PubMed Scopus (246) Google Scholar), or by a reaction of nitrous acid (HONO) (Reaction 2), a product yielded by nitrite acidification, with oxidized lipids (16O'Donnell V.B. Eiserich J.P. Chumley P.H. Jablonsky M.J. Kirk M. Barnes S. Darley-Usmar V.M. Freeman B.A. Nitration of unsatured fatty acids by nitric oxide-derived reactive nitrogen species peroxynitrite, nitrous acid, nitrogen dioxide and nitronium ion.Chem. Res. Toxicol. 1999; 12: 83-92Crossref PubMed Scopus (246) Google Scholar, 18Napolitano A. Camera E. Picardo M. D'Ischia M. Reaction of hydro(pero)xy derivatives of polyunsaturated fatty acids/esters with nitrite ions under acidic conditions. Unusual nitrosative breakdown of methyl 13-hydro(pero)xyoactadeca-9,1-dienoate to a novel 4-nitro-2-oximinoalk-3-enal product.J. Org. Chem. 2002; 67: 1125-1132Crossref PubMed Scopus (35) Google Scholar). At physiological pH, e.g., 7.4, NO2− is reasonably stable. However, in an acidic media, as in inflammatory states, gastric compartment, and neutrophil phagocytic vesicles, NO2− equilibrates with HONO (pKa = 3.25), which, in turn, is readily converted to a range of potent nitrosating/nitrating species, such as •NO2 ONOO− + H+ ← → ONOOH → •OH + •NO2 Reaction 1 NO2− + H → HONO Reaction 2 2 HONO → N2O3 + H2O Reaction 3 N2O3 → •NO + •NO2 Reaction 4 and N2O3, (Reactions 3, 4) capable of inducing modifications of polyunsaturated fatty acids (18Napolitano A. Camera E. Picardo M. D'Ischia M. Reaction of hydro(pero)xy derivatives of polyunsaturated fatty acids/esters with nitrite ions under acidic conditions. Unusual nitrosative breakdown of methyl 13-hydro(pero)xyoactadeca-9,1-dienoate to a novel 4-nitro-2-oximinoalk-3-enal product.J. Org. Chem. 2002; 67: 1125-1132Crossref PubMed Scopus (35) Google Scholar). Recent studies have demonstrated the presence of these products in biological samples. A vicinal nitrohydroxy arachidonate derivative was detected in bovine coronary arteries, which spontaneously releases •NO, causing relaxation in rat coronary aorta rings (19Balazy M. Iesaki T. Park J.L. Jiang H. Kaminski P.M. Wolin M. Vicinal nitrohydroeicosatrienoic acids: vasodilator lipids formed by reaction of nitrogen dioxide with arachidonic acid.J. Pharmacol. Exper. Ther. 2001; 299: 1-9PubMed Google Scholar). Nitrolinoleate and its nitrohydroxy derivative, nitrohydroxylinoleate, were detected in human blood plasma (20Lima E.S. Di Mascio P. Rubbo H. Abdalla D.S.P. Characterization of linoleic acid nitration in human blood plasma by mass spectrometry.Biochemistry. 2002; 41: 10717-10722Crossref PubMed Scopus (91) Google Scholar). Moreover, some studies have demonstrated biological activities of these nitrated products, such as endothelium independent vasorelaxation (19Balazy M. Iesaki T. Park J.L. Jiang H. Kaminski P.M. Wolin M. Vicinal nitrohydroeicosatrienoic acids: vasodilator lipids formed by reaction of nitrogen dioxide with arachidonic acid.J. Pharmacol. Exper. Ther. 2001; 299: 1-9PubMed Google Scholar, 21Lim 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. Nitrolinoleate, a nitric oxide-derived mediator of cell function: synthesis, characterization, and vasomotor activity.Proc. Natl. Acad. Sci. USA. 2002; 99: 15941-15946Crossref PubMed Scopus (105) Google Scholar), inhibition of platelet aggregation (22Coles B. Bloodsworth A. Eiserich J.P. Coffey M.J. McLoughlin R.M. Giddings J.C. Lewis M.J. Haslam R.J. Freeman B.A. O'Donnell V.B. Nitrolinoleate inhibits platelet activation by attenuating calcium mobilization and inducing phosphorylation of vasodilator-stimulated phosphoprotein (VASP) through elevation of cAMP.J. Biol. Chem. 2002; 277: 5832-5840Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), and antinflamatory actions, such as inhibition of superoxide generation, neutrophil degranulation, and integrin expression (23Coles B. Bloodsworth A. Clark S.R. Lewis M.J. Cross A.R. Freeman B.A. O'Donnell V.B. Nitrolinoleate inhibits superoxide generation, degranulation, and integrin expression by human neutrophils.Circ. Res. 2002; 91: 375-381Crossref PubMed Scopus (144) Google Scholar), suggesting that these nitrated lipids could have vascular-protective effects. In the present work, for the first time, is reported the presence of cholesteryl nitrolinoleate (Ch18:2NO2), a stable cholesteryl linoleate (Ch18:2)-derived nitrated product, in human blood plasma and lipoproteins, using high-pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS). The ex vivo detection of these products represents an important step in the understanding of the protective and/or deleterious effects of in vivo modified lipoproteins on blood vessels. Sodium [15N]nitrite and nitronium tetrafluoroborate were purchased from Aldrich Chemical Co. (Milwaukee, WI); [1α,2α(n)-3H] Ch18:2 was obtained from Amersham (Buckinghamshire, UK). tert-Butanol and chromatographic-grade methanol were obtained from Merck (Gibbstown, NJ). All other reagents were from Sigma Chemical Co. (St. Louis, MO). A solution of Ch18:2 (0.07 mmol) in chloroform (2 ml) was purged with nitrogen, and solid NO2BF4 was added (0.12 mmol). The mixture was kept under nitrogen atmosphere at room temperature for 1 h, and then 1 ml of 0.1 M phosphate buffer, pH 7.4 was added. The organic layer was separated, dried, and redissolved in 1 ml of 2-propanol. To isolate the nitrated lipids, the extract was passed through a 2.5 cm × 5.5 cm silica gel (200–400 mesh) column equilibrated with hexane. Nitrated lipids were separated from Ch18:2 with a hexane-diethyl ether step gradient (5% increments from 0 to 20% of diethyl ether). The content of nitrated lipid in the eluted fraction was monitored by thin-layer chromatography (TLC) using a mixture of methanol-chloroform (1:1; v/v) as solvent. Lipid fraction (1 μl) was applied to RP-18 F254s 5 × 10 cm TLC plates (Merck KgaA, Darmstadt, Germany), where the separated lipid components were detected with iodine. Fractions containing mainly nitrated lipids were separated, dried under a vacuum, redissolved in 2-propanol, maintained at −20°C, and analyzed by LC-ESI/MS/MS. Nitration was also done by adding sodium [15N]nitrite (0.1 mmol) to 0.10 mmol of Ch18:2 in 200 μl chloroform-methanol (2:1; v/v) following acidification to pH 3.0 with 1 N HCl and incubation at 25°C for 15 min under aerobic conditions. One milliliter of 0.1 M, pH 7.4, phosphate buffer was added, and extraction was done with 5 ml of diethyl ether. The organic layer was separated, dried, and maintained at −20°C until LC-ESI/MS/MS analysis. Mass spectrometry analysis was performed on a Quattro II triple quadrupole mass spectrometer (Micromass, Manchester, UK) following reversed-phase HPLC on a 20 × 4.0 mm id, 4 μ, Mercury MS column (Phenomenex, Torrance, CA) using an isocratic system with methanol-tert-butanol (3:1; v/v) containing 10 mM of ammonium acetate as mobile phase at a flow rate of 0.4 ml/min. The column eluent was totally inserted into the ion spray interface. Positive ion mass spectra were recorded with an orifice potential of 20 V. The source temperature was kept at 100°C. Daughter ion and multiple reaction monitoring (MRM) mass spectra were obtained with a collision energy of 10 eV or 20 eV, respectively, and gas (Ar) pressure at 6.0 ×10−4 mbar. The full scan was made at an interval between m/z 40 and 800. The nitrated product was detected in MRM mode as ammonium adduct [M+NH4]+, selecting the ions of m/z 711 in the first and 369 in the third quadrupole (m/z 711→369). The MRM transition for Ch18:2 and 3H-labeled cholesteryl linoleate (3H-Ch18:2) were also measured as m/z 666→369 and m/z 668→371, respectively. This detection mode was used to increase the specificity of the analysis. Quantitative yields of nitrated product (Ch18:2NO2) were calculated by elemental analysis of nitrogen content using a chemiluminescent nitrogen detector (Sievers® NOA 280, Boulder, CO) using sodium nitrite as standard. The data obtained for Ch18:2NO2 were used for its quantification in the LC-ESI/MS/MS system. A calibration curve was also performed with Ch18:2. The lipid residue obtained after treatment of Ch18:2 with NO2BF4 was used for infrared (IR) analysis. IR spectra were obtained with a Bomem MB 100 spectrometer by accumulating 32 scans between 400 and 4,000 cm−1. Human blood from four normolipidemic subjects (cholesterol <200 mg/dl) was collected after overnight fasting in tubes containing ethyldiaminetetraacetic acid (EDTA). Plasma was obtained after blood centrifugation at 2,500 rpm for 10 min at 4°C, and antiprotease inhibitors and antioxidants were immediately added to avoid lipoprotein degradation. Lipoproteins were separated from plasma by sequential ultracentrifugation using a Sorvall® Ultra Pro 80 (Sorvall Products L.P., Newtown, CT) and dialyzed against Tris buffer, pH 7.4, (150 mM NaCl, 1.0 mM EDTA, 3 mM NaN3, and 10 mM Tris) for 12 h. Lipid extraction from VLDL, LDL, and/or HDL was done by mixing 1 ml of each lipoprotein fraction with 500 μl of methanol, with vortex stirring for 30 s. Then 5 ml of hexane-diethyl ether (80:20; v/v) containing 0.02% butylated hydroxytoluene, previously treated with Chelex® to avoid further lipid oxidation during lipid extraction, was added. Samples were vortexed (2 min) and centrifuged at 2,500 rpm for 5 min at 4°C. The upper layer was collected, filtered (0.22 μ), and evaporated to dryness in a vacuum rotary evaporator. Lipids were dissolved in 100 μl of tert-butanol-methanol (1:1; v/v), and then 10 μl was immediately injected into a LC-ESI/MS/MS system using the protocol described for the standards. The same extraction system was used in the blood plasma analyses. To verify a possible artifactual nitration of Ch18:2 during the handling of the samples, the extraction procedure was also performed by the addition of an internal standard 3H-labeled Ch18:2 in plasma (100 pmol in chloroform), which can also be followed by LC-ESI/MS/MS. When Ch18:2 reacted with NO2BF4, the main reaction products, eluting at 5.9 min, were ions with m/z 711 [M+NH4]+ (Fig. 1A), characteristic of Ch18:2NO2 ammonium adduct. After fragmentation, a daughter main ion having m/z 369 was obtained, characteristic of the cholesteryl group in the parent molecule [(cholesterol)-OH]+. Ions of m/z 716, indicating a sodium adduct [M+Na]+ and of m/z 664, formed by the loss of −NO2 [M−(HNO2)+H]+, were also observed (Fig. 1B). The presence of a −NO2 group on the ion with m/z 711 was confirmed by synthesizing Ch18:2NO2 by reacting Na15NO2 with Ch18:2. This reaction yielded a molecular ion of m/z 712 that fragments given a main ion of m/z 369, which showed the same isotopic abundance pattern of those of m/z 711→369 ion (Fig. 2). The Ch18:2 used in the synthesis yielded ∼70% of Ch18:2NO2.Fig. 2Detection of Ch18:2NO2 ammonium adduct standard by LC-ESI/MS/MS analysis. MS/MS* spectra fragmentation patterns of products generated by reaction of cholesteryl linoleate (Ch18:2) with NO2BF4 (top) or sodium [15N]nitrite (lower), showing an enlargement of the [M+NH4]+ region (inside). * Selecting daughter ions mode for ions with m/z 711 or 712.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The IR spectrum of the compound with m/z 711 showed a characteristic absorption of a nitro group directly attached to the carbon chain (1,549 cm−1 and 1,374 cm−1, Fig. 3). No bands occurred in the 1,600–1,680 cm−1 region where the N = O binding of a LONO or LONO2 strongly absorbs. When human blood plasma and LDL samples were analyzed by the same LC-ESI/MS/MS method used for synthesized standards, a major product having an ion of m/z 711→369 was found. Moreover, the m/z 711 ion showed a fragmentation pattern and retention time identical to those of products synthesized by the reaction of Ch18:2 with NO2BF4 or NO2− in acid pH [Fig. 4A(trace a), B]. The product with m/z 711 that fragments in m/z 369 was detected in LDL (Fig. 5), as well as in VLDL and HDL samples (data not shown). The addition of [3H]-Ch18:2 to samples before the extraction procedure did not yield the [3H]-Ch18:2 nitrated product. This is indicated by the absence of a molecular ion of m/z 713 that fragments in a daughter ion of m/z 371 [Fig. 4A (trace b)].Fig. 5LDL samples analysis by LC-ESI/MS/MS. A: MRM chromatogram (711→369)*. B: MS/MS spectra fragmentation patterns of peak eluted at 5.9 min. * Selecting ion of m/z 711 in Q1 and m/z 369 in Q 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The Ch18:2 was also detected in plasma and lipoprotein samples in the same run of the nitrated product, eluting at ∼14.0 min. It appears as an ion with m/z 666, relative to ammonium adduct [M+NH4]+, that fragments given a main daughter ion of m/z 369, characteristic of the cholesteryl group (Fig. 6A, B). The [3H]-Ch18:2 added to plasma samples, which has the same retention time as Ch18:2 (Fig. 6C), appears as a molecular ion of m/z 668 [M+NH4]+, which fragments given a main ion of m/z 371 (Fig. 6D), characteristic of [3H]-labeled cholesterol [([3H]-cholesterol)-OH]+. The nitrated product represents ∼0.26% of the total of plasmatic Ch18:2, maintaining practically the same proportion in different lipoproteins (Fig. 7). The concentrations observed in plasma samples (mean ± SD, n = 4) were 76.9 ± 10.7 (nmol/l) and 30 ± 8.7 (μmol/l) for Ch18:2NO2 and Ch18:2, respectively.Fig. 7Presence of Ch18:2NO2 (m/z 711) in blood plasma and lipoproteins of normolipidemic subjects. The results (mean ± SD; n = 4) are expressed as the percent amount of nitrated and nonnitrated Ch18:2. The content of lipoprotein was corrected taking into consideration dilution during sample preparation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The reaction of Ch18:2 with NO2BF4 yielded a main product that exhibited a molecular ion with m/z 711, when analyzed by LC-ESI/MS/MS, characteristic of a nitrated Ch18:2 ammonium adduct. The MS/MS fragmentation spectra give a major peak with m/z 369, characteristic of the cholesteryl group [(cholesterol)-OH]+, as previously reported (24Lin Y.Y. Smith L.L. Recognition of functional groups by chemical ionization mass spectrometry.Biomed. Mass Spectrom. 1978; 11: 604-611Crossref Scopus (58) Google Scholar), indicating that nitration was occurring in the carbon chain instead of the cholesterol ring. The same product was obtained when Ch18:2 was treated with aqueous acidic sodium nitrite under aerobic conditions. The synthesis with NO2BF4 was chosen for IR and LC-ESI/MS/MS experiments due to a higher yielding of the nitrated Ch18:2 product when compared with the synthesis performed with nitrite. The nitration mechanisms for these synthesis procedures must be different (12Galon A.A. Pryor W.A. The identification of the allylic nitrite and nitro derivatives of the methyl linoleate and methyl linolenate by negative chemical ionization mass spectrometry.Lipids. 1993; 28: 125-133Crossref PubMed Scopus (43) Google Scholar, 13Galon A.A. Pryor W.A. The reaction of low levels of nitrogen dioxide with methyl linoleate in the presence and absence of oxygen.Lipids. 1994; 29: 171-176Crossref PubMed Scopus (54) Google Scholar). When NO2BF4 is used, the nitration occurs by nitronium ion (NO2+) addition in the double bond on the carbon chain (Fig. 8). In the reaction with sodium nitrite under acidic and aerobic conditions, the nitration occurs when •NO2 formed by nitrite acidification in the presence of oxygen abstracts an H atom from the allylic position of the fatty acid, forming a resonance-stabilized radical. This radical further reacts with another •NO2 molecule, forming a nitrolipid derivative (Fig. 8). In vivo, lipid nitration probably occurs by a radical-mediated mechanism in which •NO2 is generated by peroxidase-dependent oxidation of nitrite (25Carr A.C. McCall M.R. Frei B. Oxidation of LDL by mieloperoxidase and reactive nitrogen species: reaction pathways and antioxidant protection.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1716-1723Crossref PubMed Scopus (327) Google Scholar). The •NO2 can also be formed by other biological systems, such as in inflammatory states, gastric compartment, and neutrophil phagocytic vesicles (18Napolitano A. Camera E. Picardo M. D'Ischia M. Reaction of hydro(pero)xy derivatives of polyunsaturated fatty acids/esters with nitrite ions under acidic conditions. Unusual nitrosative breakdown of methyl 13-hydro(pero)xyoactadeca-9,1-dienoate to a novel 4-nitro-2-oximinoalk-3-enal product.J. Org. Chem. 2002; 67: 1125-1132Crossref PubMed Scopus (35) Google Scholar). The characterization of nitration by mass spectrometry was performed by reacting Ch18:2 with [15N]nitrite, in acidic media, obtaining a main ion with m/z 712, which showed the same fragmentation and isotopic abundance patterns of those of m/z 711 ions. The isotopic abundance pattern confirms that the spectra obtained are relative to compounds containing ∼50 carbons, such as Ch18:2 (26Havrilla C.M. Hachey D.L. Porter N.A. Coordination (Ag+) ion spray-mass spectrometry of peroxidation products of cholesterol linoleate and cholesterol arachidonate: high-performance liquid chromatography-mass spectrometry analysis of peroxide products from polyunsaturated lipid autoxidation.J. Am. Chem. Soc. 2000; 122: 8042-8055Crossref Scopus (88) Google Scholar). The increase of one mass unit using [15N]nitrite confirms that the ion contains one nitrogen atom per molecule and is compatible with the nitrated Ch18:2 structure. This evidence was reinforced by IR analyses of the nitrated product, as compared with Ch18:2, that showed novel bands at 1,549 and 1,373 cm−1, corresponding to the N = O binding of RNO2 (27Silverstein R.M. Bassler G.C. Morril T.C. Spectrometric Identification of Organic Compounds, 5th edition. John Wiley and Sons, New York1991: 65-74Google Scholar). These results clearly showed that the product formed in this synthesis is Ch18:2NO2. It was not possible to determine the position of the nitro group in the carbon chain of the fatty acid. It is possible that a mixture of stereo and positional isomers (i.e., at C9, C10, C12, or C13 and/or cis-trans isomers) is formed that is not possible to discriminate by mass spectrometry analysis. The analysis of blood plasma and lipoproteins showed an ion with m/z 666, characteristic of the Ch18:2 ammonium adduct, as previously described (28Hafidi M.E. Michel F. Bascoul J. Paulet A.C. Preparation of fatty acid cholesteryl ester hydroperoxides by photosensitized oxidation.Chem. Phys. Lipids. 1999; 100: 127-138Crossref Scopus (5) Google Scholar). Ch18:2 was chosen because it is the predominant lipid component in the LDL core (28Hafidi M.E. Michel F. Bascoul J. Paulet A.C. Preparation of fatty acid cholesteryl ester hydroperoxides by photosensitized oxidation.Chem. Phys. Lipids. 1999; 100: 127-138Crossref Scopus (5) Google Scholar) and linoleic acid (18:2) is the most abundant polyunsaturated fatty acid in human blood plasma (29Jira W. Spiteller G. Carson W. Schram A. Strong increase in hydroxy fatty acids derived from linoleic acid in human low density lipoproteins of atherosclerotic patients.Chem. Phys. Lipids. 1998; 91: 1-11Crossref PubMed Scopus (112) Google Scholar). As the presence of double-bonded carbons is an initial condition for •NO2 attack (18Napolitano A. Camera E. Picardo M. D'Ischia M. Reaction of hydro(pero)xy derivatives of polyunsaturated fatty acids/esters with nitrite ions under acidic conditions. Unusual nitrosative breakdown of methyl 13-hydro(pero)xyoactadeca-9,1-dienoate to a novel 4-nitro-2-oximinoalk-3-enal product.J. Org. Chem. 2002; 67: 1125-1132Crossref PubMed Scopus (35) Google Scholar), polyunsaturated fatty acids would be suitable substrates for •NO2-mediated nitration. Fragmentation of the ion with m/z 666 showed a main daughter ion with m/z 369, characteristic of the cholesteryl group, as previously described (28Hafidi M.E. Michel F. Bascoul J. Paulet A.C. Preparation of fatty acid cholesteryl ester hydroperoxides by photosensitized oxidation.Chem. Phys. Lipids. 1999; 100: 127-138Crossref Scopus (5) Google Scholar). Products given molecular ions with m/z 711, showing the same chromatographic characteristics and fragmentation pattern as those of synthesized standards, were found in human blood plasma and lipoproteins of normolipidemic donors. A possible artifactual nitration that could occur during the extraction procedure was tested by adding [3H]-labeled Ch18:2 to plasma samples. As the corresponding nitrated [3H]-Ch18:2 was not detected, an artifactual nitration during sample workup was excluded. The possibility that azide contributes to nitration was also excluded, because azide was used only for isolation of lipoproteins and not for plasma samples. Small differences in MS/MS fragmentation spectra patterns between synthesized standards and products of plasma or lipoproteins could be explained by the presence of different positional isomers and/or functional group orientation (e.g., −NO2 or −ONO) in vivo. Furthermore, the possibility cannot be excluded that Ch18:2-ONO (cholesteryl nitritelinoleate) could be formed in vivo and decomposed by transnitrozation or other routes during sample extraction and workup. Nitrated lipids present in plasma and lipoproteins can be considered as potential indicators of the chain-breaking antioxidant role of •NO during lipid peroxidation, as previously reported (7Rubbo H. Parthasarathy S. Barnes S. Kalyanaraman M.K.B. 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