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• W2065947716 abstract "One of the earliest steps in the development of the atherosclerotic lesion is the accumulation of monocyte/macrophages within the vessel wall. Oxidized lipids present in minimally modified-low density lipoproteins (MM-LDL) contribute to this process by activating endothelial cells to express monocyte-specific adhesion molecules and chemoattractant factors. A major focus of our group has been the isolation and characterization of the biologically active oxidized lipids in MM-LDL. We have previously characterized three oxidized phospholipids present in MM-LDL, atherosclerotic lesions of fat fed rabbits, and autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) that induced human aortic endothelial cells to adhere human monocytes in vitro. We have used sequential normal and reverse phase-high performance liquid chromatography to isolate various isomers of an oxidized phospholipid from autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine. The fatty acid in the sn-2 position of this biologically active isomer and its dehydration product was released by phospholipase A2 and characterized. Hydrogenation with platinum(IV) oxide/hydrogen suggested a cyclic moiety, and reduction with sodium borohydride suggested two reducible oxygen-containing groups in the molecule. The fragmentation pattern produced by electrospray ionization-collision induced dissociation-tandem mass spectrometry was consistent with a molecule resembling an E-ring prostaglandin with an epoxide at the 5,6 position. The structure of this lipid was confirmed by proton nuclear magnetic resonance spectroscopy analysis of the free fatty acid isolated from the dehydration product of m/z 828.5. Based on these studies, we arrived at the structure of the biologically active oxidized phospholipids as 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine. The identification of this molecule adds epoxyisoprostanes to the growing list of biologically active isoprostanes. One of the earliest steps in the development of the atherosclerotic lesion is the accumulation of monocyte/macrophages within the vessel wall. Oxidized lipids present in minimally modified-low density lipoproteins (MM-LDL) contribute to this process by activating endothelial cells to express monocyte-specific adhesion molecules and chemoattractant factors. A major focus of our group has been the isolation and characterization of the biologically active oxidized lipids in MM-LDL. We have previously characterized three oxidized phospholipids present in MM-LDL, atherosclerotic lesions of fat fed rabbits, and autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) that induced human aortic endothelial cells to adhere human monocytes in vitro. We have used sequential normal and reverse phase-high performance liquid chromatography to isolate various isomers of an oxidized phospholipid from autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine. The fatty acid in the sn-2 position of this biologically active isomer and its dehydration product was released by phospholipase A2 and characterized. Hydrogenation with platinum(IV) oxide/hydrogen suggested a cyclic moiety, and reduction with sodium borohydride suggested two reducible oxygen-containing groups in the molecule. The fragmentation pattern produced by electrospray ionization-collision induced dissociation-tandem mass spectrometry was consistent with a molecule resembling an E-ring prostaglandin with an epoxide at the 5,6 position. The structure of this lipid was confirmed by proton nuclear magnetic resonance spectroscopy analysis of the free fatty acid isolated from the dehydration product of m/z 828.5. Based on these studies, we arrived at the structure of the biologically active oxidized phospholipids as 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine. The identification of this molecule adds epoxyisoprostanes to the growing list of biologically active isoprostanes. low density lipoprotein minimally modified-LDL autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine 1-palmitoyl-2-(5)oxovaleryl-sn-glycero-3-phosphocholine 1-palmitoyl-2-glutaryl-sn glycero-3-phosphocholine electrospray ionization-mass spectrometry liquid chromatography/MS high performance liquid chromatography reverse phase normal phase butylated hydroxytoluene flow injection analysis 1-palmitoyl-2-(5,6)-epoxyisoprostane E2-sn-glycero-3-phosphocholine Atherosclerosis is a devastating disease responsible for profound human morbidity and mortality (1Braunwald E. N. Engl. J. Med. 1997; 337: 1360-1369Crossref PubMed Scopus (1200) Google Scholar, 2Fuster V. Stein B. Ambrose J.A. Badimon L. Badimon J.J. Chesebro J.H. Circulation. 1990; 82: II47-II59PubMed Google Scholar). The precursor of the atherosclerotic lesion, the fatty streak, begins to develop in the first decade of life and is characterized by the accumulation of monocyte/macrophages within the intimal layer of the blood vessel (3Strong J.P. Malcom G.T. Oalmann M.C. Wissler R.W. Ann. N. Y. Acad. Sci. 1997; 811: 226-237Crossref PubMed Scopus (44) Google Scholar,4Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19277) Google Scholar). There is evidence that oxidized lipids, primarily derived from low density lipoproteins (LDL),1contribute to all stages of atherosclerotic development (5Steinberg D. Parthasarathy S. Carew T.E. Khoo J.C. Witztum J.L. N. Engl. J. Med. 1989; 320: 915-924Crossref PubMed Google Scholar, 6Witztum J.L. Steinberg D. J. Clin. Invest. 1991; 88: 1785-1792Crossref PubMed Scopus (2473) Google Scholar, 7Parthasarathy S. Modified Lipoproteins in the Pathogenesis of Atherosclerosis. R. G. Landes Company, Austin, Texas1994: 91-119Google Scholar, 8Berliner J.A. Navab M. Fogelman A.M. Frank J.S. Demer L.L. Edwards P.A. Watson A.D. Lusis A.J. Circulation. 1995; 91: 2488-2496Crossref PubMed Scopus (1587) Google Scholar). Initially, they facilitate monocyte deposition within the subendothelial space by stimulating endothelial cells to express monocyte-specific adhesion molecules (9Berliner J.A. Territo M.C. Sevanian A. Ramin S. Kim J.A. Bamshad B. Esterson M. Fogelman A.M. J. Clin. Invest. 1990; 85: 1260-1266Crossref PubMed Scopus (768) Google Scholar, 10Weber C. Erl W. Weber P.C. Biochem. Biophys. Res. Commun. 1995; 206: 621-628Crossref PubMed Scopus (69) Google Scholar) and secrete monocyte chemoattractants (11Cushing S.D. Berliner J.A. Valente A.J. Terito M.C. Navab M. Parhami F. Gerrity R. Schwartz C.J. Fogelman A.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5134-5138Crossref PubMed Scopus (972) Google Scholar, 12Navab M. Imes S.S. Hough G.P. Hama S.Y. Ross L.A. Bork R.W. Valente A.J. Berliner J.A. Drinkwater D.C. Laks H. Fogelman A.M. J. Clin. Invest. 1991; 88: 2039-2046Crossref PubMed Scopus (646) Google Scholar). Later, highly oxidized lipids such as malondialdehyde and 4-hydroxynonenal modify the protein component of LDL so that it is recognized by the macrophage scavenger/oxidized LDL receptor rather than the native LDL receptor (13Fogelman A.M. Shechter I. Seager J. Hokom M. Child J.S. Edwards P.A. Proc. Natl. Acad. Sci., U. S. A. 1980; 77: 2214-2218Crossref PubMed Scopus (697) Google Scholar, 14Steinbrecher U.P. Parthasarathy S. Leake D.S. Witztum J.L. Steinberg D.S. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3883-3887Crossref PubMed Scopus (1418) Google Scholar, 15Morel D.W. DiCorleto P.E. Chisholm G.M. Arteriosclerosis. 1984; 4: 357-364Crossref PubMed Google Scholar). Uptake of oxidized LDL by macrophages generates foam cells that reside in the subendothelial space. It is the lipid-laden foam cells that are the hallmark of the fatty streak lesion. We have previously demonstrated that mildly oxidized LDL, which we have termed “minimally modified” (MM-LDL), stimulated human aortic endothelial cells to bind human monocytes in vitro (9Berliner J.A. Territo M.C. Sevanian A. Ramin S. Kim J.A. Bamshad B. Esterson M. Fogelman A.M. J. Clin. Invest. 1990; 85: 1260-1266Crossref PubMed Scopus (768) Google Scholar). By separating the components of MM-LDL it was found that the phospholipid fraction contained nearly all of the biological activity (9Berliner J.A. Territo M.C. Sevanian A. Ramin S. Kim J.A. Bamshad B. Esterson M. Fogelman A.M. J. Clin. Invest. 1990; 85: 1260-1266Crossref PubMed Scopus (768) Google Scholar). When the phospholipids from MM-LDL and native LDL were compared it was found that phospholipids containing arachidonic acid were preferentially oxidized compared to phospholipids containing other polyunsaturated fatty acids (16Watson A.D. Navab M. Hama S.Y. Sevanian A. Prescott S.M. Stafforini D.M. McIntyre T.M. La Du B.N. Fogelman A.M. Berliner J.A. J. Clin. Invest. 1995; 95: 774-782Crossref PubMed Google Scholar). This lead us to suspect that the biologically active phospholipids were oxidized derivatives of arachidonic acid-containing phospholipids. We then found that autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) had identical biological properties as MM-LDL, and we began using Ox-PAPC as a surrogate for MM-LDL (17Watson A.D. Berliner J.A. Hama S.Y. La Du B.N. Faull K.F. Fogelman A.M. Navab M. J. Clin. Invest. 1995; 96: 2882-2891Crossref PubMed Scopus (1040) Google Scholar). Recently, our group has described three compounds present in MM-LDL, Ox-PAPC, and rabbit atherosclerotic lesions that stimulated endothelial cells to bind monocytes in vitro (18Watson A.D. Leitinger N. Navab M. Faull K.F. Hörkkö S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). All were derived from the oxidation of arachidonic acid-containing phospholipids in LDL (16Watson A.D. Navab M. Hama S.Y. Sevanian A. Prescott S.M. Stafforini D.M. McIntyre T.M. La Du B.N. Fogelman A.M. Berliner J.A. J. Clin. Invest. 1995; 95: 774-782Crossref PubMed Google Scholar). Interestingly, we found that antibodies to these lipids were spontaneously produced in vivo by apolipoprotein E knockout mice that were genetically predisposed to develop atherosclerosis (18Watson A.D. Leitinger N. Navab M. Faull K.F. Hörkkö S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). Two of the biologically active compounds were produced by oxidative fragmentation of the arachidonic acid moiety in the sn-2 position of PAPC and were identified as 1-palmitoyl-2-(5)oxovaleryl-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC). The molecular structure of the third molecule, which gave a signal at m/z 828.5 by electrospray ionization-mass spectrometry (ESI-MS), was not determined at that time. In this study we provide evidence that this molecule contained an epoxyisoprostane in the sn-2 position of the phospholipid (SchemeFS1 A) and this molecule undergoes dehydration to form a structurally similar molecule with a mass of 810.5 [M + H+] (Scheme FS1 B). Tissue culture media, serum, and supplements were obtained from Irvine Scientific and Hyclone Laboratories, Inc. Acetonitrile, chloroform, methanol, ethyl acetate, and water (all Optima grade) were obtained from Fisher Scientific, Pittsburgh, PA. Gelatin (endotoxin-free, tissue culture grade), porcine liver esterase, calcium chloride, methoxylamine hydrochloride, ammonium acetate, sodium borohydride, platinum(IV) oxide, phospholipase A2(naja naja), and butylated hydroxytoluene (BHT) were obtained from Sigma. Authentic l-α-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL) or Sigma. Deuterated chloroform (99.99% D), deuterated methanol (99+% D), and deuterium oxide (99.9% D) were obtained from Aldrich Chemical Co. Sodium borodeuteride was obtained from Cambridge Isotope Laboratories, Inc. Human aortic endothelial cells at passages 4–7 were cultured as described in medium 199 supplemented with 10% fetal bovine serum (9Berliner J.A. Territo M.C. Sevanian A. Ramin S. Kim J.A. Bamshad B. Esterson M. Fogelman A.M. J. Clin. Invest. 1990; 85: 1260-1266Crossref PubMed Scopus (768) Google Scholar, 12Navab M. Imes S.S. Hough G.P. Hama S.Y. Ross L.A. Bork R.W. Valente A.J. Berliner J.A. Drinkwater D.C. Laks H. Fogelman A.M. J. Clin. Invest. 1991; 88: 2039-2046Crossref PubMed Scopus (646) Google Scholar). These studies were performed essentially as described previously (9Berliner J.A. Territo M.C. Sevanian A. Ramin S. Kim J.A. Bamshad B. Esterson M. Fogelman A.M. J. Clin. Invest. 1990; 85: 1260-1266Crossref PubMed Scopus (768) Google Scholar). Blood monocytes were isolated from a large pool of healthy human blood donors by a modification of the Recalde procedure (19Fogelman A.M. Sykes K. Van Lenten B.J. Territo M.C. Berliner J.A. J. Lipid Res. 1988; 29: 1243-1247Abstract Full Text PDF PubMed Google Scholar). For monocyte adhesion assays, human aortic endothelial cells were incubated with test medium for 4 h at 37 °C. The test medium was removed, the endothelial cells were washed, and a suspension of human monocytes was added for 12–15 min after which nonadherent monocytes were removed. Bound monocytes were counted and expressed as monocytes/microscopic field. PAPC was oxidized by transferring 1 mg in 100 μl of chloroform to a clean 16 × 125-mm glass test tube and evaporating the solvent under a stream of nitrogen. The lipid residue was allowed to autoxidize while exposed to air for 24–72 h at room temperature. The extent of oxidation was monitored by flow injection ESI-MS. Normal phase high performance liquid chromatography (NP-HPLC) was performed by injecting oxidized phospholipid preparations (resuspended in chloroform) onto a silica column (Adsorbosphere, 250 × 22-mm, 5 μm; Alltech Associates, Inc.) and eluting isocratically with a mobile solvent of acetonitrile/methanol/water (77:8:15, v/v/v, pH 5.0 with formic acid) at a flow rate of 18.0 ml/min. Typically, Ox-PAPC produced from 25–35 mg of PAPC was applied for each run. Reverse phase HPLC (RP-HPLC) of oxidized phospholipids was performed with a C8 column (Betasil, C8, 250 × 10-mm, 5 μm, Keystone Scientific, Inc.). Phospholipids were eluted with a mobile phase of 80% methanol that was changed linearly over a period of 60 min to 100% methanol at 5 ml/min. Fractions containing oxidized phospholipids of interest were collected by monitoring ultraviolet absorbance and ESI-MS (LC/MS). Oxidized free fatty acids were separated by RP-HPLC using a C18 column (Betasil, C18, 250 × 10-mm, 5 μm, Keystone Scientific, Inc.). A mobile phase of 60% methanol containing 1 mm ammonium acetate changed linearly over 60 min to 100% methanol containing 1 mm ammonium acetate was used. When isolating lipids for NMR analysis, solvents without ammonium acetate were used. UV absorbance was detected with a diode array detector (L-3000, Hitachi, Ltd., Tokyo, Japan) scanning from 200 to 350 nm at 2.5 nm resolution. Phospholipid fractions collected by HPLC were dried under argon to a lipid residue and resuspended in 1 ml of phosphate-buffered saline containing 5 mm CaCl2. To this solution was added 5 units of phospholipase A2. The solution was mixed and incubated at 37 °C for 45 min. After incubation, the lipids were extracted with 1 ml of ethyl acetate containing 0.01% BHT after acidification with formic acid to pH 3.0. Phospholipid fraction containing 828.5 (i2) was isolated by sequential NP-HPLC and RP-HPLC from 5 mg of Ox-PAPC. The fraction was dried under argon, and 1 ml of 0.92 mm methoxylamine hydrochloride in 1× phosphate-buffered saline was added. The solution was mixed thoroughly and incubated for 45 min at 37 °C. After incubation the lipids were extracted with CHCl3/MeOH + BHT and analyzed by positive ion ESI-MS. Chemical reduction of lipids was achieved by addition of 600 μl of a 70 mm solution of sodium borohydride or sodium borodeuteride in acetonitrile at room temperature for 30 min. Following incubation, 1 ml of ethyl acetate containing 0.01% BHT and 1 ml of water was added. The solution was mixed thoroughly and centrifuged at 2,000 × g for 5 min. The ethyl acetate phase was transferred to a clean glass tube, and 30 μl of formic acid was added to displace sodium from phospholipid sodium salts. Lipids were hydrogenated by exposure to hydrogen gas in the presence of platinum(IV) oxide (20Thomas D.W. van Kuijk F.J. Dratz E.A. Stephens R.J. Anal. Biochem. 1991; 198: 104-111Crossref PubMed Scopus (42) Google Scholar). Oxidized lipids were resuspended in 300 μl of ethyl acetate and transferred to a 25-ml round bottom flask. Platinum(IV) oxide (1 mg) was added and the flask was covered with a rubber septum. Two 18-gauge hypodermic needles were placed through the septum, and the flask was flushed by introduction of hydrogen through one of the needles. After flushing, one of the needles was removed and a balloon containing hydrogen gas was attached to the other. The samples were incubated with constant stirring at room temperature for 45 min. The reaction mixture was transferred to a 13 × 100-mm glass test tube and dried under argon gas. The lipid residue was resuspended in 1 ml of chloroform/methanol (2:1, v/v), 400 μl of water, and 20 μl of concentrated formic acid and then the lipids were recovered from the chloroform phase after mixing and centrifugation. 18Oxygen exchange experiments were performed by incubation of the free fatty acids with porcine liver esterase in H218O (21Westcott J.Y. Clay K.L. Murphy R.C. Biomed. Mass Spectrom. 1985; 12: 714-718Crossref PubMed Scopus (48) Google Scholar). To the oxidized fatty acid residue was added 100 μl of H218O and 23 units of porcine liver esterase. The contents were mixed thoroughly and incubated for 60 min at 37 °C with occasional mixing. Lipids were extracted by addition of 300 μl of chloroform/methanol (2:1, v/v) to the reaction mixture. ESI-MS was performed using an API III triple-quadrupole biomolecular mass analyzer (Perkin-Elmer Sciex Instruments, Norwalk, CT) fitted with an articulated, pneumatically assisted nebulization probe and an atmospheric pressure ionization source. Details of calibration and tuning have been described previously (18Watson A.D. Leitinger N. Navab M. Faull K.F. Hörkkö S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). Phospholipids were introduced into the mass spectrometer by direct flow injection analysis (FIA) in acetonitrile/water/formic acid (50:50:0.1, v/v/v) or via liquid chromatography (LC/MS) and analyzed as the protonated molecule [M + H+] in positive ion mode. The mass spectrometer was set to scan from m/z 450 to 950 with an orifice voltage of +65, a step size of 0.3, a dwell time of 3 msec, and a scan speed of ∼4 s. Fatty acids were analyzed as carboxylate anions [M−] by FIA in methanol/water (50:50, v/v) with 1 mm ammonium acetate or by LC/MS in chromatography solvent. For negative ion ESI-MS/MS, a solvent of 100% methanol with 1 mm ammonium acetate was used, and daughter ion spectra were obtained by colliding the Q1 selected ion of interest with argon in Q2, and scanning Q3 to analyze the fragment ion products. Reconstructed selected ion chromatograms were produced by software supplied by PE Sciex. High resolution-fast atom bombardment/MS spectra were obtained using a VG ZAB-SE fast atom bombardment mass spectrometer (Micromass, Manchester, UK) equipped with a 11/250 data system. HPLC fractions containing oxidized phospholipids of interest were dried under argon and resuspended in an aqueous solution of 0.1% trifluoroacetic acid. To the static fast atom bombardment probe containing 1–2 μl of liquid matrix (m-nitrobenzylalcohol/thioglycerol/trifluoroacetic acid, 50:50:0.5, v/v/v) was added 1–2 μl of the oxidized phospholipid solution. Spectra were recorded using a 8 kV accelerating potential, cesium bombardment at 22 kV and 1–2 μA, and a mass resolution of 3,000 (10% valley, M/ΔM). The mass spectrometer was set to scan from m/z 200–1,000, and ∼10 scans were collected into a multichannel analyzer. The data were smoothed, centroided, and mass measured using cesium iodide ion clusters for calibration. All proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Bruker ARX-500 MHz spectrometer using a microprobe (2.5 mm) NMR tube. Proton chemical shifts were reported in parts per million (ppm) on the δ scale with reference to CHCl3 (δ 7.24).1H NMR spectral data were tabulated in terms of multiplicity of proton absorption (s, singlet; d, doublet; dd, doublet of a doublet; dt, doublet of a triplet; t, triplet; q, quartet; m, multiplet; br, broad), coupling constants (Hz), and number of protons. Purified lipids were dried under argon, resuspended in CDCl3 (90 μl) and transferred to a microprobe NMR tube for analysis. Proton-proton homodecoupling experiments were performed using a power level of 55 dB. Absorbance spectra in the 190–500 nm range for various oxidized phospholipids were measured using a Shimadzu Biospec-1601 UV-visible spectrophotometer (Shimadzu Scientific Instruments, Inc, Columbia, MD). For extinction coefficient determination, the isomers of 828.5 and 810.5 were isolated by sequential normal phase and reverse phase HPLC and quantified by FIA-ESI-MS using dimyristoyl phosphatidylcholine as an internal standard (22Han X. Gubitosi-Klug R.A. Collins B.J. Gross R.W. Biochemistry. 1996; 35: 5822-5832Crossref PubMed Scopus (104) Google Scholar). The UV-visible absorbance was scanned for m/z 828.5 and 810.5 isomers in methanol (1 ml) at three different concentrations. Using these absorbance values, molar extinction coefficients were calculated (23Silverstein R.M. Bassler G.C. Morrill T.C. Spectroscopic Identification of Organic Compounds. John Wiley & Sons, Inc., New York1991: 289-315Google Scholar) for isomers of m/z 828.5 and 810.5. We have previously separated the phospholipid components of Ox-PAPC by NP-HPLC and found that the second peak enriched in m/z 828.5 induced endothelial cells to bind monocytes in vitro (18Watson A.D. Leitinger N. Navab M. Faull K.F. Hörkkö S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). We repeated these experiments and collected the active fraction between 16.5 and 18.0 min (Fig. 1 A) that contained mostly m/z 828.5 and 810.5 (mass spectrum not shown). The lipids in this fraction were then applied to a reverse phase column, which effectively separated several isomers of m/z 828.5 and 810.5 (Fig. 1 B). Each major peak was collected, dried under argon, resuspended in tissue culture medium, and tested for the ability to induce endothelial cells to bind monocytes. The only peak that showed significant biological activity above control was the second of the peaks containing m/z 828.5 (Fig. 1 C). This isomer caused a dose-dependent increase in monocyte binding reaching a statistically significant increase over control as low as 380 ng/ml. The fatty acid hydrolyzed from the sn-2 position of the biologically active isomer did not induce monocyte-endothelial interactions (data not shown). As a convention throughout this article, the five major isomers of m/z 828.5 resolved by RP-LC/MS will henceforth be abbreviated 828.5 (i1–5) and the three major isomers of m/z 810.5 will be abbreviated 810.5 (i1–3). All isomers with m/z 828.5 possessed nearly identical UV maxima at 252 nm, and all isomers of the m/z 810.5 possessed identical UV maxima at 257 nm (Fig.2). These UV maxima were consistent with a specific conjugated system within all of these molecules. Extinction coefficients (ε) at room temperature in methanol for the various isomers were calculated to be: 828.5 (i1) = 24,070; 828.5 (i2) = 18,632; 828.5 (i3) = 17,975; 828.5 (i4) = 22,275; 828.5 (i5) = 15,917; 810.5 (i1) = 20,503; 810.5 (i2) = 19,572; and 810.5 (i3) = 14,950. To confirm the molecular formula of m/z 828.5 we analyzed the molecule by high resolution-fast atom bombardment/MS. The experimental mass of the ion was determined to be 828.5391, which closely matched the mass of a molecule with the elemental composition of C44H79NO11P (calculated mass = 828.5381). Because unoxidized PAPC has an elemental composition of C44H81NO8P, we concluded that during oxidation this molecule acquired three oxygen atoms and lost two hydrogen atoms. Based on the mass of the products observed after phospholipase A2 hydrolysis of this molecule, we further concluded that the only oxidized part of the PAPC molecule was the arachidonic acid in the sn-2 position of PAPC. Thus, the molecular formula of this oxidized fatty acid in the sn-2 position was C20H30O5, compared with the elemental formula of arachidonic acid, C20H32O2. Periodically, positive ion ESI-MS was performed on stored preparations of Ox-PAPC and it was noticed that the relative ratio of 828.5 to 810.5 decreased over time, suggesting that 810.5 may be a decomposition product of 828.5. To test this, we isolated isomers of 828.5 by RP-LC/MS and allowed each of them to undergo spontaneous dehydration. After 48 h at 4 °C in chloroform, we reanalyzed the sample by RP-LC/MS using the same chromatographic conditions that were used for original isolation. Two isomers of 828.5 were collected, the biologically active isomer, 828.5 (i2), and a biologically inactive isomer, 828.5 (i5), (Fig.3 A). After 48 h at 4 °C, 828.5 (i2) had partially decomposed to a molecule that co-migrated with 810.5 (i2) eluting at 31.5 min (Fig. 3 B). In contrast, the tube containing 828.5 (i5) contained a mixture of two isomers of 810.5 that co-eluted at 30.0 min and 32.5 min with 810.5 (i1) and 810.5 (i3), respectively (Fig. 3 C). This experiment showed that 828.5 (i2) underwent dehydration to form 810.5 (i2) and that 828.5 (i5) underwent dehydration to form 810.5 (i1) and 810.5 (i3). In addition, this suggested that the molecular structure of 810.5 (i2) was similar to 828.5 (i2). Derivatization with bis(trimethylsilyl)trifluoroacetamide demonstrated the presence of one hydroxyl group in m/z 828.5, which was lost in m/z 810.5 (data not shown). As a measure of the number of carbonyl or epoxide groups we examined the derivatization of the molecule with methoxylamine hydrochloride, which adds 47 mass units to carbonyl group and reactive epoxide groups. Treatment of 828.5 (i2) with methoxylamine hydrochloride yielded several compounds (Fig.4). An ion at m/z 857.4 was produced by the addition of a methoxylamine group and subsequent loss of water ([M + H+] + 47 − 18). An ion at m/z 875.7 was produced by the addition of a methoxylamine without the loss of water ([M + H+] + 47). An ion at m/z 886.5 was produced by the addition of two methoxylamine groups with subsequent loss of two waters {[M + H+] + 2(47) − 2(18)}. An ion at m/z 904.5 was produced by the addition of two methoxylamines with the loss of one water. When the reaction with methoxylamine hydrochloride was allowed to proceed, the most abundant ion was m/z 904.5 (data not shown). These data indicate the presence of two groups on m/z 828.5, which react with methoxylamine hydrochloride. Reduction of the molecules (m/z 810.5 and 828.5) by sodium borohydride and sodium borodeuteride was used to confirm the number of reducible oxygen groups. Sodium borohydride can effectively reduce hydroperoxides, ketones, aldehydes, and some epoxides to hydroxyl groups, thereby altering the molecular weight of the molecule in a predictable manner. Individual isomers of 828.5 and 810.5 were isolated by RP-LC/MS and then treated with sodium borohydride, re-extracted, and analyzed by positive ion FIA-ESI-MS. Each reactive group adds two hydrogens. After reduction with sodium borohydride, the molecular weight of each isomer of 828.5 was increased by 4 Da to m/z 832.5. This suggested that all isomers of 828.5 possessed two reducible oxygen-containing functional groups such as aldehydes, ketones, and/or reactive epoxides. Fig.5 shows the positive ion ESI-MS of the purified biologically active isomer, 828.5 (i2), before (Fig.5 A) and after (Fig. 5 B) sodium borohydride reduction. Some 1-palmitoyl-lysophosphatidylcholine (m/z 496.2) and its corresponding sodium salt (m/z 518.1) were produced by partial saponification of the phospholipid during the reduction procedure. The reduction was likely incomplete because of the presence of a signal at m/z 830.4. Treatment with sodium borodeuteride, in addition to reducing an aldehyde, ketone, or reactive epoxide, will simultaneously add two deuterium atoms to the group, one associated with oxygen. Because ESI-MS was performed in a solvent containing H2O, the deuterium bound to the oxygen will undergo rapid exchange with protons in the solvent. Indeed, when 828.5 (i2) was treated with sodium borodeuteride the major product was an ion at m/z 834.5 rather than m/z 832.5 as seen when the molecule was treated with sodium borohydride (data not shown). These deuterium additions are consistent with the presence of two reducible oxygen functionalities. When 810.5 (i2) was treated with sodium borohydride, the mass was increased by six mass units to 816.6 (data not shown), and with sodium borodeuteride, the mass was increased by nine mass units to 819.6 (Fig.6 A). Because this molecule only possessed two reducible oxygen groups, a reactive double bond of the enone was also reduced. It is known that treatment of reactive enones with sodium borohydride results in the reduction of both carbonyl groups as well as double bonds (24Johnson M.R." @default.
• W2065947716 created "2016-06-24" @default.
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