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• W2055065992 abstract "Free radical-initiated lipid autoxidation in low density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. Oxidation of the lipid components of LDL leads to a complex mixture of hydroperoxides, bicyclic endoperoxides, monocyclic peroxides, and serial cyclic peroxides. The oxidation compounds and/or their decomposition products can modify protein components, which may lead to various diseases. A novel class of peroxides (termed dioxolane-isoprostanes) having a bicyclic endoperoxide moiety characteristic of the isoprostanes and a dioxolane peroxide functionality in the same molecule was identified in the product mixture formed from in vitro autoxidation of cholesteryl arachidonate. The same products are also detected in in vitro oxidized LDL. Various mass spectrometric techniques have been applied to characterize these new peroxides. The structure of these compounds has also been confirmed by independent synthesis. We reason, based on the free radical mechanism of the transformation, that only the 12- and 8-peroxyl radicals (those leading to 12-HPETE and 8-HPETE) of arachidonate can form these new peroxides. We also suggest that the formation of these peroxides provides a rationale to explain the fact that 5- and 15-series isoprostanes are formed in preference to 8- and 12-series. Furthermore, series of other isoprostanes, such as dioxolane A2, D2, E2, etc., can be derived from the dioxolane-isoprostane peroxides. These findings offer further insights into the oxidation products of arachidonate and the opportunity to study their potential biological relevance. Free radical-initiated lipid autoxidation in low density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. Oxidation of the lipid components of LDL leads to a complex mixture of hydroperoxides, bicyclic endoperoxides, monocyclic peroxides, and serial cyclic peroxides. The oxidation compounds and/or their decomposition products can modify protein components, which may lead to various diseases. A novel class of peroxides (termed dioxolane-isoprostanes) having a bicyclic endoperoxide moiety characteristic of the isoprostanes and a dioxolane peroxide functionality in the same molecule was identified in the product mixture formed from in vitro autoxidation of cholesteryl arachidonate. The same products are also detected in in vitro oxidized LDL. Various mass spectrometric techniques have been applied to characterize these new peroxides. The structure of these compounds has also been confirmed by independent synthesis. We reason, based on the free radical mechanism of the transformation, that only the 12- and 8-peroxyl radicals (those leading to 12-HPETE and 8-HPETE) of arachidonate can form these new peroxides. We also suggest that the formation of these peroxides provides a rationale to explain the fact that 5- and 15-series isoprostanes are formed in preference to 8- and 12-series. Furthermore, series of other isoprostanes, such as dioxolane A2, D2, E2, etc., can be derived from the dioxolane-isoprostane peroxides. These findings offer further insights into the oxidation products of arachidonate and the opportunity to study their potential biological relevance. Arachidonic acid is a ubiquitous polyunsaturated fatty acid in mammalian cellular membranes. Recent experimental observations show that enzymatic oxidation products derived from arachidonic acid play an important role in the control of organ physiology (1Funk C.D. Science. 2001; 294: 1871-1875Google Scholar, 2Rocca B. FitzGerald G.A. Int. Immunopharmacol. 2002; 2: 603-630Google Scholar, 3FitzGerald G.A. Loll P. J. Clin. Invest. 2001; 107: 1335-1338Google Scholar). For example, the bicyclic endoperoxide, prostaglandin G2 (PGG2), 1The abbreviations used are: PGG2prostaglandin G2CIDcollision-induced dissociationPGH2prostaglandin H2PGF2prostaglandin F2HPETE Chcholesteryl hydroperoxyeicosatetraenoateHPLChigh-performance liquid chromatographyLCliquid chromatographyCIScoordination ion sprayECNICIelectron capture negative ion chemical ionizationSIMselective ion monitoringSRMselective reaction monitoringPFBpentafluorobenzylTMStrimethylsilyl. is a key intermediate that serves as a precursor to a variety of biologically active compounds, such as thromboxane A2 (4Cheng Y. Austin S.C. Rocca B. Koller J. Coffman T.M. Grosser T. Lawson J.A. FitzGerald G.A. Science. 2002; 296: 539-541Google Scholar), prostaglandins PGF2α, PGD2, PGE2, and prostacyclin (PGI2) (5Pratico D. Lawson J.A. Rokach J. FitzGerald G.A. Trends Endocrinol. Metab. 2001; 12: 243-247Google Scholar, 6Porter N.A. Pryor W.A. Free Radicals in Biology. Vol. IV. Academic Press, Inc., New York1980: 261-295Google Scholar). On the other hand, oxidation of arachidonic acid or its esters can occur by a non-enzymatic free radical process. Some of the oxidation products of these non-enzymatic peroxidations have been linked to human disease states. In particular, oxidative modification of the lipid components of low density lipoprotein, the major carrier of cholesteryl esters in human blood, has been attributed to the pathogenesis and progression of atherosclerosis (7Patel R.P. Moellering D. Murphy-Ullrich J. Jo H. Bechman J.S. Darley-Usmar V.M. Free Radic. Biol. Med. 2000; 28: 1780-1794Google Scholar). prostaglandin G2 collision-induced dissociation prostaglandin H2 prostaglandin F2 cholesteryl hydroperoxyeicosatetraenoate high-performance liquid chromatography liquid chromatography coordination ion spray electron capture negative ion chemical ionization selective ion monitoring selective reaction monitoring pentafluorobenzyl trimethylsilyl. The free radical initiated oxidation of arachidonic acid or its esters is extremely complicated because of the fact that insertion of molecular oxygen may occur at C5, C8, C9, C11, C12, or C15 of the 20-carbon fatty acid chain. The resultant peroxyl radicals can undergo further reactions to give a complex mixture of primary hydroperoxides and cyclic peroxides with dozens of regioisomers and diastereomers (8Havrilla C.M. Hachey D.L. Porter N.A. J. Am. Chem. Soc. 2000; 122: 8042-8055Google Scholar). Furthermore, these unstable peroxides can decompose and give rise to other reactive products, such as malondialdehyde, γ-ketoaldehydes (such as levuglandins, LGs), and 4-hydroxy nonenal (9Esterbauer H. Zollner H. Schauer R.J. Vigo-Pelfrey, C. Membrane Lipid Oxidaiton. Vol. I. CRC Press, Inc., Boca Raton, FL1991: 239-268Google Scholar, 10Esterbauer H. Schauer R.J. Zollner H. Free Radic. Biol. Med. 1991; 11: 81-128Google Scholar, 11Boutaud O. Brame C.J. Chaurand P. Li J. Rowlinson S.W. Crews B.C. Ji C. Marnett L.J. Caprioli R.M. Roberts Jr., L.J. Oates J.A. Biochemistry. 2001; 40: 6948-6955Google Scholar). A number of oxidation products from arachidonate autoxidation have been characterized and their biological activities are a major research focus (Scheme 1). Bicyclic endoperoxides (1a), monocyclic peroxides (1b), and serial cyclic peroxides (1c) have previously been characterized from the mixture of products formed in the autoxidation of cholesteryl arachidonate (8Havrilla C.M. Hachey D.L. Porter N.A. J. Am. Chem. Soc. 2000; 122: 8042-8055Google Scholar, 12Yin H. Havrilla C.M. Morrow J.D. Porter N.A. J. Am. Chem. Soc. 2002; 124: 7745-7754Google Scholar, 13Yin H. Havrilla C.M. Gao L. Morrow J.D. Porter N.A. J. Biol. Chem. 2003; 278: 16720-16725Google Scholar). Oxidation products having a tetrahydrofuran moiety (1d) were recently characterized under conditions of reaction having increased oxygen tension (14Fessel J.P. Porter N.A. Moore K.P. Sheller J.R. Roberts Jr., L.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16713-16718Google Scholar). The epoxyisoprostanes (1e) and epoxycyclopentenones (1f) are probably derived from the bicyclic endoperoxides (15Subbangounder G. Wong J.W. Lee H. Faull K.F. Miller E. Witztum J.L. Berliner J.A. J. Biol. Chem. 2002; 277: 7271-7281Google Scholar, 16Watson A.D. Subbangounder G. Welsbie D.S. Faull K.F. Navab M. Jung M.E. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1999; 274: 24787-24798Google Scholar). Measurement of isoprostanes, the reduced compounds of bicyclic endoperoxides, in biological fluids or tissues has been regarded as one of the most useful non-invasive biomarkers for oxidative stress status (17Morrow J.D. Hill E. Burk R.F. Nammour T.M. Badr K.F. Roberts Jr., L.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9383-9387Google Scholar, 18Roberts Jr., L.J. Morrow J.D. Free Radic. Biol. Med. 2000; 28: 505-513Google Scholar). Formation of regioisomeric and diastereoisomeric isoprostanes has been extensively studied (12Yin H. Havrilla C.M. Morrow J.D. Porter N.A. J. Am. Chem. Soc. 2002; 124: 7745-7754Google Scholar, 13Yin H. Havrilla C.M. Gao L. Morrow J.D. Porter N.A. J. Biol. Chem. 2003; 278: 16720-16725Google Scholar). Previous experimental observations showed that 5- and 15-series (numbers refer to the position of hydroxyl group on the side chain) isoprostanes are more abundant than those of 8- and 12-series (12Yin H. Havrilla C.M. Morrow J.D. Porter N.A. J. Am. Chem. Soc. 2002; 124: 7745-7754Google Scholar, 19Waugh R.J. Morrow J.D. Roberts Jr., L.J. Murphy R.C. Free Radic. Biol. Med. 1997; 23: 943-954Google Scholar, 20Waugh R.J. Murphy R.C. J. Am. Soc. Mass Spectrom. 1996; 7: 490-499Google Scholar, 21Roberts Jr., L.J. Morrow J.D. Cell. Mol. Life Sci. 2002; 59: 808-820Google Scholar). We report herein that a novel class of compounds that have a bicyclic endoperoxide moiety and a cyclic peroxide functionality in the same molecule is formed in arachidonate oxidations. A variety of mass spectrometric methods have been developed to characterize these new compounds from the in vitro oxidation mixture of arachidonic acid or its esters. These compounds are named dioxolane-isoprostanes peroxides for convenience here because their structures are closely related to the isoprostanes but they have an additional dioxolane functional group. These compounds are also found as products from in vitro oxidized LDL. They may have biological relevance because of the presence of the reactive bicyclic endoperoxide functionality. All lipid autoxidation reactions were carried out under an atmosphere of oxygen unless otherwise noted. Air and argon were passed through a bed of calcium sulfate desiccant. Benzene was distilled from sodium and stored over 4A molecular sieves. Tetrahydrofuran and dichloromethane were dried by Solv-Tek (Berryville, VA) solvent purification columns using activated alumina for drying and Q-5 packing for deoxygenating the solvents. Oxygen (medical grade) was obtained from A. L. Compressed Gas (Nashville, TN). HPLC grade solvents were purchased from Burdick and Jackson (Muskegon, MI) or EM Science (Gibbstown, NJ). All lipids were purchased from NuChek Prep (Elysian, MI) and were of the highest purity (>99%). The free radical initiator di-tert-butyl hyponitrite was synthesized prior to use (22Mendenhall G.D. Tetrahedron Lett. 1983; 24: 451-452Google Scholar, 23Kiefer H. Trayler T.G. Tetrahedron Lett. 1966; 7: 6163-6168Google Scholar). 2,2′-Azobis[2-(2-imdazolin-2-yl)propane] dihydrochloride (C-0) was generously donated by Wako Chemicals USA Inc. (Richmond, VA). N,O-Bis-(trimethylsilyl)trifluoroacetamide was purchased from Supelco Inc. (Bellefonte, PA). PGF2α, PGF3α, and 1a,1b-dihomo-PGF2α were purchased from Cayman Chemicals (Ann Arbor, MI). All other reagents were purchased from Aldrich and used without further purification. Reactions involving hydroperoxides were visualized by TLC using a stain of 1.5 g of N,N′-dimethyl-p-phenylenediamine dihydrochloride, 25 ml of H2O, 125 ml of MeOH, 1 ml of acetic acid. Hydroperoxides yield an immediate pink color, whereas cyclic peroxides exhibit a pink color after mild charring. General TLC staining was accomplished by iodine or use of a phosphomolybdic acid stain prepared as a 20% (w/v) solution in EtOH. In general, hydroperoxides were stored as dilute solutions with 1 mol % butylated hydroxytoluene in either hexanes or benzene at -78 °C and were never exposed to temperatures >40 °C. Flash column chromatography was performed using 35-70-μm silica gel. Thin layer chromatography was performed using 0.2-mm layer thickness silica gel-coated aluminum (60 F254, EM Industries), and TLC plates were analyzed using UV light (254 nm) with a Mineralight UVSL-25 hand lamp. Preparative TLC was performed on Silica Gel 60ALK6D plates (Whatman). Analytical HPLC was carried out using a Waters model 600E pump with a Waters 996 Photodiode array detector. Millenium32 chromatography software (Waters Corp., Milford, MA) was used to control the 996 and to collect and process data. Cyclic peroxide analysis by analytical HPLC utilized a single Beckman Ultrasphere 5μ (4.6 mm × 25 cm) silica column. A flow rate of 1 ml/min was used for analytical NP HPLC. Preparative HPLC was performed using a Dynamax-60 Å 8-μm (83-121-C) silica column (21.4 mm × 25 cm) with a flow rate of 10 ml/min. Narrowbore HPLC for MS analysis used a single Beckman ultrasphere 5 μm (2.0 mm × 25 cm) silica column for analysis of cyclic peroxides and two Beckman Ultrasphere 5-μm (2.0 mm × 25 cm) silica columns for acyclic hydroperoxide analysis. Coordination ion spray mass spectrometry (CIS-MS) was accomplished using a Finnigan TSQ-7000 (San Jose, CA) triple quadrapole mass spectrometer operating in positive ion mode equipped with a standard API-1 electrospray ionization source. The source was outfitted with a 100-μ deactivated fused silica capillary. Data acquisition and evaluation were conducted on ICIS EXECUTIVE INST, version 8.3.2, and TSQ 7000 software INST, version 8.3. Data collected for selected reaction monitoring (SRM) experiments was also processed using Xcalibur, version 1 (Finnigan, San Jose, CA). Nitrogen gas served both as sheath gas and auxiliary gas; argon served as the collision gas. The electrospray needle was maintained at 4.6 kV and the heated capillary temperature was 200 °C. The tube lens potential and capillary voltage were optimized to maximize ion current for electrospray, with the optimal determined to be 80 and 20 V, respectively, for cholesteryl ester analysis. Positive ions were detected scanning from 100 to 1000 atomic mass units with a scan duration of 1 s. Profile data was recorded for 1 min and averaged for analysis. For CID experiments the collision gas pressure was set from 2.30 to 2.56 millitesla. To obtain fragmentation information of each compound, the dependence of offset-voltage and relative ion current was studied. The collision energy offset was varied from 10 to 40 eV depending on the compound being analyzed. Samples were introduced either by direct liquid infusion or by HPLC. For direct liquid injection, stock solutions of the lipids (100 ng/μl in 1% isopropyl alcohol in hexane) were prepared and mixed 1:1 with silver tetrafluoroborate (51.4 ng/μl in isopropyl alcohol). Samples were introduced to the ESI source by syringe pump at a rate of 10 μl/min. For HPLC sample introduction a Hewlett-Packard 1090 HPLC system was used. The auxiliary gas flow rate to the ESI interface was increased to between 5 and 10 units to assist in desolvation of the samples. For cholesteryl arachidonate hydroperoxide analysis, normal phase HPLC sample introduction was carried out using two tandem Beckman Ultrasphere narrowbore 5-μ silica columns (2.0 mm × 25 cm) operated in isocratic mode with 0.35% isopropyl alcohol in hexanes. For analysis of cyclic peroxide mixtures, sample introduction was carried out using a single Beckman Ultrasphere narrowbore 5-μ silica column (2.0 mm × 25 cm) operated in isocratic mode with 1.0% isopropyl alcohol in hexanes. The flow rate for both modes of chromatography was 150 μl/min. Column effluent was passed through an Applied Biosystems 785A programmable absorbance UV detector with detection at 234 nm. An Upchurch high pressure mixing tee was connected next in series for the post-column addition of the silver salts. The silver tetrafluoroborate (AgBF4) solution (0.25 mm in isopropyl alcohol) was added via a Harvard Apparatus (Cambridge, MA) syringe pump at a flow rate of 75 μl/min. A long section of PEEK tubing (1.04 m, 0.25-mm inner diameter) allowed time for the complexation of the silver to the lipid while delivering effluent to the mass spectrometer. A Rheodyne 7725 injector was fitted with a 100-μl PEEK loop for 20-50-μl sample injections. A PPh3-reduced oxidation mixture of 12-HPETE was analyzed by negative ion ESI-MS coupled with reverse-phase HPLC separation using a Supelco Discovery® C18 column (15 cm × 2.1 mm, 5 μ) at a flow rate of 0.2 ml/min with a linear gradient starting with 75% solvent A (0.05% acetic acid) to 20% in 40 min. Mobile phase B consists of acetonitrile/methanol (95/5). LC-APCI ECNI-MS was performed on the same instrument TSQ-7000 using an APCI ion source. The mass spectrometer operating conditions were as follows: vaporizer temperature was 475 °C, heated capillary at 300 °C, with the corona discharge needle set at 16 μA (24Singh G. Gutierrez A. Xu K. Blair I.A. Anal. Chem. 2000; 72: 3007-3013Google Scholar). The pressure of sheath gas and auxiliary gas was optimized for maximal response. For full scan and selected reaction monitoring (SRM) analyses, unit resolution was maintained for both parent and daughter ions. GC-NICI MS was performed using a Hewlett-Packard HP5989A GC/MS instrument interfaced with an IBM Pentium II computer system. GC was performed using a 30 m, 0.25-mm diameter, 0.25-μm film thickness DB-Wax column (J & W Scientific, Folsom, CA) or 15 m, 0.25-mm diameter, 0.25-μm film thickness, DB 1701 fused silica capillary column (Fison, Folsom, CA). The column temperature was programmed from 190 to 260 °C at 10 °C/min. Methane was used as the carrier gas at a flow rate of 1 ml/min. Ion source temperature was 250 °C, electron energy was 70 eV, and filament current was 0.25 mA. Lipid autoxidations were performed at 37.0 ± 0.2 °C and temperature was controlled using an I2R Thermo-watch ML6-1000SS. In a round-bottomed flask, cholesteryl arachidonate (400 mg, 0.584 mmol) was dissolved in benzene to make a solution of 0.15 m. To the mixture were added 0.5 eq of methyl trolox (methyl 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate) (25Suarna C. Dean R.T. Southwellkeely P.T. Aust. J. Chem. 1997; 50: 1129Google Scholar) and 0.1 eq of di-tert-butyl hyponitrite. The reaction was stirred under oxygen at 37 °C for 24 h. The reaction was quenched by adding 2 mg of butylated hydroxytoluene. Analytical HPLC (tandem Si column, 0.5% isopropyl alcohol in hexane, λ = 234 nm) indicated seven major fractions: A, tR = 13.80 min, 15-HPETE; B, tR = 14.54 min, 12-HPETE; C, tR = 16.11 min, 11-HPETE; D, tR = 20.91 min, 9-HPETE; E, tR = 21.29 min, 8-HPETE; F, tR = 26.11 min, 5-HPETE; G, tR = 51.30 min, methyl trolox. The excess hydrogen atom donor methyl trolox was separated by flash column chromatography on silica using solvent mixture of hexane:ethyl acetate (9:1). All the peroxide stain-positive fractions were combined and separated by semi-preparative HPLC (0.5% isopropyl alcohol in hexane, 234 nm). Components D and E were collected as a mixture. The primary hydroperoxides separated by HPLC were converted to more highly oxidized peroxides according to our previous procedure (8Havrilla C.M. Hachey D.L. Porter N.A. J. Am. Chem. Soc. 2000; 122: 8042-8055Google Scholar). Oxidized lipid derived from ∼0.13 μmol of lipid hydroperoxides was dissolved in 200 μl of ethanol and 200 μl of ethyl acetate. Approximately 5 mg of Pd-C was added to the mixture and hydrogenation was performed in a hydrogenator with a H2 pressure of 50 p.s.i. for 1 h. The mixture was filtered. The solution was subjected to basic hydrolysis using 8.0 m KOH for 60 min at 40 °C. After adjusting the pH to 1-3, the mixture was loaded on a C18 SepPak (Waters Associates) cartridge that was preconditioned with 5 ml of methanol and then 5 ml of water. The column was washed with 5 ml of water and 5 ml of heptane. The eluent of 10 ml of ethyl acetate was collected. The solution was dried over NaSO4. After evaporation of the solvent, the residue was dissolved in 20 μl of CH3CN. To the resulting solution was added 20 μl of 10% (v/v) pentafluorobenzyl bromide in acetonitrile and 10 μl of 10% (v/v) N,N-diisopropylethylamine in acetonitrile and the mixture was kept at room temperature for 30 min. The reagent was dried under nitrogen and the residue was subjected to TLC separation using the solvent ethyl acetate/methanol (98/2, v/v). Approximately 5 μg of the PFB ester of PGF2α was applied on a separate lane and visualized by spraying with a 10% solution of phosphomolybdic acid in ethanol followed by heating. Compounds migrating in the region of the PFB ester of PGF2α (R = 0.4) and the adjacent area 4 cm above and under were scrapped and extracted from the silica gel by ethyl acetate. The PFB esters after TLC separation can be directly used for LC-APCI/MS studies or further derivatized to TMS derivatives for GC-MS analysis. After evaporation of the ethyl acetate, 20 μl of N,O-bis-(trimethylsilyl)trifluoroacetamide and 10 μl of dimethylformamide were added to the residue and the mixture was incubated at 40 °C for 20 min. The reagents were dried under nitrogen and the derivatives were dissolved in 10 μl of dry undecane for analysis of GC-MS. Lipoprotein Isolation Procedures (26Havrilla C.M. Ph.D. dissertation. Duke University, Durham, NC2000Google Scholar)—Whole blood from fasting, normolipidemic healthy subjects was collected in a 440-ml ACD blood collection bag (Baxter-Fenwal). The blood bag was centrifuged at 4200 rpm for 10 min at 22 °C and the plasma was collected. LDL (1.019 < d < 1.063 g/ml) were isolated from plasma by sequential floatation ultracentrifugation (27Schmaker V.N. Pupione D.L. Methods Enzymol. 1986; 128: 155-170Google Scholar) at 14 °C using a Beckman Optima LE 80 centrifuge and a Ti 70 rotor. Each spin was performed at 504,000 × g for 5.5 h. Lipoproteins were dialyzed extensively against 50 mm phosphate-buffered saline, sterilized by passage through a Millipore Millex®-GV (0.22-μm Filter Unit), and stored at 4 °C under argon. Protein concentrations of the lipoprotein preparations were determined by the method of Lowry. LDL isolation was confirmed with the use of SDS-PAGE separation of associated apoproteins and Beckman LIPO gel electrophoresis of intact lipoproteins (28Sattler W. Mohr D. Stocker R. Methods Enzymol. 1994; 233: 269-489Google Scholar). Oxidation and Extraction of Lipoproteins—The lipoprotein concentrations were adjusted to either 1.5 mg of protein/ml or 0.75 mg of protein/ml with phosphate-buffered saline and allowed to equilibrate to 37 °C for 5 min in a round bottom flask before the initial aliquot was removed. Initiator C0 (100 mm stock solution) was added to the stirred solution to give the final C0 concentration, typically 1 mm for the 1.5 mg/ml LDL solution or 0.5 mm C0 for the 0.75 mg/ml LDL solution. Immediately after the addition of C0 (time 0) and at various time intervals, LDL aliquots (500 μl) were taken for analysis of the secondary oxidation products. Each of these aliquots was treated with 50 μl of butylated hydroxytoluene and the lipids were extracted with a Folch extraction (29Folch J. Lees M. Sloane Stanley G.H. J. Biol. Chem. 1956; 226: 497-509Google Scholar). For the Folch extraction procedure, to each 500-μl aliquot of LDL was added methanol (3.0 ml) and chloroform (6.0 ml) with a 30-s vortexing period in between each solvent addition. To the homogenous solution was added 0.74% aqueous KCl (1.75 ml) and the mixture was vortexed for 1 min to properly separate the phases. The solution was centrifuged for 10 min to separate the layers. The top phase was removed and discarded. The lower layer was treated with anhydrous sodium sulfate and placed on an ice bath for 15 min. The drying agent was filtered from the solution and the organic layer was concentrated under a stream of argon. The lipid residues were combined and transferred to a vial for storage. Separation of lipid classes of LDL was achieved by modification of a method reported by Homan et al. (30Homan R. Anderson M.K. J. Chromatogr. B Biomed. Appl. 1998; 708: 21-26Google Scholar) using an aminopropyl HPLC column by Milne (31Milne G.L. Ph.D. dissertation. Vanderbilt University, Nashville, TN2002Google Scholar). A mixed solvent systems was used, which includes Solvent A: hexanes-THF (99:1); Solvent B: acetone-CH2Cl2 (2:1); Solvent C: isopropyl alcohol/water (85:15). The separated lipid fractions, such as cholesterol esters and phospholipids, were derivatized for GC-MS analysis according to the protocol of derivatizing cholesteryl arachidonate oxidation products mentioned in the previous section. A solution of 5 mg of PGF3α was subjected to PFB esterification. The PFB ester was separated by flash chromatography using ethyl acetate and hexane (1:4). The structure of the product was characterized by 1H, 13C, and 19F NMR. The 1,3-diol on the cyclopentane ring was protected by formation of butylboronate. The C15 hydroxyl group was transformed to diethylphosphite by reaction of the protected PGF3α with triethylamine and chlorodiethylphosphite (32Nagata N. Kawakami M. Matsuura T. Saito I. Tetrahedron Lett. 1989; 30: 2817-2820Google Scholar). 15-OOH PGF3α PFB ester was synthesized by treatment of the phosphite with anhydrous H2O2. The butylboronate was removed in the same reaction. The separation of 15-OOH PGF3α PFB was achieved by HPLC using 12% isopropyl alcohol in hexane. The oxidation of 15-OOH PGF3α PFB was carried out in benzene with di-tert-butyl hyponitrite as radical initiator. The oxidation mixture was subject to MS analysis for the formation of dioxolane-isoprostane peroxide compounds. Formation of Dioxolane-Isoprostane Peroxides from 12-HPETE Ch by a Free Radical Mechanism—The oxidation of arachidonic acid gives rise to a complex mixture of products. Six different peroxyl radicals are formed from arachidonate and each of these peroxyl radicals can be converted to the corresponding HPETE by a hydrogen abstraction reaction. A set of cyclic peroxides and endoperoxides specific to each peroxyl radical can also form making analysis of the mixture difficult. The analytical problem can be significantly simplified by examination of products that form from specific peroxyl radicals. To do this, individual hydroperoxides (HPETEs) are used as starting materials for further oxidation. Thus 12-HPETE, when exposed to free radical initiators, serves as a precursor to the corresponding peroxyl radical. Consequently, the set of products derived only from the 12-peroxyl radical form when 12-HPETE is used as a starting material. Characterization of the oxidation products that derive from the individual hydroperoxides greatly simplifies the analytical problem and provides mechanistic insights for the autoxidation process (13Yin H. Havrilla C.M. Gao L. Morrow J.D. Porter N.A. J. Biol. Chem. 2003; 278: 16720-16725Google Scholar). To implement this strategy, we separated the regioisomeric hydroperoxides of cholesteryl arachidonate by semipreparative HPLC and exposed the pure regioisomeric hydroperoxides, namely the 15-, 11-, 12-, 8/9-, and 5-HPETEs to free radical initiators under air. We note that 8- and 9-HPETE Ch cannot be separated under normal phase HPLC conditions. Some preliminary results using this strategy have been previously reported for 15-, 11-, and 5-HPETE Ch (8Havrilla C.M. Hachey D.L. Porter N.A. J. Am. Chem. Soc. 2000; 122: 8042-8055Google Scholar). When 12-HPETE Ch was subjected to reaction under oxygen with free radical initiators, a complex mixture of cholesterol ester peroxides was generated and this mixture was subjected to a variety of analytical procedures, described below. Characterization of Dioxolane-Isoprostane Peroxides from Oxidation of 12-HPETE Ch by Silver CIS MS in Combination of HPLC—The characterization of organic peroxides by mass spectrometry has seen limited use because of the instability of these compounds (33Yin H. Hachey D.L. Porter N.A. Rapid Commun. Mass Spectrom. 2000; 14: 1248-1254Google Scholar, 34Rondeau D. Vogel R. Tabet J.-C. J. Mass Spectrom. 2003; 38: 931-940Google Scholar). Some structural information can be obtained from standard ionization techniques but hydroperoxides, in particular, have a propensity to dehydrate when subjected to many of the common ionization methods. Very recently, silver CIS MS has been successfully applied to study simple organic peroxides as well as the mixtures of peroxides generated from lipid autoxidation (8Havrilla C.M. Hachey D.L. Porter N.A. J. Am. Chem. Soc. 2000; 122: 8042-8055Google Scholar, 12Yin H. Havrilla C.M. Morrow J.D. Porter N.A. J. Am. Chem. Soc. 2002; 124: 7745-7754Google Scholar, 35Yin H. Hachey D.L. Porter N.A. J. Am. Soc. Mass Spectrom. 2001; 12: 449-455Google Scholar). Specific fragmentation at the peroxyl functionality occurs when silver ion is coordinated to double bonds or aromatics of unsaturated hydroperoxides. The oxidation mixture of 12-HPETE Ch was first analyzed by direct liquid infusion using a triple quadrupole mass spectrometer. The parent ion information is shown in Fig. 1. The doublet-like peaks are characteristic of silver adducts because silver has two isotopes, 107 and 109, having almost equal abundance. 12-HPETE Ch silver ion adducts are observed at m/z 811/813, whereas the peaks at m/z 793/795 and 795/797 correspond to the corresponding alcohol and ketone. The ketone and alcohol are products that result from the termination of two peroxyl radicals. The monocyclic peroxides and bicyclic endoperoxides (such as 2d and 2g, respectively) have m/z 843/845, whereas the peaks in the region m/z 827/829 would likely be because of termination products of the peroxyl radicals leading to these compounds. The peaks observed at m" @default.
• W2055065992 created "2016-06-24" @default.
• W2055065992 creator A5044445154 @default.
• W2055065992 creator A5085758047 @default.
• W2055065992 creator A5088915046 @default.
• W2055065992 date "2004-01-01" @default.
• W2055065992 modified "2023-10-03" @default.
• W2055065992 title "Identification of a Novel Class of Endoperoxides from Arachidonate Autoxidation" @default.
• W2055065992 cites W1449253847 @default.
• W2055065992 cites W1967419337 @default.
• W2055065992 cites W1971179233 @default.
• W2055065992 cites W1972153352 @default.
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• W2055065992 cites W1989984065 @default.
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• W2055065992 cites W2008532809 @default.
• W2055065992 cites W2014082305 @default.
• W2055065992 cites W2018716226 @default.
• W2055065992 cites W2020762921 @default.
• W2055065992 cites W2028927832 @default.
• W2055065992 cites W2029888633 @default.
• W2055065992 cites W2031633187 @default.
• W2055065992 cites W2035838781 @default.
• W2055065992 cites W2048018294 @default.
• W2055065992 cites W2054241405 @default.
• W2055065992 cites W2057522312 @default.
• W2055065992 cites W2063539595 @default.
• W2055065992 cites W2065502111 @default.
• W2055065992 cites W2065947716 @default.
• W2055065992 cites W2066233543 @default.
• W2055065992 cites W2068139017 @default.
• W2055065992 cites W2075256580 @default.
• W2055065992 cites W2080817989 @default.
• W2055065992 cites W2082004581 @default.
• W2055065992 cites W2083315608 @default.
• W2055065992 cites W2095014707 @default.
• W2055065992 cites W2106033501 @default.
• W2055065992 cites W2125827719 @default.
• W2055065992 cites W2168526937 @default.
• W2055065992 cites W2949615075 @default.
• W2055065992 cites W4246805334 @default.
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