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• W2059792530 abstract "Phospholipids reside in the surface layer of LDLs and constitute ∼20–25% of the particle by weight. We report a study of the primary products generated from the most abundant molecular species of phosphatidylcholines present in LDL during in vitro free radical oxidations. The 13-hydroperoxides of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC) and 1-stearoyl-2-linoleoyl-sn-glycero-phosphocholine (SLPC) and the 15-hydroperoxides of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) and 1-stearoyl-2-arachidonoyl-sn-glycero-phosphocholine (SAPC) were found to increase in a time-dependent manner and in significant amounts even in the presence of α-tocopherol. Phospholipid alcohols also formed during the course of the oxidations. Early in the LDL oxidations, while α-tocopherol was still present, the thermodynamically favored trans,trans products of PLPC and SLPC were found to form in significantly larger quantities than those formed from cholesteryl linoleate. Additionally, quantities of PAPC 11-hydroperoxide (11-OOH) decreased over time relative to PAPC 15-OOH, even while α-tocopherol was still present in the oxidation, presumably as a result of further oxidation of PAPC 11-OOH to form cyclic peroxide oxidation products.These results suggest that α-tocopherol is more closely associated with the inner cholesteryl ester-rich hydrophobic core of an LDL particle and is not as effective as an antioxidant in the outer phospholipid layer as it is in the lipid core. Phospholipids reside in the surface layer of LDLs and constitute ∼20–25% of the particle by weight. We report a study of the primary products generated from the most abundant molecular species of phosphatidylcholines present in LDL during in vitro free radical oxidations. The 13-hydroperoxides of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC) and 1-stearoyl-2-linoleoyl-sn-glycero-phosphocholine (SLPC) and the 15-hydroperoxides of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) and 1-stearoyl-2-arachidonoyl-sn-glycero-phosphocholine (SAPC) were found to increase in a time-dependent manner and in significant amounts even in the presence of α-tocopherol. Phospholipid alcohols also formed during the course of the oxidations. Early in the LDL oxidations, while α-tocopherol was still present, the thermodynamically favored trans,trans products of PLPC and SLPC were found to form in significantly larger quantities than those formed from cholesteryl linoleate. Additionally, quantities of PAPC 11-hydroperoxide (11-OOH) decreased over time relative to PAPC 15-OOH, even while α-tocopherol was still present in the oxidation, presumably as a result of further oxidation of PAPC 11-OOH to form cyclic peroxide oxidation products. These results suggest that α-tocopherol is more closely associated with the inner cholesteryl ester-rich hydrophobic core of an LDL particle and is not as effective as an antioxidant in the outer phospholipid layer as it is in the lipid core. Oxidation of LDLs is an important contributor to the pathogenesis of atherosclerosis. In vitro experimental evidence shows that oxidized LDL causes endothelial cells to recruit monocytes into the arterial wall. The monocytes are then transformed into macrophages, which take up the oxidatively modified LDL and eventually become foam cells, early-stage atherosclerotic plaques (1Steinberg D. Parthasarathy S. Carew T.E. Khoo J.C. Witztum J.L. Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity.N. Engl. J. Med. 1989; 320: 915-924Crossref PubMed Google Scholar). There is also evidence to support the presence of oxidized LDL in vivo. Oxidized LDL has been extracted from atherosclerotic lesions (2Yla-Herttuala S. Palinski W. Rosenfeld M.E. Parthasarathy S. Carew T.E. Butler S. Witztum J.L. Steinberg D. Evidence for the presence of oxidatively modified LDL in atherosclerotic lesions of rabbit and man.J. Clin. Invest. 1989; 84: 1086-1095Crossref PubMed Google Scholar), autoantibodies reactive with oxidized LDL are present in plasma and atherosclerotic plaques of humans and animals (3Palinski W. Horkko S. Miller E. Steinbrecher U.P. Powell H.C. Curtiss L.K. Witztum J.L. Cloning of monoclonal antibodies to epitopes of oxidized lipoproteins from apo-E-deficient mice. Demonstration of epitopes of oxidized LDL in human plasma.J. Clin. Invest. 1996; 98: 800-814Crossref PubMed Scopus (499) Google Scholar and references therein), and small amounts of oxidized LDL have been found in circulating plasma (4Itabe H. Yamamoto H. Imanaka T. Shimamura K. Uchiyama H. Kimura J. Sanaka T. Hata Y. Takano T. Sensitive detection of oxidatively modified LDL using a monoclonal antibody.J. Lipid Res. 1996; 37: 45-53Abstract Full Text PDF PubMed Google Scholar, 5Holvoet P. Theilmeier G. Shivalkar B. Flameng W. Collen D. LDL hypercholesterolemia is associated with accumulation of oxidized LDL, atherosclerotic plaque growth, and compensatory vessel enlargement in coronary arteries of minature pigs.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 415-422Crossref PubMed Scopus (96) Google Scholar). However, despite the vast knowledge of the proatherogenic properties of oxidized LDL, little is known about the mechanism of oxidation in vivo or about which compounds in oxidized LDL are responsible for these properties.Phospholipids are primary targets of oxidation in an LDL particle. This lipid class resides in the surface layer of LDL particles and makes up ∼20–25% of the whole particle (by weight) (6Esterbauer H. Dieber-Rotheneder M. Waeg G. Striegl G. Jurgens G. Biochemical, structural, and functional properties of oxidized low-density lipoprotein.Chem. Res. Toxicol. 1990; 3: 77-92Crossref PubMed Scopus (475) Google Scholar). The structures of a number of bioactive phospholipid oxidation products have been identified in in vitro oxidations of LDL. These products include secondary phospholipid oxidation products, such as isoprostane-containing phosphatidylcholines (PCs), epoxyisoprostane PC (7Watson A.D. Subbanagounder G. Welsbie D.S. Faull K.F. Navab M. Jung M.E. Fogelman A.M. Berliner J.A. Structural identification of a novel pro-inflammatory epoxyisoprostane phospholipid in mildly oxidized LDL.J. Biol. Chem. 1999; 274: 24787-24798Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), and PCs containing an sn-2 acyl group with a terminal γ-hydroxy(or oxo)-α,β-unsaturated carbonyl (8Podrez E.A. Poliakov E. Shen Z. Zhang R. Deng Y. Sun M. Finton P.J. Shan L. Gugiu B. Fox P.L. Hoff H.F. Salomon R.G. Hazen S.L. Identification of a novel family of oxidized phospholipids that serve as ligands for the macrophage scavenger receptor CD36.J. Biol. Chem. 2002; 277: 38503-38516Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 9Podrez E.A. Poliakov E. Shen Z. Zhang R. Deng Y. Sun M. Finton P.J. Shan L. Febbraio M. Hajjar D.P. Silverstein R.L. Hoff H.F. Salomon R.G. Hazen S.L. A novel family of atherogenic oxidized phospholipids promotes macrophage foam cell formation via the scavenger receptor CD36 and is enriched in atherosclerotic lesions.J. Biol. Chem. 2002; 277: 38517-38523Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar), as well as decomposition products of both ester- and ether-containing oxidized glycerophospholipids in which fragmentation of the sn-2 fatty acid has occurred (10, 11 and references therein).Many of these highly oxidized glycerophospholipids have been shown to be involved in biological events associated with atherosclerosis. In vitro studies show that some of these molecules can stimulate the binding of monocytes to endothelial cells (11Berliner J.A. Subbanagounder G. Leitinger N. Watson A.D. Vora D. Evidence for a role of phospholipid oxidation products in atherogenesis.Trends Cardiovasc. Med. 2001; 11: 142-147Crossref PubMed Scopus (152) Google Scholar, 12Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. Structural identification by MS of oxidized phospholipids in minimally oxidized LDL that induce monocyte/endothelial interactions and evidence for their presence in vivo.J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar, 13Leitinger N. Tyner T.R. Oslund L. Rizza C. Subbanagounder G. Lee H. Shih P.T. Mackman N. Tigyi G. Territo M.C. Berliner J.A. Vora D. Structurally similar oxidized phospholipids differentially regulate endothelial binding of monocytes and neutrophils.Proc. Natl. Acad. Sci. USA. 1999; 96: 12010-12015Crossref PubMed Scopus (227) Google Scholar), whereas other molecules cause monocyte activation and other inflammatory responses (10Marathe G.K. Prescott S.M. Zimmerman G.A. McIntyre T.M. Oxidized LDL contains inflammatory PAF-like phospholipids.Trends Cardiovasc. Med. 2001; 11: 139-142Crossref PubMed Scopus (100) Google Scholar, 14Marathe G.K. Davies S.S. Harrison K.A. Silva A.R. Murphy R.C. Casro-Faria-Neto H. Prescott S.M. Zimmerman G.A. McIntyre T.M. Inflammatory PAF-like phospholipids in oxidized LDL are fragmented alkyl phosphatidylcholines.J. Biol. Chem. 1999; 274: 28395-28404Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Also, it has been reported that PCs containing a terminal γ-hydroxy(or oxo)-α,β-unsaturated carbonyl at the sn-2 position have a high binding affinity for the macrophage scavenger receptor CD36 (8Podrez E.A. Poliakov E. Shen Z. Zhang R. Deng Y. Sun M. Finton P.J. Shan L. Gugiu B. Fox P.L. Hoff H.F. Salomon R.G. Hazen S.L. Identification of a novel family of oxidized phospholipids that serve as ligands for the macrophage scavenger receptor CD36.J. Biol. Chem. 2002; 277: 38503-38516Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). This protein is expressed on the surface of endothelial cells and macrophages and has been found to play a key role in the uptake of oxidized LDL and the onset of atherosclerosis.Not only have these bioactive phospholipids been identified in vitro, but there is also evidence suggesting that these compounds exist in vivo in atherosclerotic lesions and plasma (11Berliner J.A. Subbanagounder G. Leitinger N. Watson A.D. Vora D. Evidence for a role of phospholipid oxidation products in atherogenesis.Trends Cardiovasc. Med. 2001; 11: 142-147Crossref PubMed Scopus (152) Google Scholar, 12Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. Structural identification by MS of oxidized phospholipids in minimally oxidized LDL that induce monocyte/endothelial interactions and evidence for their presence in vivo.J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar, 15Schlame M. Haupt R. Wiswedel I. Kox W.J. Rustow B. Identification of short-chain oxidized phosphatidylcholine in human plasma.J. Lipid Res. 1996; 37: 2608-2615Abstract Full Text PDF PubMed Google Scholar, 16Itabe H. Oxidized phospholipids as a new landmark in atherosclerosis.Prog. Lipid Res. 1998; 37: 181-207Crossref PubMed Scopus (84) Google Scholar, 17Morrow J.D. Awad J.A. Boss H.J. Blair I.A. Roberts I.L.J. Non-cyclooxygenase-derived prostanoids (F2-isoprostanes) are formed in situ on phospholipids.Proc. Natl. Acad. Sci. USA. 1992; 89: 10721-10725Crossref PubMed Scopus (664) Google Scholar). Several antibodies that were developed against oxidized LDL have shown specific binding to decomposition products of 1-palmitoyl-2-linoleoyl-sn-glycero-3-PC (PLPC), a mixture of oxidation products from 1-palmitoyl-2-arachidinoyl-sn-glycero-3-PC (PAPC), or phospholipid-apolipoprotein B-100 adducts (16Itabe H. Oxidized phospholipids as a new landmark in atherosclerosis.Prog. Lipid Res. 1998; 37: 181-207Crossref PubMed Scopus (84) Google Scholar, 18Horkko S. Bird D.A. Miller E. Itabe H. Leitinger N. Subbanagounder G. Berliner J.A. Friedman P. Dennis E.A. Curtiss L.K. Palinski W. Witztum J.L. Monoclonal antibodies specific for oxidized phospholipids or oxidized phospholipid-protein adducts inhibit macrophage uptake of oxidized LDL.J. Clin. Invest. 1999; 103: 117-128Crossref PubMed Scopus (468) Google Scholar). These antibodies did not show this specific binding to oxidized free fatty acids, oxidized cholesteryl esters, or oxidized apolipoprotein B that was not modified by phospholipids. However, the antibodies did immunohistochemically stain plasma or atherosclerotic lesions from humans, demonstrating that there is accumulation of oxidized phospholipids in vivo, presumably from oxidized LDL. Electrospray ionization tandem mass spectrometry (MS/MS) analysis of phospholipids extracted from atherosclerotic lesions and plasma gives additional proof of the existence of highly oxidized phospholipids in vivo (12Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. Structural identification by MS of oxidized phospholipids in minimally oxidized LDL that induce monocyte/endothelial interactions and evidence for their presence in vivo.J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar).It seems clear from this discussion that phospholipid oxidation products are of great importance in the pathogenesis of atherosclerosis. However, most of the research reported in the literature has focused on the secondary oxidation products or the decomposition products of phospholipids. There is very little research studying the early-stage oxidation of phospholipids in LDL and the effect of naturally occurring antioxidants, primarily α-tocopherol, on this oxidation. Studying these aspects of LDL oxidation could provide insight into how one might increase the resistance of LDL to oxidation.Noguchi, Gotoh, and Niki (19Noguchi N. Gotoh N. Niki E. Dynamics of the oxidation of low density lipoprotein induced by free radicals.Biochim. Biophys. Acta. 1993; 1168: 348-357Crossref PubMed Scopus (111) Google Scholar) and Stocker and coworkers (20Stocker R. Bowry V.W. Frei B. Ubiquinol-10 protects human LDL more efficiently against lipid peroxidation than does alpha-tocopherol.Proc. Natl. Acad. Sci. USA. 1991; 88: 1646-1650Crossref PubMed Scopus (766) Google Scholar, 21Mohr D. Stocker R. Radical-mediated oxidation of isolated human VLDL.Arterioscler. Thromb. 1994; 14: 1186-1192Crossref PubMed Google Scholar) both have studied the early formation of phospholipid hydroperoxides in oxidations of human lipoproteins. These workers monitored the formation of phospholipid hydroperoxides over time using a method (22Sattler W. Mohr D. Stocker R. Rapid isolation of lipoproteins and assessment of their peroxidation by HPLC postcolumn chemiluminescence.Methods Enzymol. 1994; 233: 469-489Crossref PubMed Scopus (284) Google Scholar) adapted from one first developed by Yamamoto and colleagues (23Yamamoto Y. Ames B.N. Detection of lipid hydroperoxides and hydrogen peroxide at picomole levels by an HPLC and isoluminol chemiluminescence assay.Free Radic. Biol. Med. 1987; 3: 359-361Crossref PubMed Scopus (76) Google Scholar, 24Yamamoto Y. Brodsky M.H. Baker J.C. Ames B.N. Detection and characterization of lipid hydroperoxides at picomole levels by HPLC.Anal. Biochem. 1987; 160: 7-13Crossref PubMed Scopus (363) Google Scholar). This method does not chromatographically separate individual phospholipid oxidation products, however, and no information about the structure of these phospholipid hydroperoxide molecular species was obtained.Previously, we reported the development of an HPLC coordination ion-spray mass spectrometry (LC-CIS-MS) method for the analysis and identification of phospholipid hydroperoxides, the primary products expected during the early stages of oxidation in LDL (25Milne G.L. Porter N.A. Separation and identification of phospholipid peroxidation products.Lipids. 2001; 36: 1265-1275Crossref PubMed Scopus (38) Google Scholar). In the studies reported here, we used this method to identify and quantify the formation of hydroperoxides from the most abundant molecular species of PC in free radical-initiated in vitro LDL oxidations. Also, we compared the oxidation of the phospholipids in LDL with that of the cholesteryl esters, the most abundant lipid found in the hydrophobic core of LDL, to gain insight into how endogenous antioxidants (e.g., α-tocopherol) influence oxidation in the different regions of LDL.EXPERIMENTAL PROCEDURESMaterialsPhospholipids were purchased from Avanti Polar Lipids (Alabaster, AL) or from Sigma Chemical Co. (St. Louis, MO) and used without further purification. PLPC was purchased from Sigma as a powder, and PAPC and 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphatidylcholine (SAPC) were purchased from Sigma as chloroform solutions. 1-Stearoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (SLPC) was purchased as a powder from Avanti Polar Lipids. Soybean lipoxygenase (type I-B) and phospholipase D (from Streptomyces species; type VII) were purchased as lyophilized powders from Sigma Chemical Co. The free radical initiator 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (C-0) was generously donated by Wako Chemicals USA, Inc. (Richmond, VA). All chemicals used to make buffers for the LDL experiments were purchased from Sigma Chemical Co. and were of the highest quality (SigmaUltrapure). Solvents were HPLC quality and purchased from either Fisher Chemical (Phillipsburg, NJ) or EM Science (Gibbstown, NJ). Hexanes was purchased from Burdick and Jackson (Muskegon, MI). All other reagents were purchased from Aldrich Chemical Co. (Milwaukee, WI) and used without further purification.In general, hydroperoxides and extracts of oxidized lipids from LDL were stored as dilute solutions with 1 mol% butylated hydroxytoluene (BHT) in benzene at −78°C and never exposed to temperatures >40°C.InstrumentsHPLCAnalytical HPLC was conducted using a Waters model 2690 Alliance Separations Model instrument with a Waters 996 photodiode array detector connected to a Waters Millennium32 (version 3.20) chromatography station. Semipreparative HPLC was conducted on a Waters model 600E HPLC instrument, with either a Waters model 481 variable wavelength detector or a Waters 2487 dual wavelength absorbance detector and with output to a strip chart recorder.Mass spectrometryCIS-MS was performed using either one of two systems. 1) A ThermoFinnigan Thermoquest TSQ-7000 (San Jose, CA) triple quadrupole mass spectrometer equipped with a standard API-1 electrospray ionization source outfitted with a 100 mm deactivated fused Si capillary. Data acquisition and spectral analysis were conducted using ICIS software (version 8.3.2) running on a Digital Equipment Alpha Station 200 4/166. Nitrogen gas served as both the sheath gas and the auxiliary gas; argon served as the collision gas. The electrospray needle was maintained at 4.6 kV, and the heated capillary temperature was 250°C. The tube lens and capillary voltages were 90 and 10 V, respectively, and the sheath and auxiliary gases were at 60 and 5–10 pounds per square inch, respectively. For MS/MS experiments, collision gas was typically maintained at a pressure of 2.6–2.9 mTorr, and the offset was 25 eV. For online HPLC sample introduction, a Waters model 2690 Alliance Separations Model instrument was used. 2) A ThermoFinnigan TSQ Quantum 1.0 SR 1 mass spectrometer coupled with a Surveyor MS Pump 2.0 and a Surveyor Autosampler 1.3 (San Jose, CA). Nitrogen gas served as both the sheath gas and the auxiliary gas; argon served as the collision gas. The electrospray needle was maintained at 5 kV, and the capillary temperature was 310°C. The tube lens voltage was 194 V, and the sheath and auxiliary gases were 49 pounds per square inch and 25 units, respectively. For MS/MS experiments, the collision gas was maintained at 1.5 mTorr, and the offset was 35 eV. Data processing was conducted using Xcaliber software (version 1.3).MethodsSynthesis of d9-PLPC, d9-PAPC, d9-SLPC, and d9-SAPCPLPC, PAPC, SLPC, or SAPC (25 mg, ∼0.03 mmol) was dissolved in 1 ml of CH2Cl2. d9-Choline (>98 atom% D, 29 mg, 0.195 mmol) was dissolved in buffer (500 μl) containing 100 mM sodium acetate and 50 mM calcium chloride at pH 6.5 and added to each phospholipid solution. Phospholipase D (50 μl of a 1 U/μl solution in 100 mM sodium acetate) was then added to each reaction. The biphasic reaction mixtures were shaken at 190 rpm for 4 h at 30°C. The reactions were stopped by Folch extraction. The d9-phospholipids were purified immediately by semipreparative HPLC [Discovery C18 semi-prep column, 21.2 mm × 25 cm; mobile phase: 100% methanol, 10 ml/min; ultraviolet (UV) detection, λ = 210 nm].Synthesis of d9-PC hydroperoxidesSoybean lipoxygenase (4 mg for PLPC and SLPC, 26 mg for PAPC and SAPC) was taken up in 5.5 ml of borax buffer that contained 10 mM deoxycholate at pH 9.0. d9-PLPC, d9-PAPC, d9-SLPC, or d9-SAPC (5 mg, 0.006 mmol) was taken up in 1 ml of the same buffer and added to the enzyme solution (Fig. 1). The reaction was stirred at room temperature for 30–60 min. The progress of the reaction was monitored by UV. The reaction was stopped by Folch extraction. To synthesize the alcohol internal standards, the hydroperoxides were made using this procedure and then reduced with triphenylphosphine (PPh3). The structures of the synthesized standards are shown in Fig. 2. The isomeric purity of the d9-phospholipid hydroperoxides and alcohols were checked by analytical HPLC [Discovery C18 analytical column, 4.6 × 250 mm; mobile phase: methanol-water (95:5), 1 ml/min; UV detection, λ = 234 nm]. The standards were then purified immediately by semipreparative HPLC [Discovery C18 semi-prep column, 21.2 mm × 25 cm; mobile phase: 100% methanol, 10 ml/min; UV detection, λ = 210 nm].Fig. 2Structures of the eight internal standards designed to quantify the amount of 1-palmitoyl-2-linoleoyl-sn-glycero-3-PC (PLPC), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-PC (PAPC), 1-stearoyl-2-linoleoyl-sn-glycero-3-PC (SLPC), and 1-stearoyl-2-arachidonoyl-sn-glycero-3-PC (SAPC) hydroperoxides and alcohols formed during the autoxidation of LDL.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Quantitation and calibration of the d9-PC hydroperoxides and d9-PC alcoholsThe amount of each pure oxidized internal standard synthesized was quantified by UV using the known ε value for the 13-cis,trans-hydroperoxide (13-cis,trans-OOH) of linoleates (26Chan H. W-S. Levett G. Autoxidation of methyl linoleate. Separation and analysis of isomeric mixtures of methyl linoleate hydroperoxides and methyl hydroxylinoleates.Lipids. 1977; 12: 99-104Crossref PubMed Scopus (383) Google Scholar) (ε = 27,200) and for arachidonate hydroperoxides (27Porter N.A. Logan J. Kontoyiannidou V. The preparation and purification of arachidonic acid hydroperoxides of biological importance.J. Org. Chem. 1979; 44: 3177-3181Crossref Scopus (154) Google Scholar) (ε = 27,000). Calibration curves were done using LC-CIS-MS/MS. The mass spectrometer was operated in selected reaction monitoring (SRM) mode to monitor the precursor-to-product transition of the PC-OOHs to their corresponding Hock fragments or the loss of the phosphocholine head group from the PC alcohols (PC-OHs). The deuterated phospholipid standards eluted from the HPLC as follows: 13-cis,trans-PLPC-OOH, t = 23.40 min; 13-cis,trans-PLPC-OH, t = 23.92 min; 13-cis,trans-SLPC-OOH, t = 38.83 min; 13-cis,trans-SLPC-OH, t = 39.53 min; 15-PAPC-OOH, t = 26.08 min; 15-PAPC-OH, t = 23.38 min; 15-SAPC-OOH, t = 43.39 min; 15-SAPC-OH, t = 42.57 min.Quantitation and calibration of the unoxidized d9-PLPCAfter synthesis, the amount of d9-PLPC was determined by weighing. The calibration curve was done using LC-CIS-MS/MS. The mass spectrometer was operated in SRM mode to monitor the loss of the phosphocholine head group from the PLPC parent.Autoxidation of PAPCTo a solution of PAPC (5 mg, 0.006 mmol) in 1.5 ml of CH2Cl2 was added either 0.1 or 0.025 equivalent of 2,2,5,7,8-pentamethyl-6-chromanol (PMC; 64 or 16 μl, respectively, of a 10 mM stock solution in CH2Cl2). The solution was evaporated to dryness under a stream of argon so that the mixture formed a thin layer on the inside of a 4 ml vial. The vial was then heated to 37°C and exposed to an atmosphere of dry air. After 24 h, the mixture was dissolved in benzene and BHT (∼1–2 mg) was added to stop the reaction. The hydroperoxides were analyzed by HPLC.Isolation and oxidation of LDLLDL was isolated from the whole blood of fasting, normolipidemic healthy subjects as previously described (28Culbertson S.M. Antunes F. Havrilla C.M. Milne G.L. Porter N.A. Determination of the alpha-tocopherol inhibition rate constant for peroxidation in low-density lipoprotein.Chem. Res. Toxicol. 2002; 15: 870-876Crossref PubMed Scopus (21) Google Scholar). All blood donations were collected in accordance with guidelines established by the Institutional Review Board at Vanderbilt University (study protocol number 99073), and all donors gave written informed consent. Protein concentration was determined using the modified Lowry (29Lowry T.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) assay reported by Morton and Evans (30Morton R.E. Evans T.A. Modification of the bicinchoninic acid protein assay to eliminate lipid interference in determining lipoprotein protein content.Anal. Biochem. 1992; 204: 332-334Crossref PubMed Scopus (139) Google Scholar). SDS-PAGE gels, run on a Ciba-Corning electrophoresis system, and LipoGels, run on a Beckman Instruments Paragon LipoGel system, were used to determine the purity of the LDL.Before oxidation, the concentration of the LDL was adjusted to 0.75 mg/ml with PBS. The solution was then magnetically stirred and allowed to equilibrate at 37°C for 5 min. The initiator, either C-0 (50 mM stock solution in PBS) or 2,2′-azobis(amidinopropane) dihydrochloride (AAPH; 50 mM stock solution in methanol) was then added to give a final initiator concentration of either 1 or 0.5 mM, respectively.For phospholipid analysis, 1.0–1.5 ml aliquots were removed at various time intervals. BHT (100 μl of a 3 mM solution in methanol) was added to all aliquots to stop the oxidation. Each aliquot was immediately extracted with ice-cold CH2Cl2 (6 ml), methanol (3 ml), and 0.74% KCl (1.75ml) in sequence. The samples were vortexed after the addition of each solvent and were then centrifuged at 1,700 rpm for 5 min. The organic phase was concentrated under a stream of dry argon, and the resulting residue was stored as a dilute solution in benzene until analysis.The lipid classes in each aliquot were separated using HPLC equipped with an aminopropyl column (Supelco -NH2 column; 3.0 × 250 mm). The gradient solvent program used in this separation method employed mixtures of hexanes/tetrahydrofuran (THF) (99:1) and isopropanol-water (85:15) to elute the different lipid classes and acetone/CH2Cl2 (2:1) to wash the column. The gradient program is shown in Table 1. The cholesteryl esters and other neutral species, including the antioxidants present in LDL, elute between 2 and 5 min, whereas the phospholipids elute between 28 and 30 min.TABLE 1HPLC solvent system used to separate lipid classes on a Supelco aminopropyl analytical columnTimeFlow RateSolvent ASolvent BSolvent Cminml/min% by volume01.610000201.610000251.640060361.640060402.001000452.001000501.610000601.610000Solvent A, hexanes/tetrahydrofuran (THF) (99:1); solvent B, acetone/CH2Cl2 (2:1); solvent C, isopropanol-water (85:15). Open table in a new tab For cholesteryl ester analysis, LDL was oxidized as described above and 200 μl aliquots were removed at various time points. To each aliquot, BHT (20 μl, 3.0 mM in ethanol) was added. Because many of the cholesteryl linoleate and cholesteryl arachidonate hydroperoxides coelute, the hydroperoxides were converted to the corresponding alcohols by the addition of PPh3 (20–30 μl, 25 mM in ethanol) to each aliquot. Aliquots were extracted with ice-cold methanol (1.0 ml) and ice-cold hexane (5.0 ml) in sequence, vortexing vigorously after the addition of each solvent (15 s), and then centrifuged at 1,700 rpm for ∼1 min. The hexane phase was retained, concentrated under argon, and reconstituted in 200–300 μl of mobile phase for analysis.Quantification of PC-OOHs and PC-OHs in LDL oxidationsFor these experiments, 1 ml aliquots were taken from the LDL oxidation. A known amount of a mixture containing the eight oxidized internal standards was added to aliquots at later times. After work-up and separation of the lipid classes, each phospholipid aliquot was taken up in 2 ml of methanol and subsequently analyzed by LC-CIS-MS/MS [Discovery C-18 microbore column, 1.0 mm × 30 cm, 5-μm (Supelco); mobile phase: 100% methanol, 0.085 ml/min; 16 μl injections] operating in the SRM mode to monitor the precursor-to-product transition of either the PC-OOHs fragmenting to form their corresponding Hock fragments, a common Ag+ CIS-MS/MS fragmentation pathway for hydroperoxides (31Hock H. Schrader O. Der Mechanismus der autoxydation einfacher Kohlenwasserstoffe als beitrag zur Kentnis der Autoxydation von brennstoffen.Angew. Chem. 1936; 49: 595-596Crossref Google Scholar, 32Havrilla C.M. Hachey D.L. Porter N.A. Coordination (Ag+) ion spray-mass spectrometry of peroxidation products of cholesteryl linoleate and cholesteryl arachidonate: HPLC-MS analysis of peroxide products from polyunsaturated lipid autoxidation" @default.
• W2059792530 created "2016-06-24" @default.
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• W2059792530 date "2005-02-01" @default.
• W2059792530 modified "2023-09-26" @default.
• W2059792530 title "Identification and analysis of products formed from phospholipids in the free radical oxidation of human low density lipoproteins" @default.
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