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• W2047026433 abstract "Numerous studies have suggested relationships between myeloperoxidase (MPO), inflammation, and atherosclerosis. MPO-derived reactive chlorinating species attack membrane plasmalogens releasing α-chloro fatty aldehydes including 2-chlorohexadecanal (2-ClHDA), which have been found to accumulate in activated neutrophils, activated monocytes, infarcted myocardium and human atheromas. The present study employed synthetically prepared 2-Cl-[3H]-HDA as well as stable isotope-labeled 2-ClHDA to elucidate the metabolism of 2-ClHDA. The results herein demonstrate that human coronary artery endothelial cells oxidize and reduce 2-ClHDA to its respective chlorinated fatty acid (α-ClFA) and chlorinated fatty alcohol (α-ClFOH). Within the first hour of incubations of human coronary artery endothelial cells with 2-Cl-[3H]-HDA, the label was incorporated into the α-ClFOH and α-ClFA pools. After 1 h, the radiolabel was predominantly found in the α-ClFOH pool. Cell-derived α-ClFOH and α-ClFA were also released into the cell culture medium. Additionally, chlorinated fatty acid was incorporated into complex endothelial cell glycerolipids, including monoglycerides, triglycerides, phosphatidylcholine, and phosphatidylethanolamine. The oxidation and reduction of 2-ClHDA to α-ClFA and α-ClFOH, respectively, was further supported by mass spectrometric analyses of human coronary artery endothelial cells incubated with either 2-ClHDA or stable isotope-labeled 2-ClHDA (2-Cl-[d4]-HDA). 2-ClHDA was also oxidized to α-ClFA and reduced to α-ClFOH in both control and phorbol 12-myristate 13-acetate-stimulated neutrophils. Taken together, these results show that a family of chlorinated lipidic metabolites is produced from α-chloro fatty aldehydes derived from reactive chlorinating species targeting of plasmalogens. These metabolites are incorporated into complex lipids and their biological roles may provide new insights into MPO-mediated disease. Numerous studies have suggested relationships between myeloperoxidase (MPO), inflammation, and atherosclerosis. MPO-derived reactive chlorinating species attack membrane plasmalogens releasing α-chloro fatty aldehydes including 2-chlorohexadecanal (2-ClHDA), which have been found to accumulate in activated neutrophils, activated monocytes, infarcted myocardium and human atheromas. The present study employed synthetically prepared 2-Cl-[3H]-HDA as well as stable isotope-labeled 2-ClHDA to elucidate the metabolism of 2-ClHDA. The results herein demonstrate that human coronary artery endothelial cells oxidize and reduce 2-ClHDA to its respective chlorinated fatty acid (α-ClFA) and chlorinated fatty alcohol (α-ClFOH). Within the first hour of incubations of human coronary artery endothelial cells with 2-Cl-[3H]-HDA, the label was incorporated into the α-ClFOH and α-ClFA pools. After 1 h, the radiolabel was predominantly found in the α-ClFOH pool. Cell-derived α-ClFOH and α-ClFA were also released into the cell culture medium. Additionally, chlorinated fatty acid was incorporated into complex endothelial cell glycerolipids, including monoglycerides, triglycerides, phosphatidylcholine, and phosphatidylethanolamine. The oxidation and reduction of 2-ClHDA to α-ClFA and α-ClFOH, respectively, was further supported by mass spectrometric analyses of human coronary artery endothelial cells incubated with either 2-ClHDA or stable isotope-labeled 2-ClHDA (2-Cl-[d4]-HDA). 2-ClHDA was also oxidized to α-ClFA and reduced to α-ClFOH in both control and phorbol 12-myristate 13-acetate-stimulated neutrophils. Taken together, these results show that a family of chlorinated lipidic metabolites is produced from α-chloro fatty aldehydes derived from reactive chlorinating species targeting of plasmalogens. These metabolites are incorporated into complex lipids and their biological roles may provide new insights into MPO-mediated disease. Phagocytes are important mediators of host defense mechanisms against microbes (1Daugherty A. Dunn J.L. Rateri D.L. Heinecke J.W. J. Clin. Invest. 1994; 94: 437-444Crossref PubMed Scopus (1128) Google Scholar, 2Harrison J.E. Schultz J. J. Biol. Chem. 1976; 251: 1371-1374Abstract Full Text PDF PubMed Google Scholar, 3Sepe S.M. Clark R.A. J. Immunol. 1985; 134: 1896-1901PubMed Google Scholar). The concomitant release of myeloperoxidase (MPO) 2The abbreviations used are: MPO, myeloperoxidase; RCS, reactive chlorinating species; HOCl, hypochlorous acid; FA, fatty acid; α-ClFA, α-chloro fatty acid; α-Cl-FALD, α-chloro fatty aldehyde; α-ClFOH, α-chloro fatty alcohol; 2-ClHDA, 2-chlorohexadecanal; HCAEC, human coronary artery endothelial cells; GC-MS, gas chromatographymass spectrometry; LC-MS, liquid chromatography-mass spectrometry; SIM, selected ion monitoring; SRM, selected reaction monitoring; PC, phosphatidylcholine; PFB, pentafluorobenzoyl; FAME, fatty acid methyl ester; eNOS, endothelial nitric-oxide synthase; TLC, thin layer chromatography. with its substrate, H2O2, by phagocytes results in the production of the reactive chlorinating species (RCS), hypochlorous acid (HOCl), which is a bactericidal agent (4Klebanoff S.J. Waltersdorph A.M. Rosen H. Methods Enzymol. 1984; 105: 399-403Crossref PubMed Scopus (209) Google Scholar). The production of HOCl by MPO amplifies the oxidizing potential of reactive oxygen species produced by the phagocyte respiratory burst (2Harrison J.E. Schultz J. J. Biol. Chem. 1976; 251: 1371-1374Abstract Full Text PDF PubMed Google Scholar). HOCl elicits its antimicrobial actions and cytotoxicity by targeting multiple critical molecules including proteins, nucleic acids, and lipids (5Albrich J.M. McCarthy C.A. Hurst J.K. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 210-214Crossref PubMed Scopus (387) Google Scholar, 6Weiss S.J. Klein R. Slivka A. Wei M. J. Clin. Invest. 1982; 70: 598-607Crossref PubMed Scopus (677) Google Scholar, 7Thomas E.L. Jefferson M.M. Grisham M.B. Biochemistry. 1982; 21: 6299-6308Crossref PubMed Scopus (108) Google Scholar, 8Winterbourn C.C. van den Berg J.J. Roitman E. Kuypers F.A. Arch. Biochem. Biophys. 1992; 296: 547-555Crossref PubMed Scopus (233) Google Scholar, 9Heinecke J.W. Li W. Mueller D.M. Bohrer A. Turk J. 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Invest. 1997; 99: 2075-2081Crossref PubMed Scopus (753) Google Scholar, 16Domigan N.M. Charlton T.S. Duncan M.W. Winterbourn C.C. Kettle A.J. J. Biol. Chem. 1995; 270: 16542-16548Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). In addition to the antimicrobial actions of MPO-derived HOCl, it also targets host tissues and likely participates in the pathophysiological sequelae of several cardiovascular diseases, including atherosclerosis and myocardial ischemia-reperfusion injury (17Thukkani A.K. McHowat J. Hsu F.F. Brennan M.L. Hazen S.L. Ford D.A. Circulation. 2003; 108: 3128-3133Crossref PubMed Scopus (176) Google Scholar, 18Thukkani A.K. Martinson B.D. Albert C.J. Vogler G.A. Ford D.A. Am. J. Physiol. 2005; 288: H2955-H2964Crossref PubMed Scopus (69) Google Scholar, 19Sugiyama S. Okada Y. Sukhova G.K. Virmani R. Heinecke J.W. Libby P. Am. J. Pathol. 2001; 158: 879-891Abstract Full Text Full Text PDF PubMed Scopus (611) Google Scholar, 20Vasilyev N. Williams T. Brennan M.L. Unzek S. Zhou X. Heinecke J.W. Spitz D.R. Topol E.J. Hazen S.L. Penn M.S. Circulation. 2005; 112: 2812-2820Crossref PubMed Scopus (148) Google Scholar, 21Askari A.T. Brennan M.L. Zhou X. Drinko J. Morehead A. Thomas J.D. Topol E.J. Hazen S.L. Penn M.S. J. Exp. Med. 2003; 197: 615-624Crossref PubMed Scopus (205) Google Scholar, 22McMillen T.S. Heinecke J.W. LeBoeuf R.C. Circulation. 2005; 111: 2798-2804Crossref PubMed Scopus (147) Google Scholar). Recently, RCS have been shown to target the plasmalogen molecular subclass of phospholipids, resulting in the unmasking of the plasmalogenic vinyl ether aliphatic group as an α-chloro fatty aldehyde (23Albert C.J. Crowley J.R. Hsu F.F. Thukkani A.K. Ford D.A. J. Biol. Chem. 2001; 276: 23733-23741Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). This halogenated aldehyde accumulates in both human atherosclerotic lesions and infarcted rat myocardium (17Thukkani A.K. McHowat J. Hsu F.F. Brennan M.L. Hazen S.L. Ford D.A. Circulation. 2003; 108: 3128-3133Crossref PubMed Scopus (176) Google Scholar, 18Thukkani A.K. Martinson B.D. Albert C.J. Vogler G.A. Ford D.A. Am. J. Physiol. 2005; 288: H2955-H2964Crossref PubMed Scopus (69) Google Scholar). Furthermore, at physiological concentrations, α-chloro fatty aldehyde is a chemoattractant and has been shown to elicit cardiac injury and ventricular dysfunction (18Thukkani A.K. Martinson B.D. Albert C.J. Vogler G.A. Ford D.A. Am. J. Physiol. 2005; 288: H2955-H2964Crossref PubMed Scopus (69) Google Scholar, 24Thukkani A.K. Hsu F.F. Crowley J.R. Wysolmerski R.B. Albert C.J. Ford D.A. J. Biol. Chem. 2002; 277: 3842-3849Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The accumulation of α-chloro fatty aldehyde in atherosclerotic lesions and in infarcted myocardium, coupled with the potential role this lipid may have on cardiovascular function, underscore the importance of determining the mechanisms responsible for α-chloro fatty aldehyde catabolism. Many aldehydes found in vivo are produced through free radical mechanisms. The cytotoxicity of aldehydes is attenuated by reduction, oxidation, or conjugation with glutathione (25O'Brien P.J. Siraki A.G. Shangari N. Crit. Rev. Toxicol. 2005; 35: 609-662Crossref PubMed Scopus (515) Google Scholar, 26Jakoby W.B. Ziegler D.M. J. Biol. Chem. 1990; 265: 20715-20718Abstract Full Text PDF PubMed Google Scholar, 27Lindahl R. Evces S. J. Biol. Chem. 1984; 259: 11991-11996Abstract Full Text PDF PubMed Google Scholar, 28Berhane K. Widersten M. Engstrom A. Kozarich J.W. Mannervik B. J. Clin. Invest. 1994; 91: 1480-1484Google Scholar, 29Hartley D.P. Ruth J.A. Petersen D.R. Arch. Biochem. Biophys. 1995; 316: 197-205Crossref PubMed Scopus (223) Google Scholar, 30Siems W.G. Zollner H. Grune T. Esterbauer H. J. Lipid Res. 1997; 38: 612-622Abstract Full Text PDF PubMed Google Scholar). Aldehyde dehydrogenase oxidizes the aldehyde to its carboxylic acid. Human liver microsomal aldehyde dehydrogenase is specific for medium- and long-chain fatty aldehydes (31Kelson T.L. Secor McVoy J.R. Rizzo W.B. Biochim. Biophys. Acta. 1997; 1335: 99-110Crossref PubMed Scopus (118) Google Scholar). A mutation in this fatty aldehyde dehydrogenase has been linked to Sjögren-Larsson syndrome (32De Laurenzi V. Rogers G.R. Hamrock D.J. Marekov L.N. Steinert P.M. Compton J.G. Markova N. Rizzo W.B. Nat. Genet. 1996; 12: 52-57Crossref PubMed Scopus (231) Google Scholar). Alternatively, long-chain fatty aldehydes can be reduced to their respective fatty alcohols (33Kawalek J.C. Gilbertson J.R. Arch. Biochem. Biophys. 1976; 173: 649-657Crossref PubMed Scopus (34) Google Scholar, 34Takahashi N. Saito T. Goda Y. Tomita K.J. J. Biochem. (Tokyo). 1986; 99: 513-519Crossref PubMed Scopus (6) Google Scholar, 35Rizzo W.B. Craft D.A. Dammann A.L. Phillips M.W. J. Biol. Chem. 1987; 262: 17412-17419Abstract Full Text PDF PubMed Google Scholar). Additionally, aldehydes can diffuse within, or escape from, their cells of origin and form covalent adducts with proteins and phospholipids. Reactive aldehydes have been shown to react with primary amines on proteins to form Schiff base adducts and Michael addition products (formed by the nucleophilic attack on an α,β-unsaturated aldehyde such as 4-hydroxynonenal). In fact, 4-hydroxynonenal has been shown to covalently modify low density lipoprotein apolipoproteins contributing to the formation of a high uptake form of low density lipoprotein and to form protein adducts during ischemia-reperfusion injury (36Eaton P. Li J.M. Hearse D.J. Shattock M.J. Am. J. Physiol. 1999; 276: H935-H943Crossref PubMed Google Scholar, 37Uchida K. Toyokuni S. Nishikawa K. Kawakishi S. Oda H. Hiai H. Stadtman E.R. Biochemistry. 1994; 33: 12487-12494Crossref PubMed Scopus (230) Google Scholar). Another aldehyde, p-hydroxyphenylacetaldehyde, a product of MPO oxidation of l-tyrosine, forms Schiff base adducts with aminophospholipids of low density lipoprotein (38Heller J.I. Crowley J.R. Hazen S.L. Salvay D.M. Wagner P. Pennathur S. Heinecke J.W. J. Biol. Chem. 2000; 275: 9957-9962Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), which are present in human atherosclerotic tissues (39Hazen S.L. Gaut J.P. Crowley J.R. Hsu F.F. Heinecke J.W. Biochem. J. 2000; 352: 693-699Crossref PubMed Scopus (42) Google Scholar). Understanding the metabolism of α-chloro fatty aldehyde may be critical in revealing the role of this novel plasmalogen oxidation product in cardiovascular disease. Accordingly, the present study was designed to elucidate the metabolism of the α-chloro fatty aldehyde, 2-chlorohexadecanal (2-ClHDA). The results herein demonstrate that both endothelial cells and neutrophils oxidize and reduce 2-ClHDA to its respective chlorinated fatty acid and chlorinated fatty alcohol, which are subsequently incorporated into complex glycerolipids. Synthesis and Purification of 2-Cl-[d4]-HDA and 2-Cl-[3H]-HDA—2-Cl-[d4]-HDA was synthesized and purified as previously described (24Thukkani A.K. Hsu F.F. Crowley J.R. Wysolmerski R.B. Albert C.J. Ford D.A. J. Biol. Chem. 2002; 277: 3842-3849Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). 2-Cl-[3H]-HDA was synthesized and purified by a modification of the synthetic scheme employed for 2-Cl-[d4]-HDA (24Thukkani A.K. Hsu F.F. Crowley J.R. Wysolmerski R.B. Albert C.J. Ford D.A. J. Biol. Chem. 2002; 277: 3842-3849Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). For this scheme, [9,10-3H]-hexadecanol was prepared by the reduction of [9,10-3H]-hexadecanoic acid (0.256 Ci/mmol, PerkinElmer Life Sciences). The purity of the synthetic 2-Cl-[3H]-HDA was determined by TLC of underivatized 2-Cl-[3H]-HDA and TLC of its corresponding dimethylacetal and pentafluorobenzyl oxime derivatives using petroleum ether/ethyl ether/acetic acid (90/10/1, v/v/v) as the mobile phase. Synthesis of 2-Chlorohexadecanoic Acid and 2-Chlorohexadecanol—Hexadecanoic acid (16:0 fatty acid (FA)) and [d4]-16:0 FA were subjected to α-chlorination with chlorine(g) using the Hell-Volhard-Zelinsky reaction and phosphorous as the catalyst (40Van den Bergen H. Daloze D. Braekman J.C. J. Braz. Chem. Soc. 1999; 10: 1-12Crossref Scopus (4) Google Scholar). Briefly, 16:0 FA was melted at 80 °C before an equimolar amount of phosphorous trichloride in dichloromethane was added to the reaction vial. Chlorine(g) was then slowly bubbled into the reaction mixture for 1 h. The crude product was sequentially extracted using a modified Bligh and Dyer method (41Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42875) Google Scholar), TLC-purified (silica gel G TLC plates with a mobile phase composed of petroleum ether/ethyl ether/acetic acid (70/30/1, v/v/v) (RF = 0.24)), and then further purified on a Thermo Finnigan Surveyor liquid chromatograph equipped with a Beckman RP Ultrasphere™ ODS (5 μ, 4.6 mm × 25 cm) column coupled to a TSQ Quantum Ultra triple quadrupole mass spectrometer. Products were eluted at a flow rate of 2 ml/min from the stationary phase with a mobile phase composed of 85/15 MeOH/H2O containing 0.02% formic acid (A) for 3 min followed by a linear gradient from 100% A to 100% MeOH (B) over 7 min. The solid phase was further eluted with 100% B for 10 min. The LC eluate was split 10:1, and the LC-purified reaction products were detected by electrospray ionization-mass spectrometry using selected ion monitoring (SIM) of m/z 289 ([M – H]– of 2-Cl-16:0 FA) or m/z 293 ([M – H]– of [d4]2-Cl-16:0 FA) and selected reaction monitoring (SRM) for the loss of HCl of m/z 289 → m/z 253 or m/z 293 → m/z 257 in the negative ion mode (electrospray needle voltage = 5 kV, capillary T = 320 °C, collision energy = 15 eV) (tr for the 2-Cl-16:0 FA and [d4]2-Cl-16:0 FA were 8.55 and 8.53 min, respectively). 2-ClHDA and 2-Cl-[d4]-HDA were resuspended in 2 ml of radical free ethyl ether and 0.5 ml of benzene and treated with Vitride™ reagent (sodium bis(2-methoxyethoxy)aluminum hydride) for 30 min at 37 °C (42Ford D.A. Gross R.W. Biochemistry. 1994; 33: 1216-1222Crossref PubMed Scopus (24) Google Scholar). The resultant 2-chlorohexadecanol (α-ClFOH) was purified by TLC (petroleum ether/ethyl ether/acetic acid (70/30/1, v/v/v)) (RF = 0.41). 2-ClHDA Metabolism in Human Coronary Artery Endothelial Cells—Human coronary artery endothelial cells (HCAEC) (Cell Applications, Inc.) were grown to confluency on 60-mm tissue culture plates (Corning) in EGM®-2-MV medium (Cambrex) supplemented with 5% fetal bovine serum following the supplier's instructions at 37 °C with 100% humidity and 5% CO2 (passages 4–10). Confluent HCAEC were incubated with selected concentrations of 2-Cl-[3H]-HDA (0.1 μm (256 mCi/mmol), 1 μm (256 mCi/mmol), or 10 μm (25.6 mCi/mmol)) in 5 ml of normal growth medium at 37 °C for selected time intervals. At the end of each experimental interval, lipids in the cell culture medium were extracted by the method of Bligh and Dyer (41Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42875) Google Scholar). The cell culture plates containing the cells were then washed with phosphate-buffered saline. Subsequently, the cells were scraped with methanol:water (1:1, v/v) prior to lipid extraction by the method of Bligh and Dyer (41Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42875) Google Scholar). Lipids extracted from the cells or culture medium lipids were stored in chloroform under nitrogen prior to analyses by chromatography. For pulse-chase experiments, HCAEC were incubated (pulse) with 1 μm 2-Cl-[3H]-HDA (256 mCi/mmol) for 30 min. Following this 30-min pulse radiolabeling interval, the cell culture medium was removed from HCAEC, and the cells were washed once with phosphate-buffered saline and then incubated with cell culture medium containing 10 μm 2-ClHDA for selected time intervals (chase). At the end of each chase interval, radiolabeled lipid metabolites of 2-Cl-[3H]-HDA associated with the HCAEC, as well as the cell culture medium, were prepared for analyses (see above). Parallel experiments were performed using stable isotope-labeled 2-ClHDA. In these experiments, HCAEC were incubated with 10 μm 2-Cl-[d4]-HDA for either 3 or 24 h. Extracting the lipids from the cells and cell culture medium terminated the incubations, as described above. Stable isotope-labeled metabolites of 2-ClHDA were subsequently purified by TLC and analyzed by electrospray ionization-mass spectrometry or GC-MS. 2-Cl-[3H]-HDA Metabolism in Human Neutrophils—Whole blood (50 ml) was taken from healthy volunteers and anti-coagulated with EDTA (final concentration 5.4 mm) prior to the isolation of neutrophils using a Ficoll-Hypaque gradient as previously described (43Ferrante A. Thong Y.H. J. Immunol. Methods. 1980; 36: 109-117Crossref PubMed Scopus (447) Google Scholar). Pelleted neutrophils (5 × 106 cells/condition) were resuspended in Hanks' balanced salt solution (pH 7.3) supplemented with both MgSO4 and CaCl2 at 1 mm and immediately incubated with 1 μm 2-Cl-[3H]-HDA (256 mCi/mmol) in the presence or absence of 200 nm phorbol 12-myristate 13-acetate for 0, 15, 30, or 60 min at 37 °C. The cells were pelleted and washed once with Hanks' balanced salt solution before the lipids were extracted by the method of Bligh and Dyer (41Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42875) Google Scholar). Thin Layer Chromatographic Analyses of 2-ClHDA Metabolites—Neutral lipid metabolites of 2-Cl-[3H]-HDA were purified from crude lipid extracts using silica gel 60 TLC plates (Whatman) as a solid phase with a mobile phase composed of petroleum ether/ethyl ether/acetic acid (70/30/1, v/v/v) (44Ford D.A. Gross R.W. J. Biol. Chem. 1988; 263: 2644-2650Abstract Full Text PDF PubMed Google Scholar). Alternatively, polar lipid metabolites of 2-Cl-[3H]-HDA were purified on the same solid phase but with a mobile phase composed of chloroform/methanol/water (65/35/4, v/v/v). Lipid metabolites were further characterized by two-dimensional TLC plates using silica gel 60 TLC plates and an initial mobile phase composed of chloroform/methanol/ammonia (65/25/5, v/v/v) followed by development in the second dimension with a mobile phase composed of chloroform/acetone/methanol/acetic acid/water (3/4/1/1/0.5, v/v/v/v/v). Radioactivity associated with lipid metabolites of 2-Cl-[3H]-HDA that was resolved on TLC plates was detected by fluorography. In brief, the developed TLC plates were treated with EN3HANCE spray (PerkinElmer Life Sciences) prior to exposure to x-ray film (Kodak) at –80 °C. Alternatively, parallel TLC plates or lanes (on TLC plates) were developed with lipid standards, and silica from regions associated with specific lipids was scraped from the plate. Radioactivity was quantified by liquid scintillation spectrometry. Additionally, silica associated with regions of TLC plates detected by fluorography was also scraped from the plate and quantified by liquid scintillation spectrometry. Chlorinated Fatty Alcohols—TLC-purified chlorinated fatty alcohols were converted to their respective pentafluorobenzoyl esters using 2,3,4,5,6-pentafluorobenzoyl chloride (PFB-Cl) at 60 °C for 45 min (45Wildsmith K.R. Albert C.J. Hsu F.-F. Kao J.L.-F. Ford D.A. Chem. Phys. Lipids. 2006; 139: 157-170Crossref PubMed Scopus (31) Google Scholar). GC-MS analysis of PFB esters was performed using a Hewlett Packard (Palo Alto, CA) 6890 gas chromatograph and 5973 mass spectrometer using the negative ion chemical ionization mode with methane as the reagent gas. The source temperature was set at 150 °C. The electron energy was 240 eV, and the emission current was 300 mA. The PFB derivatives were separated on a J & W Scientific (Folsom, CA) DB-1 column (12.5 m, 0.2 mm inner diameter, 0.33 mm film thickness). The injector and the transfer line temperatures were maintained at 250 °C. The GC oven was maintained at 150 °C for 3.5 min, increased at a rate of 30 °C/min to 270 °C, and held at 270 °C for an additional 2 min. Chlorinated Fatty Acids—For fatty acid analyses, cells treated with 2-Cl-[d4]-HDA and [d4]-chlorinated fatty acids were first TLC-purified and subsequently subjected to LC-MS as described above for the synthesis of 2-Cl-16:0 FA. For these analyses, fatty acid molecular species were resolved using a Supelco Discovery® HS C18 5 μ column (15 cm × 2.1 mm) as the stationary phase at a flow rate of 0.2 ml/min. The 2-Cl-16:0 FA was detected by SRM as described above, and [d4]-16:0 FA was detected by SIM m/z 259 in the negative ion mode (electrospray needle voltage = 3.5 kV, capillary T = 320 °C). Chlorinated Fatty Acid Residues in Phosphatidylcholine—TLC-purified phosphatidylcholine (PC), from lipid extracts of cells treated with either 2-Cl-[3H]-HDA or 2-Cl-[d4]-HDA, was incubated with lipase from Rhizopus arrhizus (Sigma) for 1 h at 37°C in a bilayer consisting of 0.1 m borate buffer (pH 6.5) and ethyl ether. Lipase reaction products were then extracted by the Bligh-Dyer method and were purified using silica gel 60 TLC plates with a mobile phase composed of petroleum ether/ethyl ether/acetic acid (70/30/1, v/v/v). TLC-purified reaction products were first visualized by fluorography. Silica associated with regions of the TLC plate containing radioactivity (visualized by fluorography) were subsequently scraped and quantified by scintillation spectroscopy. For experiments employing stable isotope labeling, bands corresponding to authentic FA and α-ClFA were extracted and analyzed by LC-MS for [d4]-16:0 FA and [d4]2-Cl-16:0 FA content. Additionally, radiolabeled TLC-purified PC was subjected to either base or acid methanolysis. The resultant 2-ClFAME and FAME was analyzed by TLC and fluorography. Synthetic 2-Cl-[3H]-HDA—2-Cl-[3H]-HDA was synthesized, purified, and used to elucidate α-chloro fatty aldehyde metabolism in both HCAEC and neutrophils. Fig. 1A demonstrates that synthetic 2-Cl-[3H]-HDA migrates to the same region as that of unlabeled authentic 2-ClHDA. Furthermore, conversion of the synthetic 2-Cl-[3H]-HDA as well as authentic 2-ClHDA to their dimethylacetal derivatives or their pentafluorobenzyl oxime derivatives further demonstrated the purity of the radiolabel. Additionally, Fig. 1B is an overdevelopment of a fluorograph of the synthetic 2-Cl-[3H]-HDA, which demonstrates the purity of this radiolabeled aldehyde. Metabolism of 2-Cl-[3H]-HDA in HCAEC—HCAEC were incubated with cell culture medium containing 1 μm 2-Cl-[3H]-HDA for either 1 min, 3 or 24 h, and the incorporation of the radiolabel into the lipid metabolites was analyzed by two-dimensional TLC (Fig. 2). These experiments demonstrated that 2-ClHDA was taken up rapidly by HCAEC, and radiolabel derived from 2-ClHDA was eventually incorporated into complex glycerolipids in the HCAEC, including phosphatidylcholine and phosphatidylethanolamine. Radiolabel in these complex glycerolipids remained in the HCAEC and was not released. Additionally, radiolabel derived from 2-ClHDA was found in the α-chloro fatty alcohol (α-ClFOH) and α-chloro fatty acid (α-ClFA) pools of the cells, as well as these same lipid pools in the cell culture medium (Fig. 2). 2-Cl-[3H]-HDA was not metabolized by cell culture medium without cells present (data not shown). Thus, these results revealed that radiolabeled 2-ClHDA is readily oxidized and reduced to fatty acids and fatty alcohols, respectively, in the cells and that these metabolites are released from the cells. It is likely that the radiolabeled fatty acid is incorporated into the complex glycerolipids. The temporal course of the incorporation of radiolabel from 2-Cl-[3H]-HDA into cellular lipid pools is shown in greater detail in Fig. 3. For these experiments, lipid classes were separated by one-dimensional TLC using mobile phases that separate either neutral or polar lipid classes. After 2-Cl-[3H]-HDA enters the HCAEC, it is rapidly reduced to α-ClFOH within the first hour of labeling (Fig. 3, inset). Radiolabel also is found in the α-ClFA pool within the first hour. Fig. 3B shows the incorporation of the radioactivity derived from 2-Cl-[3H]-HDA into the polar lipid pools. The majority of the radioactivity in the polar lipid pools is associated with phosphatidylcholine, and this appears after the first hour of radiolabeling, which suggests that 2-Cl-[3H]-HDA is first oxidized to fatty acids prior to its incorporation into phosphatidylcholine. It should be noted that the migrations of α-ClFA and α-ClFOH using TLC are considerably different from non-chlorinated FA and FOH. The assignment of radiolabel to α-ClFA and α-ClFOH is based on the co-migration of radiolabel during TLC with regions of authentic α-ClFA and α-ClFOH. Using a mobile phase of petroleum ether/ethyl ether/acetic acid (70/30/1) and a silica gel 60 solid phase, the RF for FA and α-ClFA are 0.34 and 0.24, respectively. The attenuation of α-ClFA migration on TLC compared with FA is likely because of the electron-withdrawing properties of the α-chlorine on the carbanion of the carboxylic group, thus reducing its pKa.FIGURE 3Temporal course of 2-ClHDA incorporation into HCAEC lipids. HCAEC were incubated with 1 μm 2-Cl-[3H]-HDA, and the cellular lipids were extracted at the indicated time points and subsequently subjected to one-dimensional TLC and analyzed as described under “Experimental Procedures.” A and B are the neutral and polar lipids, respectively, which were separated in two different solvent systems as described under “Experimental Procedures.” Values represent the mean ± S.D. for three independent experiments. Under some conditions, the S.D. was within the size of the symbol for the mean. MAG, monoacylglycerol; CE, cholesterol ester; SM, sphingomyelin; PS/PI, phosphatidylserine/phosphatidylinositol.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Additional studies were performed to determine the metabolism of 2-ClHDA within the range of concentrations that have been observed in neutrophils (24Thukkani A.K. Hsu F.F. Crowley J.R. Wysolmerski R.B. Albert C.J. Ford D.A. J. Biol. Chem. 2002; 277: 3842-3849Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Fig. 4 shows that the temporal course of 0.1, 1, and 10 μm 2-ClHDA metabolism in HCAEC is very similar. At each concentration of 2-ClHDA, radiolabel was incorporated into both the cellular α-ClFOH and α-ClFA pools followed by the incorporation of radiolabel into complex glycerolipids. At 10 μm 2-ClHDA compared with 0.1 and 1 μm 2-ClHDA, there was in" @default.
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• W2047026433 title "Metabolism of Myeloperoxidase-derived 2-Chlorohexadecanal" @default.
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