Selective plasmenylcholine oxidation by hypochlorous acid: formation of lysophosphatidylcholine chlorohydrins

Chemistry and Physics of Lipids 144 (2006) 34–44

Selective plasmenylcholine oxidation by hypochlorous acid: formation of lysophosphatidylcholine chlorohydrins Maria C. Messner a , Carolyn J. Albert a , Fong-Fu Hsu b , David A. Ford a,∗ a

Department of Biochemistry and Molecular Biology, St. Louis University Health Sciences Center, St. Louis, MO 63104, United States b Department of Internal Medicine, Washington University, St. Louis, MO 63110, United States Received 24 April 2006; received in revised form 9 June 2006; accepted 9 June 2006 Available online 3 July 2006

Abstract The plasmalogen sn-1 vinyl ether bond is targeted by hypochlorous acid (HOCl) produced by activated phagocytes. In the present study, the attack of the plasmalogen sn-1 vinyl ether bond by HOCl is shown to be preferred compared to the attack of double bonds present in the sn-2 position aliphatic chain (sn-2 alkenes) of both plasmenylcholine and phosphatidylcholine. Lysophosphatidylcholine (LPC) is a product from the initial HOCl attack of plasmenylcholine and the sn-2 alkene bonds present in this LPC product are secondary targets of HOCl leading to the production of LPC–chlorohydrins (ClOH). The aliphatic ClOH was demonstrated in both the positive and negative ion mode using collisionally-activated dissociation (CAD) of the molecular ion of LPC–ClOH. Furthermore, HOCl treatment of endothelial cells led to the preferential attack of plasmalogens in comparison to that of diacyl choline glycerophospholipids. Taken together, plasmenylcholine is oxidized preferentially over phosphatidylcholine and leads to the production of LPC–ClOH. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Myeloperoxidase; Electrospray ionization-mass spectrometry; Choline glycerophospholipids; Plasmalogen

1. Introduction

Abbreviations: GPC, sn-glycero-3-phosphorylcholine; GC–MS, gas chromatography–mass spectrometry; GC-FID, gas chromatography flame ionization detection; TLC, thin layer chromatography; PFBBr, pentafluorobenzoyl bromide; ␣-ClFALD, ␣-chloro-fatty aldehydes; 2-ClHDA, 2-chlorohexadecanal; LPC, lysophosphatidylcholine; pPOPC, 1-O-hexadec-1 -enyl-2-octadec-9 -enoyl-GPC; POPC, 1hexadecanoyl-2-octadec-9 -enoyl-GPC; ClOH, chlorohydrin; ESIMS, electrospray ionization-mass spectrometry; CAD, collisionallyactivated dissociation; RCS, reactive chlorinating species; SRM, selected reaction monitoring; HCAEC, human coronary artery endothelial cells ∗ Correspondence to: Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104, United States. Tel.: +1 314 977 9264; fax: +1 314 977 9205. E-mail address: [email protected] (D.A. Ford).

The potent oxidant, hypochlorous acid (HOCl), is produced by myeloperoxidase from hydrogen peroxide and chloride. Myeloperoxidase-derived hypochlorous acid is a potent antimicrobial agent produced by leukocytes of the innate immune system (Klebanoff, 1980; Klebanoff et al., 1984). Additionally, leukocytes can cause host cell injury and are likely participants in inflammatory mechanisms of atherosclerosis and organ ischemia/reperfusion injury (Daugherty et al., 1994; Hazell et al., 1996; Kaminski et al., 2002; Malle et al., 2000; Thukkani et al., 2003a,b, 2005). HOCl is in equilibrium with its conjugate base anion OCl− and Cl2(g) and collectively these are the reactive chlorinating species (RCS) produced by activated leukocytes. RCS are highly reactive, targeting primary amines (Thomas

0009-3084/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2006.06.003

M.C. Messner et al. / Chemistry and Physics of Lipids 144 (2006) 34–44

et al., 1982; Weiss et al., 1982) and oxidizing iron–sulfur centers of heme-containing proteins (Albrich et al., 1981). Plasma membranes of many mammalian cells are enriched with plasmalogens (Gross, 1984; Han and Gross, 1994; Post et al., 1988). Plasmalogens contain a vinyl ether linkage between the sn-1 aliphatic chain and the glycerol backbone. The plasmalogen vinyl ether bond has anti-oxidant properties since it terminates freeradical flux and likely protects cells and their plasma membranes from oxidative stress (Morand et al., 1988; Vance, 1990; Zoeller et al., 1988). Recently, the vinyl ether bond of plasmalogens was shown to be targeted by RCS resulting in the release of ␣-chloro fatty aldehydes (␣-ClFALD) with concomitant production of an sn-1 lysophospholipid (Albert et al., 2001; Thukkani et al., 2002). Furthermore, levels of ␣-ClFALD and lysophosphatidylcholine molecular species are elevated in human atherosclerotic lesions (Thukkani et al., 2003a,b). ␣ClFALD produced from HDL plasmalogens has been shown to decrease eNOS expression in endothelial cells (Marsche et al., 2004). Additionally, ␣-ClFALD are neutrophil chemoattractants (Thukkani et al., 2002). Previous studies have shown that RCS attack alkenes within aliphatic chains of phospholipids (Winterbourn et al., 1992) and cholesterol (Hazen et al., 1996). While the production of chloramines is favored over the production of aliphatic ClOH (Pattison et al., 2003), the appearance of ␣-ClFALD in activated neutrophils (Thukkani et al., 2002), activated monocytes (Thukkani et al., 2003a,b), ischemic/reperfused myocardium (Thukkani et al., 2005) and atherosclerotic aorta (Thukkani et al., 2003a,b) demonstrates that the vinyl ether bond of plasmalogens is a preferred target of RCS produced by phagocytes. Accordingly, the present study was designed to compare the relative reactivity of the vinyl ether bond of plasmalogens to double bonds present in the sn-2 position aliphatic chain (sn-2 alkenes) of diacyl phospholipids. The results show that the vinyl ether bond of plasmalogens is preferentially targeted by RCS compared to that of aliphatic sn-2 alkenes of diacyl phospholipids. Furthermore, the unsaturated lysophosphatidylcholine produced from RCS attack of plasmalogens undergoes a secondary attack by RCS yielding lysophosphatidylcholine (LPC)–ClOH molecular species. 2. Experimental procedures

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fied as described previously (Han et al., 1992). The diacyl choline glycerophospholipid, 1-hexadecanoyl-2octadec-9 -enoyl-GPC (POPC), was purchased from Avanti Polar Lipids (Alabaster, MA). The plasmalogen choline glycerophospholipid, 1-O-hexadec-1 -enyl2-octadec-9 -enoyl-GPC (pPOPC), was synthesized by an anhydrous reaction utilizing 1-O-hexadec-1 -enylGPC and octadec-9 -enoyl chloride as precursors with dimethylaminopyridine as a catalyst (Han et al., 1992). pPOPC was purified first by normal phase HPLC and then further purified by reverse phase HPLC (Han et al., 1992). Purity of the synthetic pPOPC was confirmed by ESI-MS and the mass was quantitated by GC following acid methanolysis. Arachidic acid was added as an internal standard. 1-O-hexadec-1 enyl-2-heptadec-10 -enoyl-GPC (16:0–17:1) and 1-Ohexadec-1 -enyl-2-nonadec-10 -enoyl-GPC (16:0–19:1) plasmenylcholines were synthesized using heptadec10 -enoyl and nonadec-10 -enoyl chloride, respectively. 16:0–17:1 and 16:0–19:1 plasmenylcholine molecular species were treated with 10-fold molar excess HOCl to simultaneously cleave the vinyl ether bond and form 17:0- and 19:0-LPC–ClOH, respectively. These synthetic LPC–ClOH were purified by HPLC and quantitated by Bartlett (Dittmer and Wells, 1969). All other materials were of the highest grade available and were purchased from Sigma–Aldrich or Fisher. 2.2. Phospholipid treatments with hypochlorous acids HOCl was prepared from NaOCl (Hazen et al., 1996; Thomas et al., 1986). Concentrations were determined spectrophotometrically at ε292 = 350 M−1 cm−1 HOCl (Morris, 1966). Binary mixtures and homogeneous lipid vesicles were prepared by vigorously mixing 0.4 mM phospholipid in 6 ␮l ethanol and resuspending in a 20 mM sodium phosphate buffer (pH 4 or 7) containing 0.1 M NaCl. Lipid vesicles were treated with 0.1–2 mM HOCl for 1–5 min. Alternatively, lipids were resuspended in a solution containing a 20 mM sodium phosphate buffer (pH 4), 0.1 M NaCl, 1 mM H2 O2 , and either 1 unit myeloperoxidase or water as a control. The reactions were stopped by the addition of chloroform/methanol (2:1, v/v) and lipids were extracted into the organic layer by the method of Bligh and Dyer (1959). Extracted reaction products were dried under N2 and were stored in chloroform under N2 .

2.1. Lipids

2.3. Cell culture of HCAEC

Lysoplasmenylcholine (1-O-hexadec-1 -enyl-GPC) was prepared from bovine heart lecithin and puri-

Human coronary artery endothelial cells (HCAEC) (Cell Applications) were plated onto T75 culture flasks

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and maintained in EGM-2-MV medium (Cambrex) at 37 ◦ C in an atmosphere of 95% air and 5% CO2 . Eighty percent confluent cells were washed with modified Hanks balanced salt solution (HBSS) supplemented with 1 mM each of CaCl2 and MgSO4 and 5 mM monobasic H2 PO4 (pH 7.35). The cells were incubated with 0.1, 0.5, or 3.0 mM HOCl for 1–10 min as indicated (water vehicle was added to control conditions). At the end of each experimental interval, cells were scraped in the presence of 1:1 methanol/saline (v/v) and lipids were extracted by the method of Bligh and Dyer (1959) in the presence of 17:0- and 19:0-LPC–ClOH, 17:0 LPC and 2-Cl-[d4 ]HDA (internal standards). 2.4. Electrospray ionization-mass spectrometry Extracted lipids were resuspended in methanol or methanol/chloroform (4/1) and analyzed by ESI-MS in the direct infusion mode at a flow rate of 1–3 ␮l/min using either Finnigan TSQ-7000 or Thermo Electron TSQ Quantrum Ultra instrumentation. 20–50 ␮M NaOH was added to samples prior to injection (unless indicated) and samples were run in the positive ion mode (unless otherwise noted). Tandem mass spectrometry was performed on selected ions (typical collision energies were ∼35 eV) and spectra were averaged 3–5 min and processed utilizing either ICIS (Finnigan) or Xcalibur (Thermo Electron) software. To detect LPC and LPC–ClOH molecular species, neutral loss scanning of 59 and 95, respectively, was employed. Spectra were averaged for 3–5 min and processed utilizing Xcalibur software (Thermo Electron). Individual molecular species were quantified by comparing the ion intensity of individual molecular species to that of the internal standards. Values are expressed as mass of each individual molecular species normalized to the mass of total lipid inorganic phosphate present in the sample. 2.5. Reverse phase-liquid chromatography/mass spectrometry HCAEC lipid extracts were suspended in methanol and subjected to LC/MS. Lipid molecular species were separated using a 15 cm × 2.1 mm, 5 um Discovery® HS C18 HPLC column (Supelco) and an initial mobile phase comprised of methanol/water/acetonitrile (60/20/20, v/v/v) containing 1 mM ammonium acetate with a gradient to 1 mM methanolic ammonium acetate solution over 5 min followed by isocratic elution in this mobile phase for an additional 35 min. The flow rate was 0.2 ml/min. For detection, 10 ␮M NaOH in methanol was infused into the post-column eluent prior to detec-

tion by ESI in the positive ion mode using selected reaction monitoring (SRM) for m/z 834 → 739 (16:0–18:0 PC–ClOH), 610 → 515 (19:0 LPC–ClOH, internal standard), 596 → 501(18:0 LPC–ClOH) and 582 → 487 (17:0 LPC–ClOH, internal standard). 2.6. Preparation of PFB oximes of α-chloro fatty aldehydes and GC–MS analysis Lipid extracts isolated from HCAEC were evaporated to dryness under N2 and ␣-chloro fatty aldehydes were converted to their respective PFB oxime derivatives prior to analysis by GC–MS as previously described (Albert et al., 2001; Thukkani et al., 2002). GC–MS analysis of PFB oximes of ␣-chloro fatty aldehydes was performed on a Hewlett HP 5973 mass spectrometer coupled to a HP 6890 gas chromatograph using the negative ion chemical ionization (NICI) 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 A. 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 ␮m 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. Quantitation of 2-chlorohexadecanal (2-ClHDA) was performed utilizing selected ion monitoring GC–MS (SIM GC–MS) as previously described (Thukkani et al., 2002). Specifically, the total integrated peak area arising from m/z 288, the structurally informative fragment ion produced from the PFB oxime of 2-ClHDA, was compared to the total integrated peak area produced by m/z 292, the structurally informative fragment ion of the PFB oxime of 2-Cl-[d4 ]-HDA. Also, 2-chlorooctadecanal (2-ClODA), the 18 carbon-containing ␣-chloro fatty aldehyde, was monitored by detection of m/z 316, the structurally informative fragment ion produced from the PFB oxime of 2-ClODA. 2-ClODA was quantified by comparison of the integrated peak area produced by m/z 316 to that produced by m/z 292 from the PFB oxime of 2-Cl-[d4 ]HDA. 3. Results Initial studies were performed to determine the relative targeting of alkene bonds present in the sn-2 aliphatic chain of both plasmalogen and diacyl choline glycerophospholipids to that of the vinyl ether bond linking the sn-1 aliphatic chain of plasmalogens to the glycerol backbone. The ESI-MS spectra of the lipid extract from

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Fig. 1. Preferential attack of the plasmalogen vinyl ether bond compared to sn-2 alkenes of phospholipids by HOCl. Lipid vesicles comprised of a binary mixture of 100 nmol pPOPC and 100 nmol POPC were treated with either no HOCl (A), equimolar HOCl (B), or 5-fold molar excess HOCl (C) for 10 min as described in Section 2. Following treatments, lipids were extracted into chloroform and subjected to electrospray ionization in the positive ion mode as described in Section 2.

vesicles comprised of a binary mixure of 0.2 mM pPOPC and 0.2 mM POPC shows both molecular ions at m/z 744 and 766 corresponding to the protonated and sodiated adducts of pPOPC and molecular ions at m/z 760 and 782 corresponding to the protonated and sodiated adducts of POPC (Fig. 1A). Analyses of this binary mixture following treatment with equimolar HOCl revealed the loss of molecular ions corresponding to plasmenylcholine (pPOPC at m/z 744 and 766) while the diacyl molecular species (POPC) was relatively spared attack by RCS (Fig. 1B). The loss of plasmalogen was accompanied by the appearance of molecular ions at m/z 522 and 544, corresponding to the protonated and sodiated adducts of 2-octadec-9 -enoyl-GPC (18:1 LPC) (Fig. 1B). Also molecular ions are observed at m/z 574 and 596, which are the putative chlorohydrin species of 2-octadecanoylGPC (18:0 LPC–ClOH) (Fig. 1B). In comparison to treatments with equimolar HOCl in which 18:1 LPC was the most abundant ion from the plasmalogen, incubation of the binary mixture with 5-fold molar excess HOCl resulted in the chlorohydrin species (m/z 596 and 834) being the most abundant ions (Fig. 1C). Also with 5-fold excess of HOCl, all of the plasmalogen was consumed by the RCS and the phosphatidylcholine was converted to its respective chlorohydrin (molecular ions at m/z 812 and 834). To demonstrate the precursor-product relation-

ship between the components of this binary mixture, these two choline glycerophospholipids were treated with HOCl individually. Treatment of the plasmalogen, pPOPC, with equimolar HOCl demonstrated that pPOPC gave rise to 18:1 LPC (m/z 544) and the putative chlorohydrin molecular species, 18:0 LPC–ClOH (m/z 596 and 598, Fig. 2B). It should be appreciated that the ions at m/z 596 and 598 are present at a 3:1 ratio, which suggests this is a monochlorinated molecule due to the 3:1 isotopic abundance of 35 Cl and 37 Cl. (Fig. 2B, inset). In contrast, the ESI/MS analyses of POPC treated with equimolar HOCl showed that the putative chlorohydrin at m/z 834 and 836 is a product of the attack of POPC (m/z 782, Fig. 2D). Again it should be noted that the ions at m/z 834 and 836 are present in a 3:1 ratio, which suggests these are monochlorinated species (Fig. 2D, inset). Additional experiments demonstrated that treating a binary mixture of POPC and pPOPC with an RCSgenerating system comprised of MPO/H2 O2 /NaCl (pH 4) for 5 min also leads to 18:1 LPC and its chlorohydrin, 18:0 LPC–ClOH, produced from pPOPC and the production of a phosphatidylcholine chlorohydrin species from POPC (Fig. 3). Collisionally-activated dissociation (CAD) analyses were used to confirm the identities of the molecular ions produced from RCS attack of plasmenylcholine. CAD

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Fig. 2. HOCl targeting of plasmenylcholine and phosphatidylcholine in lipid vesicles. Lipid vesicles comprised of either 100 nmol pPOPC (A and B) or 100 nmol POPC (C and D) were treated with either no HOCl (A and C), or equimolar HOCl (B and D) for 10 min as described in Section 2. Following treatments lipids were extracted into chloroform and subjected to electrospray ionization in the positive ion mode in the presence of 10 ␮M NaOH in methanol as described in Section 2.

analyses of the molecular ion at m/z 544 confirms that this ion corresponds to the sodiated adduct of 18:1 LPC (Fig. 4A). These CAD spectra are similar to the fragmentation schemes that have previously been demonstrated (Han and Gross, 1996; Hsu et al., 1998, 2003). The fragment ion at m/z 485 is from the loss of trimethylamine and the fragment ions at m/z 339 (H+ ) and 361 (Na+ ) are

from the rearrangement ions following the loss of the phosphocholine head group (Han and Gross, 1996; Hsu et al., 1998, 2003). The fragment ion at m/z 104 is derived from the choline head group and the fragment ion at m/z 147 is the sodiated ethylene phosphate ion derived from the choline head group. Additionally, the predominance of the m/z 147 ion as compared to that at m/z 104 is

Fig. 3. Preferential attack of the plasmalogen vinyl ether bond compared to sn-2 alkenes of phospholipids by RCS-generating system. Lipid vesicles comprised of a binary mixture of 50 nmol pPOPC and 50 nmol POPC were treated with either H2 O2 /NaCl (A) or with MPO/H2 O2 /NaCl (B) for 5 min as described in Section 2. Following treatments lipids were extracted into chloroform and subjected to electrospray ionization in the positive ion mode as described in Section 2.

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Fig. 4. ESI-MS/MS analysis of plasmalogen-derived lipidic products following HOCl treatment. Lipid vesicles comprised of 100 nmol pPOPC were treated with equimolar HOCl for 10 min and lipidic reaction products were sequentially extracted into chloroform and subjected to electrospray ionization in the positive ion mode as described in Section 2. CAD was performed on ions at m/z 544 (A); 596 (B); and 598 (C) as described in Section 2.

indicative of an sn-1 lysophosphatidylcholine molecular species (Han and Gross, 1996; Hsu et al., 2003). The CAD spectra for the ions at m/z 596 and 598 are shown in Fig. 4B and C, respectively. Both of these parent ions gave rise to an ion fragment resulting from the loss of the trimethylamine, m/z 537 and 539, respectively. Additionally, CAD of both m/z 596 and 598 gave the common ion fragment m/z 501 that is from the loss of both the trimethylamine and H35 Cl and H37 Cl, respectively. For CAD of m/z 596, structural support of a chlorohydrin in the aliphatic chain is provided by fragment ions at m/z 413, 391, 377, 355 and 337 (Fig. 4B). The fragment ions at m/z 413 (Na+ ) and 391 (H+ ) are from the loss of the phosphocholine head group and this ion contains the intact aliphatic structure at the sn-2 position (Fig. 4B). Fragment ions at m/z 377 and 355 are derived from the fragment ions at m/z 413 and 391 due to the loss of HCl from the chlorohydrin. Furthermore, m/z 337 is derived from the further loss of H2 O from the fragment ion m/z 355. Parallel fragments are observed from the

CAD of m/z 598, which contains 37 Cl in the aliphatic chain (Fig. 4C). The putative lysophosphatidylcholine chlorohydrin, 18:0 LPC–ClOH, was also subjected to CAD analyses in the negative ion mode to elucidate the aliphatic constituents. Fig. 5 shows that the chloride adduct in the negative ion mode (m/z 608) fragments into a fatty acid containing the chlorohydrin (m/z 333), the chlorohydrin with the loss of HCl (m/z 297) and the aliphatic group with the loss of both HCl and H2 O (m/z 279). Also shown in the negative ion CAD spectra is the ion peak fragment from the loss of methylchloride from the choline headgroup (m/z 558) and the further loss of HCl from the m/z 558 ion (e.g., m/z 522). Taken together these CAD analyses are consistent with molecular ions at m/z 596 and 598 in the positive ion mode being 18:0 LPC–ClOH derived from RCS attack of plasmenylcholine. Based on the CAD data of the positive ion at m/z 596 corresponding to 18:0 LPC–ClOH (Fig. 4), a strategy was developed to identify choline glycerophospholipid

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Fig. 5. ESI-MS/MS analysis of plasmalogen-derived lysophosphatidylcholine–chlorohydrin products following HOCl treatment. Lipid vesicles comprised of 100 nmol pPOPC were treated with equimolar HOCl for 10 min and lipidic reaction products were sequentially extracted into chloroform and subjected to electrospray ionization in the negative ion mode as described in Section 2. CAD was performed on the chloride adduct ion at m/z 608 as described in Section 2.

molecular species that contained chlorohydrins (ClOH) in a complex mixture utilizing neutral loss scanning of 95 (loss of both trimethylamine and H35 Cl). Comparisons of the positive ion spectra of lipid extracts from control and 3 mM HOCl-treated HCAEC indicated that several new molecular species were present following HOCl treatment (Fig. 6A and C). These new molecular species included molecular ions at m/z 596, 806, 834, 862 and 882. Further analyses using neutral loss scanning of 95 of these same lipid extracts specifically identified these molecular ions (e.g., m/z 596, 806, 834, 862 and 882) as choline glycerophospholipid chlorohydrins (Fig. 6D). Panel D shows major ions at m/z 596 and 834, which appear to be 18:0 LPC–ClOH and 16:0–18:0 PC–ClOH arising from the cell-derived lipids pPOPC and POPC, respectively. It should be noted that the internal standards 17:0- and 19:0 LPC–ClOH are observed in Panels B and D as well. CAD analyses of the ions at m/z 834/836 and 848/850 show that they lose H35 Cl and H37 Cl, respectively. 16:0–18:0 PC–ClOH (m/z 834) produced from HOCl incubation of synthetic POPC (Avanti) yields the same fragmentation pattern (Hsu et al., 2003). LPC and PC chlorohydrins were also identified under these conditions by LC/MS analyses using SRM. Fig. 7 shows the SRM analyses of PC and LPC–chlorohydrins

produced in HOCl-treated HCAEC are similar to that observed by the neutral loss analyses of chlorohydrins shown in Fig. 6. Table 1 summarizes data for the production of 16:0–18:0 PC–ClOH (m/z 834), 18:0 LPC–ClOH (m/z 596) and 16:0 LPC–ClOH (m/z 568) from HCAEC treated with increasing amounts of HOCl. 18:0 LPC–ClOH formation occurs at lower concentrations of HOCl treatments compared to that of the phosphatidylcholine chlorohydrin, 16:0–18:0 PC–ClOH. In fact, 18:0 LPC–ClOH was found in HCAEC treated with as little as 100 ␮M HOCl (Table 1). Concomitant with the appearance of 18:0 LPC–ClOH at 100 ␮M HOCl, both 2-ClHDA and 2-ClODA accumulated. However, it is important to note that the differences in mass of the 2-Cl fatty aldehydes in comparison to the LPC–ClOH and the 18:1 LPC pool indicate that other plasmenylcholine and plasmenylethanolamine molecular species contribute to the accumulation of 2-Cl fatty aldehydes in these cells. Since 18:1 LPC is present in control-treated cells it is likely that LPC–ClOH detected in cells treated with 100 ␮M HOCl is derived both from the free LPC pool as well as the LPC that is released from plasmenylcholine pools. Only with HCAEC treated with 3 mM HOCl were chlorohydrins of choline glycerophospho-

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Fig. 6. ESI-MS analysis of choline glycerophospholipids from HOCl-treated human coronary artery endothelial cells. Human coronary artery endothelial cells were treated with either no HOCl (A and B) or 3 mM HOCl (C and D) for 10 min. Following these treatments, lipids were extracted with the addition of the internal standards, 17:0- and 19:0 LPC–ClOH (m/z 582 and 610, respectively), and analyzed by ESI-MS in the positive ion mode by either total ion scanning (A and C) or neutral loss scanning of 95 (B and D) as described in Section 2.

lipids, e.g., 16:0–18:0 PC–ClOH, observed (Table 1) thus, demonstrating the selective attack of plasmalogens at lower HOCl concentrations compared to diacyl glycerophospholipids. Higher concentrations of HOCl (3 mM) led to a greater abundance of 18:0 LPC–ClOH as well as 16:0 LPC–ClOH (Table 1).

4. Discussion The vinyl ether bond of plasmalogens has previously been shown to be oxidized to yield ␣-ClFALD and LPC (Albert et al., 2001). In comparison to alkene bonds present in the aliphatic chain at the sn-2 position of

Table 1 HOCl-modified HCAEC lipid oxidation products Lipid oxidation product

No treatment

100 ␮M HOCl

500 ␮M HOCl

3 mM HOCl

18:1 LPC 16:0 LPC–ClOH 18:0 LPC–ClOH 16:0–18:0 PC–ClOH 2-ClHDA 2-ClODA

3.37 ± 0.19 N.D. N.D. N.D. N.D. N.D.

3.15 ± 0.31 N.D. 0.05 ± 0.03 N.D. 15.07 ± 0.83 5.48 ± 0.23

2.88 ± 0.09 N.D. 0.33 ± 0.08 N.D. 55.03 ± 1.68 21.30 ± 0.86

N.D. 1.42 ± 0.23 10.50 ± 1.24 11.14 ± 2.76 55.37 ± 0.93 21.03 ± 0.80

HCAEC were incubated with selected concentrations of HOCl for 10 min and incubations were terminated by Bligh–Dyer extraction of lipids in the presence of internal standards as described in Section 2. Lipids were quantified by mass spectrometric techniques and are expressed as mass of each individual molecular species normalized to the mass of total lipid inorganic phosphate in pmol/nmol Pi for the mean ± S.E.M. for at least three independent measurements. N.D. indicates levels that were not detectable.

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Fig. 7. LC/MS/MS ESI-MS analysis of choline glycerophospholipids from HOCl-treated human coronary artery endothelial cells. Human coronary artery endothelial cells were treated with either 0, 0.1, 0.5 or 3 mM HOCl (A–D, respectively) for 10 min. Following these treatments, lipids were extracted with the addition of the internal standards, 17:0- and 19:0 LPC–ClOH (m/z 582 and 610, respectively), and analyzed by LC/MS/MS ESI-MS in the positive ion mode using SRM as described in Section 2. 17:0-, 18:0-, and 19:0 LPC–ClOH as well as 16:0–18:0 PC–ClOH were monitored by m/z 582 → 487, 596 → 501, 610 → 515, and 834 → 739, respectively. Values in parentheses are the total ion current at 100% relative intensity for each analyte.

phospholipids, the vinyl ether bond of plasmalogens represents a unique chemical and physical target for the RCS produced by myeloperoxidase. In biological membranes, the vinyl ether bond is exposed to the hydrophilic domain of membranes, which would make it susceptible to targeting by RCS. In comparison, the aliphatic alkenes present in the sn-2 chain of diacyl glycerophospholipids, is embedded in the hydrophobic domain of membranes. Furthermore, in comparison to the carbon–carbon double bond of an alkene, the carbon–carbon double bond of the vinyl ether bond should be preferentially oxidized due to electrons donated by the oxygen of the vinyl ether bond. Additionally, double bonds present in the aliphatic chain at the sn-1 position may be preferentially oxidized since double bonds present in the aliphatic chain at the sn-1 and sn-3 positions of triglycerides have been shown to be more readily oxidized than those present at the sn-2

chain (Endo et al., 1997). Our results from experiments using binary mixtures of plasmenylcholine and phosphatidylcholine support this paradigm by showing that the vinyl ether bond of plasmenylcholine, as opposed to the alkene bond of diacyl choline glycerophospholipids, is preferentially attacked by HOCl. Furthermore, the LPC product of plasmalogen oxidation by RCS is an amphipathic molecule. LPC can diffuse out of membranes as monomers in aqueous phase and thus, have a higher likelihood of RCS attack at the aliphatic alkene, as compared to aliphatic alkenes present in intact phospholipids that are embedded in the hydrophobic domain of membranes. Again the data herein bear out this prediction by showing a secondary attack of unsaturated molecular species of lysophosphatidylcholine released from the initial attack of the vinyl ether bond of plasmenylcholine. The resultant LPC–chlorohydrins were

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identified through mass spectrometric analyses in both negative and positive ion modes. CAD analyses of LPC–ClOH revealed that these molecules have a signature neutral loss of 95. This neutral loss is from the combined loss of trimethylamine (loss of 59, a conventional loss observed in choline glycerophospholipids) and HCl (loss of 36 from the aliphatic chain). Another signature neutral loss of LPC–ClOH was 219, which is from the loss of HCl from the ClOH of the aliphatic chain as well as phosphorylcholine (loss of 183). These unique neutral losses of chlorohydrins from choline glycerophospholipids provide a unique mechanism to identify and quantify these molecules in complex lipid mixtures. We exploited neutral loss scanning of 95 to identify LPC–ClOH in lipid extracts from HCAEC treated with HOCl and thus, demonstrate the targeting of endogenous plasmalogens in HCAEC. These studies in HCAEC demonstrated that plasmenylcholine is selectively targeted by HOCl compared to phosphatidylcholine molecular species and in fact, comparisons of phosphatidylcholine ClOH formation to that of 2-ClHDA derived from oxidation of plasmalogens was striking. It should however, be appreciated that much of the 2-ClHDA and 2-ClODA observed in these experiments was likely derived from plasmenylethanolamine, but the LPC–chlorohydrins were likely derived from monounsaturated molecular species of LPC produced from plasmenylcholine oxidation as well as free LPC in these cells. McHowat and co-workers have demonstrated approximately three to four times more plasmenylethanolamine present in HCAEC in comparison to plasmenylcholine (Meyer et al., 2005). Plasmenylethanolamine in HCAEC is enriched with polyunsaturated aliphatic groups at the sn-2 position (Meyer et al., 2005) suggesting that it may also lead to chlorohydrin formation following the attack of the vinyl ether bond. We have not detected lysophosphatidylethanolamine chlorohydrins in our studies. It is likely that RCS oxidation of plasmenylethanolamine is considerably more complex in comparison to that of plasmenylcholine due to competing RCS targets in plasmenylethanolamine including the vinyl ether, the primary amine and multiple conjugated dienes in the sn-2 aliphatic chain. The only chlorinated lipid products of RCS targeting that have been shown in biological systems are ␣-chloro fatty aldehydes (Thukkani et al., 2002, 2003a,b, 2005). The present study demonstrates in both model binary lipid systems, as well as in intact cells, that the vinyl ether bond of plasmalogens is more reactive to RCS attack compared to aliphatic alkenes of phospholipids. Recently, a role of these aldehydes has been suggested by the demonstration that 2-ClHDA inhibits eNOS activity

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