Activated Platelets and Monocytes Generate Four Hydroxyphosphatidylethanolamines via Lipoxygenase
2007; Elsevier BV; Volume: 282; Issue: 28 Linguagem: Inglês
10.1074/jbc.m611776200
ISSN1083-351X
AutoresBenjamin H. Maskrey, Alexandra Bermúdez-Fajardo, Alwena H. Morgan, Esther Stewart-Jones, Vincent Dioszeghy, Graham W. Taylor, Paul R.S. Baker, Barbara Coles, Marcus J. Coffey, Hartmut Kühn, Valerie B. O’Donnell,
Tópico(s)Sphingolipid Metabolism and Signaling
Resumo12/15-Lipoxygenase (LOX) mediates immune-regulatory activities not accounted for by its known free acid eicosanoids, suggesting that additional lipids may be generated by activated cells. To characterize novel LOX-derived lipids, a lipidomic approach was utilized. Ionophore-activated interleukin-4-treated human peripheral monocytes generated up to 10-fold more esterified 15-hydroxyeicosatetraenoic acid (15-HETE) than free in a phosphatidylinositol 3-kinase- and protein kinase C-sensitive manner. Precursor scanning electrospray ionization/tandem spectroscopy for m/z 319 (HETE, [M-H]–) showed 4 ions at m/z 738, 764, 766, and 782 that were identified using tandem spectroscopy and MS3 as specific diacyl and plasmalogen 15-HETE phosphatidylethanolamines. Using H 182O water, the compounds were shown to form by direct oxidation of endogenous phosphatidylethanolamine (PE) by 15-LOX, with PE being the preferred phospholipid pool containing 15-HETE. Similarly, human platelets generated 4 analogous PE lipids that contained 12-HETE and increased significantly in response to ionophore, collagen, or convulxin. These products were retained in the cells, in contrast to free acids, which are primarily secreted. Precursor scanning of platelet extracts for the major platelet-derived prostanoid, thromboxane B2 (m/z 369.2), did not reveal PE esters, indicating that this modification is restricted to the LOX pathway. In summary, we show formation of PE-esterified HETEs in immune cells that may contribute to LOX signaling in inflammation. 12/15-Lipoxygenase (LOX) mediates immune-regulatory activities not accounted for by its known free acid eicosanoids, suggesting that additional lipids may be generated by activated cells. To characterize novel LOX-derived lipids, a lipidomic approach was utilized. Ionophore-activated interleukin-4-treated human peripheral monocytes generated up to 10-fold more esterified 15-hydroxyeicosatetraenoic acid (15-HETE) than free in a phosphatidylinositol 3-kinase- and protein kinase C-sensitive manner. Precursor scanning electrospray ionization/tandem spectroscopy for m/z 319 (HETE, [M-H]–) showed 4 ions at m/z 738, 764, 766, and 782 that were identified using tandem spectroscopy and MS3 as specific diacyl and plasmalogen 15-HETE phosphatidylethanolamines. Using H 182O water, the compounds were shown to form by direct oxidation of endogenous phosphatidylethanolamine (PE) by 15-LOX, with PE being the preferred phospholipid pool containing 15-HETE. Similarly, human platelets generated 4 analogous PE lipids that contained 12-HETE and increased significantly in response to ionophore, collagen, or convulxin. These products were retained in the cells, in contrast to free acids, which are primarily secreted. Precursor scanning of platelet extracts for the major platelet-derived prostanoid, thromboxane B2 (m/z 369.2), did not reveal PE esters, indicating that this modification is restricted to the LOX pathway. In summary, we show formation of PE-esterified HETEs in immune cells that may contribute to LOX signaling in inflammation. Activated platelets and monocytes generate four hydroxyphosphatidylethanolamines via lipoxygenase.Journal of Biological ChemistryVol. 284Issue 37PreviewVOLUME 282 (2007) PAGES 20151–20163 Full-Text PDF Open Access Mammalian LOXs 2The abbreviations used are: LOX, lipoxygenase; HETE, hydroxyeicosatetraenoic acid; PE, phosphatidylethanolamine; MS, mass spectrometry; ESI, electrospray ionization; HPLC, high pressure liquid chromatography; PBS, phosphate-buffered saline; IL, interleukin; MRM, multiple reaction monitoring; PI, phosphatidylinositol; PC, phosphatidylcholine; SAPE, 1-stearyl-2-arachidonyl-phosphatidylethanolamine; SAPC, 1-stearyl-2-arachidonyl-phosphatidylcholine. oxidize arachidonate to form hydro(pero)-xy-eicosatetraenoic acids (H(p)ETE). There are several isoforms expressed in circulating vascular cells, including 12-LOX (platelets), 12/15-LOX (monocytes/macrophages, eosinophils), and 5-LOX (neutrophils). Overall, they play immune modulatory roles in diseases such as asthma, atherosclerosis, diabetes, and hypertension (1Anning P.B. Coles B. Bermudez-Fajardo A. Martin P.E. Levison B.S. Hazen S.L. Funk C.D. Kuhn H. O'Donnell V.B. Am. J. Pathol. 2005; 166: 653-662Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 2Cyrus T. Witztum J.L. Rader D.J. Tangirala R. Fazio S. Linton M.F. Funk C.D. J. Clin. Investig. 1999; 103: 1597-1604Crossref PubMed Scopus (465) Google Scholar, 3Bleich D. Chen S. Zipser B. Sun D. Funk C.D. Nadler J.L. J. Clin. Investig. 1999; 103: 1431-1436Crossref PubMed Scopus (131) Google Scholar, 4Kim D.C. Hsu F.I. Barrett N.A. Friend D.S. Grenningloh R. Ho I.C. Al-Garawi A. Lora J.M. Lam B.K. Austen K.F. Kanaoka Y. J. 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Witztum J.L. J. Biol. Chem. 2001; 276: 19431-19439Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). This suggests that H(p)ETE metabolites formed within or close to the membrane during LOX turnover may contribute; however, the identities of such lipids are currently unknown. Given the recent studies suggesting an important anti-inflammatory and pro-resolving function for 12/15-LOX, identification of novel products of this pathway is a clinically relevant goal (24Middleton M.K. Rubinstein T. Pure E. J. Immunol. 2006; 176: 265-274Crossref PubMed Scopus (46) Google Scholar, 25Gronert K. Maheshwari N. Khan N. Hassan I.R. Dunn M. Laniado Schwartzman M. J. Biol. Chem. 2005; 280: 15267-15278Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Herein, we utilized a lipidomic technique, precursor ESI/MS/MS to identify complex pools of endogenous H(p)ETE in immune cells. The studies identify specific esterified eicosanoids that form after immune cell activation and, furthermore, show the powerful nature of precursor scanning as a lipidomic tool for identifying novel endogenously generated lipid adducts. Materials—15(S)-Hydroxy-[S-(E,Z,Z,Z)]-5,8,11,13-eicosatetraenoic acid (15(S)-HETE), 12(S)-hydroxy-[S-(E,Z,Z,Z)]-5,8,10,14-Eicosatetraenoic acid (12(S)-HETE), 15S-hydroxy-11Z,13E-eicosadienoic acid, palmitoyltrifluoromethyl ketone, and oleyloxyethyl phosphocholine were from Alexis Chemicals Ltd., Nottingham, UK. Human recombinant interleukin 4 (IL-4) was from Promega, wortmannin and bisindolylmaleimide were from Calbiochem. LymphoprepTM was from Axis-Shield, Oslo, Norway. H 182O was from Cambridge Isotope Laboratories, Andover, MA. All other reagents were from Sigma unless otherwise stated. Isolation and Activation of Human Monocytes—50 ml of buffy-coat blood was diluted 1:1 (v/v) with PBS/citrate/dextran (0.8% w/v citrate, 2% w/v dextran 400, pH 7.4). Red cells were allowed to sediment for 1–2 h. The straw-colored supernatant was collected and underlayed with Lymphoprep 2:1 (v/v, supernatant:Lymphoprep) and centrifuged 800 × g for 20 min at 4 °C. The interface was collected and diluted 1:1 (v/v) with ice-cold PBS containing 0.4% (w/v) citrate, pH 7.4, and spun at 400 × g, 10 min at 4 °C. The supernatant was discarded, and the cell pellet was washed 5× with ice-cold PBS/citrate buffer, 400 × g for 5 min at 4 °C. The cell pellet was finally resuspended in a small volume of RPMI 1640 (10% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm glutamine). Approximately 108 cells were seeded per T75 flask and incubated at 37 °C for 2 h to allow monocytes to adhere. The medium was replaced, and cells were cultured for 72 h with 700 pm IL-4 to induce 15-LOX1. Monocytes were harvested by centrifugation and resuspended in Krebs buffer (50 mm HEPES, 100 mm NaCl, 5 mm KCl, 1 mm NaH2PO4·2H2O, 1 mm CaCl2, 2 mm glucose). 4 × 106 cells in 1 ml were stimulated with A23187 (10 μm) with/without 0.8 μm phorbol 12-myristate 13-acetate at 37 °C for 10 min. In some experiments H 182O was used in place of water in Krebs buffer. Experiments using signaling inhibitors included a 10-min preincubation step at 37 °C before the addition of stimulus. In some experiments 15-HETE-d8 (330 ng) was included during activation of cells. Murine-resident peritoneal cells were isolated from wild-type or 12/15-LOX–/– mice by peritoneal lavage into 2 ml of PBS and used without further purification (26Sun D. Funk C.D. J. Biol. Chem. 1996; 271: 24055-24062Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Isolation and Activation of Washed Human Platelets—Whole blood was collected from healthy volunteers free from nonsteroidal anti-inflammatory drugs for at least 14 days into acid-citrate-dextrose (ACD; 85 mm trisodium citrate, 65 mm citric acid, 100 mm glucose) (blood:ACD, 8.1:1.9 v/v) and centrifuged at 250 × g for 10 min at room temperature. The platelet-rich plasma was recentrifuged at 900 × g for 10 min, and the pellet was resuspended in Tyrode buffer (134 mm NaCl, 12 mm NaHCO3, 2.9 mm KCl, 0.34 mm Na2HPO4, 1 mm MgCl2, 10 mm Hepes, 5 mm glucose, pH 7.4) containing acid-citrate-dextrose (1:9 v/v). The platelets were washed by centrifuging at 800 × g for 10 min then resuspended in Tyrode buffer. Platelets were activated at 37 °C in the presence of 1 mm CaCl2 for 15 min, with 10 μm A23187, 10 μg/ml collagen, or 60 ng/ml convulxin before lipid extraction as below. Lipid Extraction—15(S)-Hydroxyeicosadienoic acid (10 μg for HPLC-UV or 10 ng for LC/MS/MS) and/or 10 ng of di-14: 0-phosphatidylethanolamine was added to each sample before extraction as internal standards. Hydroperoxides were then reduced to their corresponding stable alcohols by adding 1 mm SnCl2 (27Zhang R. Brennan M.L. Shen Z. MacPherson J.C. Schmitt D. Molenda C.E. Hazen S.L. J. Biol. Chem. 2002; 277: 46116-46122Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar). Lipids were extracted by adding a solvent mixture (1 m acetic acid, 2-propanol, hexane (2:20:30, v/v/v) to the sample at a ratio of 2.5 ml of solvent mixture/1 ml of sample, vortexing, and then adding 2.5 ml of hexane (27Zhang R. Brennan M.L. Shen Z. MacPherson J.C. Schmitt D. Molenda C.E. Hazen S.L. J. Biol. Chem. 2002; 277: 46116-46122Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar). After vortex and centrifugation, lipids were recovered in the upper hexane layer. The samples were then re-extracted by the addition of an equal volume of hexane followed by vortex and centrifugation. The combined hexane layers were dried under N2 flow and analyzed for free 15-HETE using LC-UV or LC/MS/MS or esterified compounds using LC/MS/MS (as described below). To calculate esterified HETEs, free HETE was subtracted from total measured after saponification. For this, N2-dried lipids were resuspended in 1.5 ml of 2-propanol, and then fatty acids were released by base hydrolysis with 1.5 ml of 0.2 m NaOH at 60 °C for 30 min under argon atmosphere. Hydrolyzed samples were acidified to pH 3.0 with 0.5 m HCl, and then fatty acids were extracted twice with 3 ml of hexane. The combined hexane layers were dried under N2 flow, resuspended in 100 μl of methanol, and stored under argon at 80 °C until analysis by LC-UV or LC/MS/MS. HETE Quantitation Using LC/UV or LC/MS/MS—Samples were separated on a C18 ODS2, 5-μm, 150 × 4.6-mm column (Waters Ltd) using a gradient of 50–90% B over 20 min (A, water:acetonitrile:acetic acid, 75:25:0.1; B, methanol:acetonitrile: acetic acid, 60:40:0.1) at 1 ml/min. Products were quantitated either by UV absorbance (235 nm) or by LC/ESI/MS/MS on a Q-Trap (Applied Biosystems 4000 Q-Trap) using specific parent to daughter transitions of m/z 319.2 (HETE, [M-H]–) to m/z 219 (15-HETE) or 179 (12-HETE) and 323.2 to 223 for 15-hydroxyeicosadienoic acid with collision energies of –20 or –28 V, respectively. Products were identified and quantified using either 15(S)- or 12(S)-HETE, and 15(S)-hydroxyeicosadienoic acid standards run in parallel under the same conditions. Precursor Scanning Mass Spectrometry—Electrospray mass spectra were obtained on a Q-Trap instrument (Applied Biosystems 4000 Q-Trap) operating in the negative mode. Lipid extracts were diluted (1:50–1:100) and introduced at 10 μl/min in methanol using a Hamilton syringe. Instrument settings were determined by tuning on an oxidized phosphatidylethanolamine (PE) standard and run with declustering potential –140 V and collision energy –45 V. Spectra were obtained from 550 –1000 atomic mass units over 12 s, with typically 10 scans acquired and averaged. For determination of –fold changes in specific 15-HETE-containing lipids, 10 μl of diluted extracts were injected under flow (1 ml/min) in a methanol:water (50:50) mixture with specific multiple reaction monitoring (MRM) transitions monitored using m/z 319.2 as daughter ion and comparing ion intensity to the internal standard, di-14:0-PE (MRM m/z 634.5 → 227.2). For 15-HETE-d8, m/z 327.2 was used instead. Normal Phase HPLC-UV of Phospholipid Classes—To fractionate monocyte phospholipid classes before MS analysis, extracts were separated on a Spherisorb S5W 4.6 × 150-mm column (Waters Ltd) using a gradient of 50 –100% B over 25 min (A, hexane:2-propanol, 3:2; B, solvent A:H2O, 94.5:5.5) at a flow rate of 1.5 ml min–1 (28Dugan L.L. Demediuk P. Pendley Jr., C.E. Horrocks L.A. J. Chromatogr. 1986; 378: 317-327Crossref PubMed Scopus (89) Google Scholar). Absorbance was monitored at 205 nm, and products were identified using a mixture of standard phospholipids (Sigma). 1-min fractions were collected for subsequent analysis by ESI/MS/MS. After drying down and resuspending in 100 μl of methanol, 10-μl samples of each fraction were injected under flow (1 ml/min) in a methanol:water (50:50) mixture into the electrospray source, with specific MRM transitions monitored using m/z 319.2 as the daughter ion, and areas for each transition were determined in each fraction by integration of the peaks. These were then replotted versus time to obtain normal phase elution profiles for each HETE-containing ion. Reverse Phase LC/MS/MS of Phospholipids—Online reverse phase separation of phospholipids to separate based on acyl chain was carried out using a Luna 3-μm C18 (2Cyrus T. Witztum J.L. Rader D.J. Tangirala R. Fazio S. Linton M.F. Funk C.D. J. Clin. Investig. 1999; 103: 1597-1604Crossref PubMed Scopus (465) Google Scholar) 150 × 2-mm column (Phenomenex, Ltd) with a gradient of 0–100% B over 30 min (A, acetonitrile:methanol, 35:65; B, acetonitrile:methanol:triethylamine, 35:65:1.5) at a flow rate of 200 μl/min. MS/MS scans using the ion trap mode of the Q-Trap were triggered at the apex of an MRM transition (parent → 219 or 179 for 15-or 12-HETE, respectively). The scan was from 150 to 800 atomic mass units over 0.65 s, with a linear ion trap fill time of 200 ms and Q0 trapping. MS3 was carried out using the electrospray source, with direct infusion of the normal phase PE fraction diluted 1:20 in methanol at 10 μl/min. The linear ion trap fill time was 250 ms with excitation time 200 ms, excitation energy 200 V, with a mass range scanned from 100 to 320 atomic mass units. HETE Isomer Determination—Lipid extracts were separated by normal phase HPLC, the PE-containing fraction (5.5–8 min) was collected, saponified, then resuspended in 50 μl of methanol, and HETE isomers were analyzed by reverse phase LC/MS/MS. For chiral phase analysis 15-HETE was collected, resuspended in the chiral phase mobile phase (hexane:2-propanol:acetic acid, 100:5:0.1), and injected onto a Chiralcel OD 0.46 × 25-cm column (Chiral Technologies Ltd, Exton, PA) with isocratic separation at 1 ml/min with absorbance monitored at 235 nm. 15-HETE Quantitation in Phospholipid Classes—Lipid extracts from A23187-activated monocytes were separated by normal phase LC-UV, and 1-min fractions were collected from 0 to 20 min. Fractions were dried under N2 and resuspended in methanol. Fractions containing each phospholipid class were identified by head-group precursor scanning (PE = 7–9 min, phosphatidylcholine (PC) = 18–20 min, phosphatidylinositol = 11–13 min, phosphatidylglycerol 6–7 min, phosphatidylserine 14–16 min) and hydrolyzed as already described, then analyzed by reverse phase LC/MS/MS monitoring for 15-HETE. Amounts of PE and PC were determined using normal phase LC-UV (205 nm). Generation of 15-HETE-containing Phospholipids by Soybean LOX Oxidation of Commercial Phospholipid Preparations—5 mg/ml l-α-phosphatidylethanolamine (egg (Sigma) or brain, porcine plasmalogen (Avanti Polar Lipids, Alabaster, AL)) was incubated for 30 min at 37 °C in PBS, pH 7.4, 4% sodium cholate, with 50 kilounits of soybean lipoxygenase type IV (Sigma). Samples were then reduced using 1 mm SnCl2 for 10 min, 20 °C, spiked with 10 ng of internal standard (di-14:0-PE), and extracted as before. For experiments comparing the effects of SnCl2 and H2 18O, SAPE or SAPC (500 μm) was oxidized using 8 kilounits of soybean 15-LOX in 0.2 m borate, 10 mm deoxycholate buffer, pH 9, for 30 min at 37 °C either in buffer composed of H 162O or H 182O. Samples were separated by reverse phase LC/MS/MS monitoring the MRM transitions 782–319 (15-HETE-16O-PE) and 784–321 (15-HETE-18O-PE) in negative mode and 826 → 184 (15-HETE-16O-PC) and 828 →184 (15-HETE-18O-PC) in positive mode. Ion intensities were related to the internal standard to reveal -fold changes. Statistical Analysis—Data are representative of at least three separate donors, with samples run in triplicate for each experiment (mean ± S.E.). Significance was examined using an unpaired t test, where p < 0.05 was considered significant (denoted by an asterisk on the figures). Monocytes Generate Predominantly Esterified 15-H(p)ETE after Ionophore Activation—To define pathways that regulate calcium-dependent activation of 15-LOX1, generation of 15-H(p)ETEs after stimulation of IL-4-treated human monocytes with A23187 was examined. Samples were reduced using SnCl2 before lipid extraction to convert all HpETE into the more stable HETE (27Zhang R. Brennan M.L. Shen Z. MacPherson J.C. Schmitt D. Molenda C.E. Hazen S.L. J. Biol. Chem. 2002; 277: 46116-46122Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar). Most 15-H(p)ETE generated on activation was esterified to complex lipids of unknown structure (Fig. 1A). Also, basal levels of esterified 15-H(p)ETE were detected without ionophore activation. Esterified H(p)ETE generation occurred early and continued after free H(p)ETE had plateaued (Fig. 1B). Murine peritoneal macrophages that express 12/15-LOX, the functional equivalent of human 15-LOX1, generated esterified 12-H(p)ETE, and this was absent in macrophages from 12/15-LOX–/– mice (Fig. 1C). These data show that the predominant H(p)ETEs generated by monocytes and macrophages on calcium activation are not free acid products, but complex lipids. Regulation of 15-H(p)ETE Release by Intracellular Signaling Pathways—Calcium facilitates membrane association of 12/15-LOX (29Brinckmann R. Schnurr K. Heydeck D. Rosenbach T. Kolde G. Kuhn H. Blood. 1998; 91: 64-74Crossref PubMed Google Scholar). However, it is unknown whether additional signaling pathways participate in monocyte 12/15-LOX regulation by calcium and whether differences exist between regulation of free versus esterified product formation. Inhibition of both H(p)ETE forms was observed on the addition of wortmannin, implicating phosphatidylinositol 3-kinase (Fig. 1D). The protein kinase C inhibitor bisindolylmaleimide did not significantly affect ionophore stimulation of 12/15-LOX; however, activation of protein kinase C using phorbol 12-myristate 13-acetate significantly promoted generation of both forms in a bisindolylmaleimide-sensitive manner (Fig. 1E). This indicates that stimulation of protein kinase C potentiates calcium-dependent activation of 12/15-LOX. H(p)ETE Is Esterified to PE in Activated Human Monocytes and Mouse Peritoneal Macrophages—We sought to determine the specificity of 15-H(p)ETE esterification in monocytes using an MS-based lipidomic approach. Precursor ESI/MS/MS scanning for 319.2 (HETE [M-H]–) of crude lipid extracts from IL-4-treated human monocytes activated with A23187 revealed 4 dominant ions at m/z 738.5, 764.5, 766.5, and 782.5 (Fig. 2A). Also, precursor ESI/MS/MS of murine peritoneal macrophage extracts demonstrated identical ions (Fig. 2B). Next, MRM transitions of the parent-HETE daughter ion were monitored in activated and unactivated human monocytes and compared with an internal standard (di14:0-PE). All four ions increased significantly after calcium mobilization (Fig. 2C). These m/z values are consistent with nitrogen-containing HETE derivatives of two phospholipid species, PE and PC (e.g. m/z 766.5 could be 16:0p/15-HETE-PC or 18:0p/15-HETE-PE). However, the m/z values are not consistent with other phospholipids, including phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, or phosphatidic acids (30Murphy R. Mass Spectrometry of Phospholipids: Tables of Molecular and Product Ions. Illuminati Press, Denver, CO2002Google Scholar). To determine phospholipid class, normal phase LC was undertaken that separates phospholipids according to head group. Fractions were collected, and aliquots of each 1-min fraction were monitored for the parent → m/z 319.2 transition using MS/MS. Greater than 95% of ion intensity of the transition for all 4 peaks co-eluted with di-14:0-PE standard (5–6-min fraction), with the remainder ( 92% of the 15-HETE was found in PE (Fig. 3). The preference of PE over PC as the site of HETE synthesis is observed whether it is expressed as a function of total phospholipid class or per μg of phospholipids, with the level equating to ∼1.5% of the PE pool in activated monocytes containing 15-HETE (based on detecting 0.7125 nmol of 15-HETE in 48 nmol of purified PE). Structural Identification of Individual 15-HETE Ions—Because of isobaric peaks in the PE fraction, further LC was required to separate different 15-HETE-containing PEs. Reverse phase-LC/MS/MS, which separates based on acyl chain composition, was used on normal phase-purified PE fractions (see "Experimental Procedures"). The parent → m/z 219 transition (15-HETE ion) was monitored with an MS/MS spectrum triggered during elution of the MRM transition. The spectrum shown is obtained at the apex of the peak of elution for each compound, which was at the same retention time (4.6 min) for all 4 ions, likely due to the presence of identical sn-2 lipids. Using this approach, spectra were obtained that could be compared with PE oxidized in vitro using soybean 15-LOX. The product ion spectrum of m/z 782.5 (Fig. 4A) contains major ions at m/z 283 (18:0, stearic acid), m/z 319, 301, and 257 (HETE) along with 219 and 175 (characteristic of the 15-HETE isomer only). Comparison of this with that obtained for m/z 782.5 from soybean 15-LOX-oxidized egg PE shows an identical spectrum (Fig. 4E). Not only are the same ions observed, but the ratios of ions to each other are consistent. This together with an identical retention time on both normal phase and reverse-phase HPLC, supports the identification of m/z 782.5 as 18:0/15-HETE-diacyl-PE (Scheme 1A).SCHEME 1Proposed structures for the four 15-HETE PE compounds identified in activated human monocytes. A–D show proposed structures, based on comparison with synthetic standards. For platelets, 15-HETE is replaced by 12-HETE.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The product ion spectra for m/z 766.5 (Fig. 3B), 764.5 (Fig. 4C) and 738.5 (Fig. 4D) reveal ions characteristic of the HETE carboxylate anion at m/z 319 and its fragments at m/z 301 and 257, with minor 15-HETE ions at m/z 219 and 175 atomic mass units. There was also neutral loss of the free acid [M-H-320]– and also the ketene [M-H-302] but no ions characteristic of an sn-1 fatty acid. These spectra suggest that PE possesses either ether or plasmalogen bonds at sn-1. The samples were examined in positive mode, as previously done for plasmalogen PC, but sensitivity was lower, and good quality spectra could not be obtained (Ref. 32Zemski Berry K.A. Murphy R.C. J. Am. Soc. Mass Spectrom. 2004; 15: 1499-1508Crossref PubMed Scopus (220) Google Scholar; data not shown). Plasmalogen phospholipids are sensitive to acidic conditions and can be hydrolyzed by exposure to HCl fumes (32Zemski Berry K.A. Murphy R.C. J. Am. Soc. Mass Spectrom. 2004; 15: 1499-1508Crossref PubMed Scopus (220) Google Scholar). However, not only did ions at m/z 738.5, 764.5, and 768.5 degrade relative to the diacyl internal standard (di14:0-PE) after acid exposure but also the m/z 782.5, which is not a plasmalogen (data not shown), degraded. This suggests that HETE-containing lipids, by virtue of their -OH group on the 20:4, are sensitive to acid hydrolysis. Brain PE, which contains ∼50% plasmalogen and little ether-linked lipids was, therefore, oxidized using soybean 15-LOX. Spectra obtaine
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