Susceptibility of plasmenyl glycerophosphoethanolamine lipids containing arachidonate to oxidative degradation

Free Radical Biology & Medicine, Vol. 26, Nos. 3/4, pp. 275–284, 1999 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/99/$–see front matter

PII S0891-5849(98)00211-1

Original Contribution SUSCEPTIBILITY OF PLASMENYL GLYCEROPHOSPHOETHANOLAMINE LIPIDS CONTAINING ARACHIDONATE TO OXIDATIVE DEGRADATION NONA KHASELEV

and

ROBERT C. MURPHY

Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO, USA (Received 8 May 1998; Accepted 8 July 1998)

ABSTRACT—Plasmenyl phospholipids (1-alk-19-enyl-2-acyl-3-glycerophospholipids, plasmalogens) are a structurally unique class of lipids that contain an a-unsaturated ether substituent at the sn-1 position of the glycerol backbone. Several studies have supported the hypothesis that plasmalogens may be antioxidant molecules that protect cells from oxidative stress. Because the molecular mechanisms responsible for the antioxidant properties of plasmenyl phospholipids are not fully understood, the oxidation of plasmalogens in natural mixtures of phospholipids was studied using electrospray tandem mass spectrometry. Glycerophosphoethanolamine (GPE) lipids from bovine brain were found to contain six major molecular species (16:0p/18:1-, 18:1p/18:1-, 18:0p/20:4-, 16:0p/20:4, 18:0a/20:4-, and 18:0a/22:6GPE). Oxidation of GPE yielded lyso phospholipid products derived from plasmalogen species containing only monounsaturated sn-2 substituents and diacyl-GPE with oxidized polyunsaturated fatty acyl substituents at sn-2. The only plasmalogen species remaining intact following oxidation contained monounsaturated fatty acyl groups esterified at sn-2. The mechanism responsible for the rapid and specific destruction of plasmalogen GPE may likely involve unique reactivity imparted by a polyunsaturated fatty acyl group esterified at sn-2. This structural feature may play a central role determining the antioxidant properties ascribed to this class of phospholipids. © 1998 Elsevier Science Inc. Keywords—Plasmalogens, Molecular species, Phospholipid oxidation, Mass spectrometry, Electrospray, Antioxidant, Arachidonate, Oxidant stress, Free radical

INTRODUCTION

rosis [8], ischemic injury [9], and carcinogenesis [10]. Initial targets of phospholipid peroxidation are thought to be polyunsaturated fatty acyl substituents [11] and a high proportion of plasmalogen molecular species contain such fatty acids, in particular arachidonic acid [1,2]. Few biochemical functions for plasmalogens have been proposed in spite of extensive investigations. Recently, a unique susceptibility of plasmalogen phospholipids to attack by oxygen radicals has been suggested to be related to the presence of the enol ether double bond at sn-1 position. Raetz and coworkers [12,13] reported that 12-(19-pyrene)dodecanoic acid treated mutant cells lacking plasmalogen phospholipids showed increased cytotoxicity on ulraviolet (UV) irradiation, whereas the supplementation of the mutants with plasmalogen phospholipids increased the resistance of these cells to oxidative degradation. In vitro enrichment of LDL with plasmalogen phospholipids was found to increase the lag phase of formation of conjugated double bonds induced by oxidation with copper ions [14], indicating a possible antioxidant role for plasmalogens. The oxidative degra-

Plasmalogens are a unique subclass of glycerophospholipids characterized by the presence of a vinyl ether substituent at the sn-1 position of the glycerol backbone (Fig.1). These unique phospholipids are found in high concentration in cellular membranes of numerous mammalian tissues [1]. The percentage of plasmalogen lipids in general has been reported to lie between 15% and 20% of total phospholipids, but can reach more than 50% in heart [2], brain [3,4], neutrophils [5], and macrophages [6]. In LDL particles, the content of plasmalogen phospholipids is approximately 30 mmol/mmol of LDL cholesterol [7]. The oxidation of phospholipids in biological tissues has been suggested to be a significant chemical event in a variety of pathological conditions such as atheroscleAddress correspondence to: Dr. Robert C. Murphy, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206, USA; Tel: 303-398-1849; Fax: 303-398-1694; E-Mail: [email protected]. 275

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anolic HCl. The mixture was allowed to stand for 30 min at room temperature, and then the solvent was evaporated to dryness under nitrogen. The residue was dissolved in 100 ml of methanol containing 1 mM ammonium acetate (pH 7.4) and an aliquot (1 ml) was injected into the electrospray mass spectrometer (flow injection at 50 ml/min).

Fig. 1. Generic structure of glycerophosphoethanolamine (GPE) plasmalogen molecular species. R1 corresponds to an alkyl chain, e.g., C16H33. R2 would correspond to the alkyl chain such as C19H31 for an arachidonoyl molecular species and C17H33 for an oleoyl molecular species esterified at sn-2.

dation of phospholipids as analyzed by NMR [15] indicated delayed disappearance of diacyl phospholipids when plasmalogens were present. The products of these plasmalogen oxidations were not structurally characterized. The potential importance of cell membrane phospholipid protection from oxidative degradation prompted us to investigate the major products of plasmalogen oxidation utilizing mass spectrometric techniques. MATERIALS AND METHODS

Materials L-a-phosphatidylethanolamine (bovine brain) was purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). HPLC solvents were purchased from Fisher Scientific (Fair Lawn, NJ, USA) and used for HPLC, extraction, hydrolysis, and oxidation. Other chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA). HPLC separation of lipid mixture Reverse phase HPLC (RP-HPLC) separation of phospholipid mixtures was performed using Phenomenex Ultremex 5m C18 (250 3 4.6 mm) and monitoring UV absorbance of the effluent at 206 nm. The mobile phase consisted of an isocratic elution of methanol/water/acetonitrile (90.5/7/2.5, v/v/v) containing 1 mM ammonium acetate adjusted to pH 7.4. A flow rate of 1.4 mL/min was used and fractions (0.5 min) were collected. Fractions were analyzed by tandem mass spectrometry for phospholipid molecular species analysis. Acid hydrolysis The vinyl ether double bond of ethanolamine plasmalogen (100 mg of phospholipid mixture) was hydrolyzed by dissolving the lipid in 200 ml of 2.5 N meth-

Oxidation procedure and solid phase extraction (SPE) Bovine brain GPE (10 mmol) mixture was suspended in 6.5 ml of 50 mM PBS (pH 7.4) by vortexing and sonication. To the solution, 30% H2O2 and 6 mM CuCl2 were added to result in a final concentration of 70 mM and 100 mM, respectively. The reaction vessel was capped and shaken gently at 37°C for time periods up to 3 h. The formation of conjugated dienes on oxidation of phospholipids led to the increase of absorbance at 234 nm, that was continuously monitored at 30-min intervals. The incubation was stopped by immersion into an ice bath and the addition of one volume of CHCl3 containing 50 mg/ml butylhydroxytoluene (BHT). Lipids were then extracted according to Bligh and Dyer [15]. The reaction mixture was loaded onto a C-18 (1 ml, 100 mg) reverse phase 40 mm silica packed Sep-Pack cartridge (solid phase extraction) that was preconditioned with 10 ml of methanol and 10 ml of PBS. The inorganic salts and excess of H2O2 were eluted with 10 ml of water. The phospholipids fraction was eluted first with 10 ml of 80% methanol and secondly by the combined eluates of 10 ml of 90% methanol, and 10 ml of pure methanol. The two fractions were taken to dryness by rotary evaporation, and 1 ml of chloroform/methanol (2:1 v/v) was added to each residue. Aliquots (5 ml) were taken for direct analysis by electrospray tandem mass spectrometry. Electrospray ionization mass spectrometry and tandem mass spectrometry (MS/MS) RP-HPLC fractions of bovine brain GPE were analyzed using electrospray ionization and tandem mass spectrometry. Flow injection of 2–5 ml of the HPLC and SPEfractions was carried out with a flow rate of 10 ml/min, 90% methanol, 1 mM ammonium acetate as a mobile phase. The Sciex API III1 (Perkin-Elmer Sciex, Toronto, Canada) was operated in the negative and positive modes with the orifice at 2100 V and 170 V, respectively. Collision induced dissociation (CID) was performed by using 30 eV and a collision gas thickness (argon) of 230 3 1013 molecules/ cm2 . A curtain gas flow of 1.6 l/min and spray temperature of 400°C were used. On-line RP-HPLC/MS analysis of bovine brain GPE

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Fig. 2. Reverse phase HPLC separation of the molecular species of glycerophosphatidylethanolamine derived from bovine brain. Elution of the individual molecular species containing unsaturated fatty acyl substituents detected by UV absorbance at 206 nm. Identification of the individual components corresponding to each peak was carried out using LC/MS/MS techniques as described in the Results section.

was performed in the negative mode by using Ultremex C-18 5m (250 3 4.6 mm) column connected to a UV monitor (206 nm) in line just prior to the electrospray interface. The HPLC was operated at a flow rate of 1 ml/min with an isocratic elution of the same mobile phase as described previously and a postcolumn split that yielded 20 ml/min flow into the mass spectrometer. Phospholipids obtained after oxidation and subsequent solid phase extraction were analyzed by ESI/MS negative mode. Aliquots (5 ml) were applied for the analysis by flow injection with mobile phase of 90% methanol and flow rate of 20 ml/min. RESULTS

Identification of molecular species of GPE mixture On-line RP/HPLC-MS analysis of brain phosphatidylethanolamine showed that the phospholipid mixture contained a large number of different species as shown in Fig.2. The elution profile (absorbtion at 206 nm) of GPE mixture from RP-HPLC column revealed predominant components eluting at 44, 46, 48, 64, 71, 73, and 76 min corresponding to plasmalogen molecular species containing unsaturated fatty acyl groups. The other abundant components eluting at 37, 38, 58, and 60 min represented diacyl phospholipid species. The identification of the phospholipid molecular species was performed by electrospray ionization mass spectra where each individual component yielded prominent ions [MH]1 and [M-H]- obtained as positive and negative ions, respectively (Figs. 3A and B). Comparison of these two spectra confirmed the molecular weight of each of the major GPE molecular species (Table 1). However, to determine the fatty acyl constituents in each

individual molecular species, negative ion tandem mass spectrometry was used (Table 2). As shown in Fig. 4, there were significant differences in the abundant collisional induced decomposition (CID) product ions from different subtypes of phospholipids. CID spectra of [M-H]- ions of diacyl phospholipids (e.g., Fig. 4A) resulted in two carboxylate anions obtained from both acyl groups esterified at sn-1 and sn-2 positions of phospholipid. With some exceptions [17], the abundance of the sn-2 carboxylate anion was more prominent than the abundance of the sn-1 carboxylate anion that permitted assignment of the esterification site on the glycerol backbone of the diacyl phospholipid species. In the case of plasmenyl or alkyl ether phospholipids, the CID spectra of [M-H]- ions resulted in a single carboxylate anion obtained from the acyl group esterified at sn-2 position of phospholipid glycerol backbone (e.g., Fig. 4B and C). In order to distinguish plasmenyl GPE molecular species (plasmalogens) from alkyl ether glycerophospholipids, the phospholipid mixture was hydrolyzed under mild acidic conditions that did not attack alkyl ether or ester bonds. The disappearance of [M-H]- of acid-labile plasmenyl species and the appearance of a corresponding sn-1 lyso phospholipid in the mass spectra of the hydrolyzed mixture provided a means to identify and determine relative distribution of the brain phosphatidylethanolamine subclasses (Fig. 3C). Therefore, the high plasmalogen content of the brain-GPE mixture was supported by abundant [M-H]- ions observed at m/z 700, 726, 728, 748, 750, 774, and 776, which were not found following exposure to mild acid (Table 1) and appearance of a new set of [M-H]- ions corresponding to the sn-1 lyso-phospholipid products [M-H]- at m/z 478, 500,

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Fig. 3. Electrospray ionization mass spectrometric analysis of bovine brain GPE molecular species. (A) Positive ions corresponding to [MH] 1 for each molecular species. Identification of each of the major component was carried out through a combination of collision induced dissociation experiments and treatment of phospholipid mixture with HCl and reanalysis. (B) Negative ions corresponding to [M-H]- for each molecular species. (C) Negative ion electrospray mass spectra of bovine brain GPE following treatment with 0.1 N HCl to degrade plasmalogen molecular species. Plasmalogens yield lyso products retaining the sn-2 ester group while diacyl GPE is unaffected.

506, 524, 528, and 544 corresponding to the loss of the sn-1 alkenyl chain by acidic hydrolysis (Fig. 3C). This level of mass spectrometric analysis of brain

phosphatidylethanolamine revealed that diacyl phospholipids were mainly comprised of species with 18:0 fatty acyl groups at the sn-1 position, whereas the plasmenyl

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Table 1. Glycerophosphoethanolamine Lipids (GPE) Molecular Species Present in Commercial Bovine Brain GPE and Identification of Plasmenyl Subclass as Determined by Electrospray Mass Spectrometry Molecular Ion Species GPE Molecular Speciesa,b

m/z [M-H]2/[M1H]1

Relative Abundancec (%)

Relative Abundance After HCl-treatmentd,e

700/702 702/704 716/718 722/724 726/728 728/730 730/732 738/740 740/742 742/744 744/746 746/748 748/750 750/752

60 9 9 11 100 56 5 10 15 17 18 12 31 59 7 18 20 5 12 5 33 29 24 26 23 9

N.D. 18 51 N.D. N.D. N.D. 19 20 15 68 78 15 N.D. N.D. N.D. 21 N.D. 26 29 12 100 N.D. N.D. N.D. 82 39

16:0p/18:1 18:1e/16:0 16:0a/18:1 16:0p/20:4 18:1p/18:1 18:0p/18:1 18:0e/18:1 16:0a/20:4 18:1a/18:2 18:1a/18:1 18:0a/18:1 18:0a/18:0 18:1p/20:4 18:0p/20:4 major 16:0p/22:4 minor 18:0e/20:4 18:1p/20:1 16:0a/22:5 16:0a/22:6 18:1a/20:4 18:0a/20:4 18:0p/22:6 18:1p/22:4 18:0p/22:4 18:0a/22:6 18:0a/22:4

752/754 754/756 762/764 764/766 766/768 774/776 776/778 778/780 790/792 794/796

a Abbreviations for individual GPE molecular species used in this paper: n:jk/s:t GPE (e.g., 16:0p/20:4 GPE), where n is the number of carbons in the sn-1 substituent and j is the number of double bonds in the sn-1 hydrocarbon chain; k represents the type of sn-1 linkage (where a 5 1-O-acyl, e 5 1-O-alkylether, p 5 1-O-alk-19-enyl (plasmalogen)); s is the number of carbons and t is the number of double bonds at the sn-2 fatty acyl substituent. b Assignment of sn-1/sn-2 from tandem LC/MS/MS experiments (Table 2). c Average of the relative abundances observed in positive and negative ion modes. d Relative abundance of [M 2 H]2 ions following exposure to methanolic HCl (see Materials and Methods) prior to negative ion electrospray mass spectrometry. e N.D., not detected.

phospholipids were comprised of species with 18:0 and 18:1 carbon chains at the sn-1 position. Those molecular species with 18:1 carbon chain substituents at sn-1 were more abundant in plasmenyl GPE compared to diacyl GPE (Fig 5A). The plasmalogen subfraction of brain GPE was esterified at sn-2 predominantly with oleic (18:1) and arachidonic acid (20:4). The content of arachidonic acid in plasmenyl GPE was approximately twice that observed in diacyl GPE and about five times more for docosatetraenoic acid (22:4) (Fig. 5B). Analysis of oxidized phospholipid fractions Oxidation of the bovine brain GPE phospholipid mixture in the presence of H2O2 and CuCl2 (70 mM and 100 mM, respectively) was carried out for 3 h at 37°C. Reverse phase solid phase extraction was used to partially separate products of the reaction. The first fraction

was eluted with 80% methanol and was identified as a mixture of sn-1 lyso products (Fig. 6A), whereas the second fraction eluted with 90% and pure methanol was identified as a mixture of intact phospholipids (Fig. 7). Major sn-1 lyso products were observed at m/z 478, 506, and 534, which resulted from the loss of the sn-1 alkenyl chain during the oxidation of phospholipids yielding the corresponding lyso/18:1-, lyso/20:1-, and lyso/22:1GPE. These species were observed as lyso products after HCl-treatment of the starting bovine brain GPE- mixture. However, abundant ions at m/z 500, 524, and 528 (Fig. 6B) corresponding to the sn-1 lyso-phospholipids with polyunsaturated carbon chains at the sn-2 position (OH/ 20:4-, OH/22:6-, and OH/22:4-GPE, respectively) were observed as plasmalogen hydrolysis products, but not as sn-1 lyso products after oxidation. The formation of sn-1 lyso compounds and corresponding fatty aldehyde following oxidation has been previously reported [13].

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N. KHASELEV and R. C. MURPHY Table 2. Assignment of Fatty Acyl Position in Glycerophosphoethanolamine Lipids (GPE) Using Tandem Mass Spectrometry of Individual Molecular Species Separated by Reverse Phase HPLC Compounda

m/z, [M 2 H]2

16:0a/22:6 16:0a/20:4 18:1a/20:4 16:0p/22:6 16:0p/20:4 16:0p/22:5 18:1p/20:4 16:0a/18:1 18:0a/22:6 18:1a/18:1 18:1a/18:2 18:0a/20:4 16:0p/22:4 16:0p/18:1 18:1p/22:4 18:0p/22:6 18:1p/18:1 18:0p/20:4 18:1e/16:0 18:0e/20:4 18:0a/22:4 18:0p/22:5 18:0a/18:1 18:0p/22:4 18:0e/18:1 18:1p/20:1 18:0p/18:1

762 738 764 746 722 764 748 716 790 742 740 766 750 700 776 774 726 750 702 752 794 776 744 778 730 754 728

Observed Carboxylate Anionb 255 (6), 327 (17) 255 (6), 303 (15) 281 (7), 303 (18) 327 303 329 303 255 (18), 281 (32) 283 (9), 327 (10) 281, 281 (25) 281 (15), 279 (15) 283 (7), 303 (16) 331 281 331 327 281 303 255 303 283 (9), 331 (15) 329 283 (9), 281 (21) 331 281 309 281

Retention Time Minc 37 38 39 44 46 47 48 58 60 61 62 63 64 71 72 73 74 77 79 83 91 92 95 109 112 113 115

a

For abbreviations of molecular species, see Table 1. A single carboxylate anion resulted from plasmenyl and alkyl ether GPE molecular species. Assignment of sn-1 and sn-2 esterification positions derived from the relative abundance (listed in parentheses) of the two carboxylate anions when compared to previously published ratios [17]. c Reverse phase HPLC retention time using conditions described in the Materials and Methods section. b

However, a comparison of oxidation and HCl treatment of the natural mixture of GPE molecular species revealed a somewhat more complicated series of reactions than a simple involvement vinyl ether oxidation (Fig. 8). The acid hydrolysis of plasmalogen GPE yielded a mixture of unsaturated fatty acyl substituents at the sn-2 position (Fig. 8A). When the plasmalogen sn-1 vinyl ether group was cleaved during oxidation, lyso products were formed, but the sn-2 position retained only monounsaturated fatty acid chains (Fig. 8B). No sn-1 lyso products containing sn-2 polyunsaturated fatty acyl groups were observed after oxidation of bovine brain GPE. The second solid phase extraction fraction contained intact phospholipids as determined by tandem mass spectrometric analysis of the major ions at m/z 716, 742, and 744. Each of these diacyl GPE species contained the 18:1 fatty acyl group esterified at the sn-2 position (Fig. 7). The other abundant ions at m/z 700, 726, and 728 corresponded to the only remaining plasmalogen species and each of these plasmalogen species were esterified with 18:1 at the sn-2 position (16:0p/18:1-, 18:1p/18:1-, and 18:0p/18:1-GPE, respectively). Ions at m/z 756, 772, and 798 corresponded to diacyl GPE species retaining

sn-2 esterified 18:2 and 20:4, but with the addition of one oxygen atom (m/z 756) and two oxygen atoms (m/z 772 and 798). DISCUSSION

Free radical-induced lipid peroxidation is known to be a complex process in which components of biological membranes, and in particular glycerophospholipids containing polyunsaturated fatty acyl groups react with molecular oxygen [11,18]. Such events are known to propagate during various pathological conditions including ischemia-reperfusion, chronic inflammation, aging, cancer, and other pathological conditions [19,20]. Polyunsaturated fatty acyl groups esterified to membrane glycerophospholipids, which have one or more bisallylic methylene groups can have a hydrogen atom removed in a radical reaction leading to the relatively long lived carbon-centered radical species [21]. Such radical intermediates then react with molecular oxygen leading to the formation of conjugated diene lipid hydroperoxides. Additional reactions are known to occur from such radical species as well as hydroperoxy radical species, including

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Fig. 4. Representative collision induced decomposition (CID) of [M-H]- ions of three different GPE phospholipid molecular species present in bovine brain GPE. (A) CID of m/z 716.6 corresponding to a diacyl GPE molecular species, which yields two carboxylate anions at m/z 255 and 281. This molecular species was identified as 16:0a/18:1-GPE. (B) CID of m/z 700.6 corresponding to [M-H]of a plasmalogen-GPE molecular species. This component was identified as 16:0p/18:1-GPE. (C) CID of m/z 702 corresponding to an alkyl ether GPE molecular species. The single carboxylate anion at m/z 255 corresponded to palmitate esterified at the sn-2 position. This molecular species was identified as 18:1e/16:0-GPE and was present after treatment with acid.

formation of aldehydes, chain-shortened alkyl groups as well as more complex covalent modification of the polyunsaturated fatty acyl group. These reactions include the formation of compounds isomeric to prostaglandins and leukotrienes, termed isoeicosanoids [22]. Interest in reactions that can terminate lipid peroxidation events has led to a large number of studies of antioxidant substances within membranes including vitamin E, glutathione as

well as other species [11]. A compelling case has been made for the role of plasmalogen glycerophospholipids as antioxidant substances that protect cells from damage due to radical-initiating reactions [12,13,15]. In the present study, brain GPE, which existed as a complex mixture of diacyl and plasmenyl phospholipids was used to study covalent modifications taking place during the oxidation of natural mixtures of glycerophos-

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Fig. 5. Summary of the relative abundance of the major molecular species in bovine brain GPE normalized to (A) the most abundant substituent at the sn-1 position and (B) the most abundant fatty acyl group esterified to the sn-2 position. The abundance of each molecular species was determined using electrospray ionization mass spectrometry and the abundance of the corresponding [M1H]1 ions (see Fig. 3). Diacyl molecular species are indicated by the closed field columns and plasmalogen molecular species indicated by the gray columns.

pholipids molecular species. The analysis of the individual molecular species was carried out using reverse phase HPLC separation and analysis using electrospray mass spectrometry with collision induced dissociation and tandem mass spectrometric experiments. The bovine brain GPE mixture consisted primarily of diacyl and plasmenyl glycerophospholipids (Tables 1 and 2), consistent with previous findings [2– 4,23] that plasmalogens were major components of the GPE fraction found in brain. The mass spectrometric investigation of the molecular species additionally revealed that diacyl phospholipids were comprised primarily of molecular species with 18:0 carbon atoms at the sn-1 position, whereas the plasmenyl GPE was comprised primarily of species with 18:0 and 18:1 substituents the sn-1 position although the molecular species with 16:0 substituent at sn-1 were still relatively abundant. The plasmalogen subclass was particularly enriched with polyunsaturated fatty acids, primarily arachidonic acid, when compared to the arachidonate content in diacyl molecular species. Previous detailed analysis of molecular species of GPE within various tissues including the heart [2], pancreatic islet cells [24], as well as red blood cells [25] have also shown that arachidonic acid is highly enriched in the plasmenyl GPE molecular species in many cell types. The report [12–15] that plasmalogen molecular species were uniquely susceptible to free radical oxidation suggested that such compounds may serve a protective role within cellular membranes. Results of the present study confirmed these previous findings that there is an involvement of plasmalogens in the initial oxidation reactions of cellular membranes, which consist of diacyl and plasmenyl phospholipid mixtures. A pronounced

loss of specific plasmalogen molecular species were observed following exposure of bovine brain GPE phospholipids to oxidative conditions compared to the loss of diacyl phospholipids [15]. When bovine brain GPE was treated with Cu21/H2O2 for various times (data not shown), major changes were observed in the phospholipid components compared to that of the starting molecular species. In addition, the results of the oxidation reactions of GPE plasmalogen resulted in the formation of sn-1 lyso GPE. Lyso phospholipids have been reported previously to accumulate in the ischemic myocardium [26,27] and the formation of these compounds were attributed to the activation of phospholipase A1 and/or phospholipase A2. The procedures used in these previous studies could not differentiate between a sn-1 or sn-2 lyso phospholipids. In the present study, a detailed molecular species analysis of the lyso products derived from oxidation of bovine brain GPE was only consistent with sn-1 lyso-GPE derived specifically from plasmenyl GPE precursors. The oxidation of plasmalogen phospholipids and the identification of the lyso phospholipid products is consistent with the results found in the present study with an oxidative degradation origin for sn-1 lyso glycerophospholipids based on a reactivity of the vinyl ether double bond present at the sn-1 position. However, all plasmalogen molecular species were not equally reactive to oxidative degradation into sn-1 lyso GPE. Intact plasmalogens remained following oxidation of bovine brain GPE, but these species contained only monounsaturated fatty acyl groups esterified to the sn-2 position. Additionally, the sn-1 lyso phospholipids observed as products of oxidation also contained only monounsaturated substituents at the sn-2 position. Taken together, these

Plasmenyl GPE lipids

Fig. 6. (A) Electrospray ionization mass spectrometric analysis (negative ions) of the lyso-GPE products obtained following oxidation of bovine brain GPE. The identity of the remaining sn-2 fatty acyl group indicated in the figure was obtained following CID of each of the observed negative ions. (B) Electrospray mass spectrometric analysis (negative ions) of the sn-1 lyso-GPE products following HCl treatment of unoxidized bovine brain GPE revealing the plasmalogen molecular species and fatty acyl distribution.

results suggest that polyunsaturated fatty acids esterified to the sn-2 position of plasmalogen glycerophospholipids may be uniquely susceptible to oxidative degradation and that participation of the polyunsaturated fatty acyl group in the oxidative process is an important feature of these degradation reactions. Some oxidation of plasmalo-

Fig. 7. Electrospray ionization mass spectrometry (negative ions) in the high mass region indicative of intact brain GPE molecular species that remained following oxidation. The acyl groups were assigned using CID mass spectrometry of each indicated [M-H]-.

283

Fig. 8. (A) Relative abundance of all lyso-GPE molecular species containing indicated sn-2 fatty acyl substituent following HCl treatment of brain GPE phospholipid. (B) Relative abundance of all lysoGPE molecular species containing indicated sn-2 fatty acyl substituent following free radical oxidation (see Materials and Methods).

gen molecular species that contain monounsaturated fatty acyl groups at the sn-2 position were observed, yet these products were considerably more stable and could be isolated as intact compounds. Following oxidation of bovine brain glycerophosphoethanolamine, the abundant plasmenyl GPE species, which contain arachidonic acid could no longer be found nor a corresponding sn-1 lyso-GPE containing sn-2 arachidonate (Fig. 8B). The ultimate fate of the arachidonic acid as well as these arachidonate-containing GPE molecular species is currently under investigation. In summary, the molecular species of bovine brain GPE as determined by electrospray ionization mass spectrometry were found to be a mixture of diacyl and plasmenyl phospholipids, the latter containing a relatively high abundance of esterified polyunsaturated fatty acid at the sn-2 position. Following oxidation, the diacyl phospholipids were considerably more stable than the plasmalogen molecular species and the composition of the remaining intact glycerophospholipids was substantially altered. These observations suggest that specific

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plasmenyl GPE species undergo oxidative decomposition in a more facile manner than do the diacyl analogs. In concordance with this observation, was the appearance of new phospholipid components comprised primarily of sn-1 lyso GPE molecular species derived from the plasmenyl phospholipids. None of these major products contained polyunsaturated fatty acids esterified to the phospholipid backbone. Plasmalogens exhibit antioxidant properties in that their polyunsaturated species are more susceptible to lipid peroxidation than diacyl components or saturated plasmenyl species present in complex mixtures. The participation of a polyunsaturated group at the sn-2 position appears to be an important mechanism responsible for the antioxidant character of plasmalogens. Acknowledgement — This work was supported in part by a grant from the National Institutes of Health (HL34303).

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GPE— glycerophosphoethanolamine lipids RP-HPLC/MS—reverse phase high pressure liquid chromatography on line to an electrospray ionization mass spectrometer CID— collision induced decomposition of mass selected ions in a tandem mass spectrometer [M-H]-—molecular anion (negative ion) of phospholipid corresponding to molecular weight minus a proton [MH]1—molecular cation (positive ion) of phospholipid corresponding to molecular weight plus an additional proton