Free-Radical-Induced Oxidation of Arachidonoyl Plasmalogen

Chem. Res. Toxicol. 2001, 14, 463

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Invited Review Free-Radical-Induced Oxidation of Arachidonoyl Plasmalogen Phospholipids: Antioxidant Mechanism and Precursor Pathway for Bioactive Eicosanoids Robert C. Murphy* Department of Pediatrics, Division of Cell Biology, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, Colorado 80206 Received December 14, 2000

Introduction Free-radical chemistry plays an important role in chemical toxicology since various agents and their metabolites generate radical species which initiate complex chemical reactions within the living cell. Furthermore, normal biochemical processes can lead to the formation of extracellular reactive oxygen species such as hydrogen peroxide or O2-. When these forms of oxygen are present along with trace quantities of transition metals such as Fe(II) and Cu(II), formation of the highly reactive oxygen species, hydroxyl radical, can result (1). The phospholipid bilayer is the defining limit of the cell and cellular organelles and, as such, protects the contents from reactive free-radical entities by being one of the important targets of free-radical chemical events (Figure 1). Nevertheless, radical reactions do propagate through the complex mixture of lipids that make up the membrane bilayer leading to products of lipid peroxidation. Freeradical production can also be initiated within the cell and there are numerous antioxidant entities and antioxidant mechanisms that protect intracellular proteins and membranes from free-radical reactions. Classically, radical reactions begin with an initiation process to form a radical species (a molecular fragment with a single unpaired electron), such as the reaction of hydrogen peroxide with transition metals (Fenton chemistry) to form hydroxyl radical (2). Reactive radical entities can interact with neutral molecules, and the products of these reactions also yield radicals. This is the propagation step of radical reactions which often proceed through tens to thousands of molecular intermediates. Radicals are eliminated when termination reactions occur in which two radical entities react together or alternatively when the process of propagation results in the reduction in the product radical energy to such an extent that it cannot further react (e.g., abstract a hydrogen atom) with another molecule. In fact, the driving force for radical reactions is the difference between the free energy of bond formation and bond dissociation, typically hydrogen atom abstraction. While it is not easy to obtain free energies for such complex molecules, one estimate would be to use the bond dissociation enthalpy and ignore entropy changes as a result of these reactions (Table 1). * To whom correspondence should be addressed. Phone: (303) 3981849. Fax: (303) 398-1694. E-mail: [email protected].

Figure 1. A model of the membrane lipid bilayer showing the polar region composed of the phosphoester polar headgroups (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine) and the hydrophobic region with structured interaction of the hydrocarbon chains of the phospholipid radyl substituents. The lipid bilayer serves as an initial barrier to a reactive chemical radical such as hydroxyl radical.

Thus, the magnitude (and sign) of the difference between the bond formation free energy (-∆GBF) and the enthalpy required for bond dissociation (hydrogen atom abstraction, ∆HBDE) for target molecules interacting could be a first approximation as to whether a reaction will proceed. Radical reactions within the cell can be effectively terminated through the formation of free radicals with such low bond formation energies that they do not react with other entities and thus have extremely long halflives, facilitating eventual termination reactions with another free radical. A detailed step by step understanding of the radical reactions taking place during lipid peroxidation is of fundamental interest. Yet, there can be other events taking place such as the direct or indirect formation of novel lipid products which can exert profound biological effects through G-protein-linked receptors that, when occupied, activate signal transduction processes and facilitate a cellular response to tissue oxidative stress. Thus, secondary lipid signaling molecules can play a critical role in the more global responses of tissues to freeradical chemistry. A consideration of free-radical chemistry would not be complete without an understanding of the mechanism of formation of biologically active

10.1021/tx000250t CCC: $20.00 © 2001 American Chemical Society Published on Web 05/01/2001

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Chem. Res. Toxicol., Vol. 14, No. 5, 2001 Table 1. Radical Strengths of Oxygen Radicals and Bond Dissociation Energies for Hydrogen Atom Abstractiona

a

oxygen radical

(-∆GBF)

hydrogen atom abstraction

(∆HBDE)

hydroxyl radical HO‚ alkoxyl radical R-O‚ hydroperoxy radical R-OO‚ molecular oxygen‚O2‚

111 97 82 51

alkyl R-CH2-H allylic R-CHdCH-CH2-H Bis(allylic) (R-CHdCH2)C-H allylicb R-O-CHdCH-CHR1-Hc

103 ( 2 88 78 ( 3 81 ( 8

Aqueous solutions (adapted from ref 1). b Allylic to a vinyl ether. c Calculated from CH3O-CHdCH-CH3 according to ref 50.

Scheme 1

molecules during the process of free-radical propagation within a cell. Free-radical reactions taking place in the lipid bilayer are not always possible to predict because of the complex mixture of reactants present within the membrane, the population of radical species, and the diversity of possible reaction pathways. Within the bilayer, there exist regions of high polarity (at the polar headgroup of the phospholipids) as well as areas of high hydrophobicity where alkyl chains interact (Figure 1). Phospholipids are very ordered species within the lipid bilayer, keeping these regions rather separate while maintaining fluidity of molecular movement within the plane of the bilayer. Furthermore, deep within the hydrophobic region of the bilayer, polyunsaturated fatty acyl substituents of glycerophospholipids are present, which have reasonably low bond dissociation energies for the abstraction of specific bisallylic hydrogen atoms (Table 1). Common fatty acyl groups having such bisallylic methylene groups are linoleate (one bisallylic group), arachidonate (three bisallylic groups), and docosahexaenoate (five bisallylic groups). Lipid peroxidation often involves propagation of radical intermediates and the reaction of dissolved oxygen (a triplet diradical itself) in the lipophilic region of the lipid bilayer. In addition to polyunsaturated fatty acyl substituents, a subclass of phospholipids termed plasmenyl or plasmalogen phospholipids have a methylene group with low hydrogen atom bond dissociation energy adjacent to a vinyl ether substituent (Table 1) and are additional targets for free-radical chemistry. Plasmalogens are a unique class of choline and ethanolamine glycerophospholipids in that they contain a vinyl ether moiety (Scheme 1) rather than an ester group at the designated first carbon atom of the glycerol backbone (sn-1). These ether phospholipids, properly termed plasmenyl glycerophospholipids, are quite abundant in certain cells and tissues including the red blood

cell (3), cardiac tissue (4), nervous tissue (5), spermatozoa (6), as well as inflammatory cells (7). Plasmenyl phospholipids are also found to be reservoirs of polyunsaturated fatty acids such as arachidonic acid and docosahexaenoic acid. However, an understanding of the biochemical role played by plasmalogens in vivo that warrants the additional complexity in biosynthesis of these molecules has been wanting. Plasmalogens have been considered to play an important role in diseases such as cancer (8), atherosclerosis (9), and aging (10) facilitating ion channels in the cardiac sarcolemma (11), and as intermediates driving lipid mediator biosynthesis (12). Yet, none of these specific roles has been broadly accepted to explain why these unique phospholipids are found distributed in specific cells and tissues. In this article, the proposal that plasmalogens can play an important role in limiting freeradical propagation in the lipid membranes of cells will be examined. The initial suggestion that plasmalogens were antioxidants (13, 14) was attributed to the unique chemistry of the vinyl ether substituent, and this concept will be expanded to include involvement of the sn-2 polyunsaturated radyl group (arachidonic or docosahexaenoic acid) and the ordered structure of the bilayer that confines the hydroperoxy radical intermediates formed during lipid peroxidation close to the polar region of the phospholipid bilayer adjacent to the glycerol backbone. As a further consequence, oxygenation of arachidonate at carbon-5 is facilitated with concomitant formation of biologically active eicosanoids that may mediate biological responses within nearby cells.

Antioxidants Plasmalogens In 1988, Raetz and co-workers (13, 14) described a series of experiments with mutant Chinese hamster ovary cells (CHO)1 deficient in plasmenyl phospholipid biosynthesis and their response to challenge with reactive oxygen species. CHO cells had been mutagenized with ethyl methane sulfonate and screened for deficiency in peroxisomal dihydroxyacetone phosphate (DHAP)-acyl transferase (15). When these cells were incubated with 12-(1′-pyrene)dodecanoic acid (P12) and exposed to UV irradiation (> 300 nm), mutant cells were rapidly killed relative to wild-type CHO cells. Furthermore, when 1-Ohexadecyl-sn-glycerol was added to the incubation medium (0-20 µM), there was a dose-dependent accumulation of plasmenyl glycerophosphoethanolamine lipids 1Abbreviations: 18:1, oleic acid (9-octadecenoic acid); 20:1, 11eicosenoic acid; 22:1, 13-docosenoic acid; 20:4, arachidonic acid (5,8,11,14-eicosatetraenoic acid); 22:4, 7,10,13,16-docosatetraenoic acid; 22:6, 4,7,10,13,16,19-docosahexaenoic acid; AAPH, 2,2′-azo-bis(2-amidinopropane hydrochloride); CHO, Chinese hamster ovary cells; DHAP, dihydroxyacetone phosphate; diHETE, dihydroxyeicosatetraenoic acid; GPEtn, glycerophosphoethanolamine lipids; GPCho, glycerophosphocholine lipids; HpETE, hydroperoxyeicosatetraenoic acid; HETE, hydroxyeicosatetraenoic acid; LC/MS/MS, combined liquid chromatography tandem mass spectrometry; LDL, low-density lipoproteins; 5-oxoETE, 5-oxo-6,8,11,14-eicosatetraenoic acid; P12, 12-(1′pyrene)dodecanoic acid.

Invited Review

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Figure 2. Plasmalogen reaction proposed by Morand et al. (14) to explain the antioxidant properties of ethanolamine plasmalogens. The identity of the initiating radical X‚ and that of YH were not established by these investigators. The action of a purported peroxidase on the putative allylic hydroperoxide would result in the formation of the unsaturated aldehyde and lyso-phosphatidylethanolamine observed by these investigators.

(GPEtn) phospholipids as well as an increased resistance to 12-(1′-pyrene)dodecanoic acid and subsequent UV exposure. Analysis of products obtained from wild-type and mutant cells after P12/UV treatment led to the identification of sn-1 lyso-GPEtn, formic acid, and pentadecenal from wild-type CHO cells, but not from mutant cells deficient in plasmenyl phospholipid biosynthesis (14). There was no structural characterization of products derived from the sn-2 fatty acyl substituent of the plasmenyl phospholipid. While the radical oxygen species involved in this photosensitized-mediated toxicity in CHO cells were not unambiguously established, the authors suggested that the facile decomposition of plasmalogens was the result of formation of a hydroperoxy hemiacetal intermediate based on the products isolated from the normal CHO cells (Figure 2). That plasmenyl phospholipids might protect the membranes of cells from reactive oxygen species has emerged as an attractive hypothesis (14). Additional investigations have further supported that plasmalogen phospholipids play a role in reducing membrane free-radical chemistry. Engelmann and co-workers (9) carried out experiments with in vitro oxidation of lowdensity lipoprotein (LDL) by 2,2′-azo-bis(2-amidinopropane hydrochloride) (AAPH), which decomposes in aqueous solutions to yield carbon centered radicals, and Cu(II) and found that both R-tocopherol (vitamin E) as well as plasmalogen phospholipids were decreased in a parallel manner. When LDLs were supplemented with plasmenyl phosphatidylcholine, a lag phase for the oxidation of polyunsaturated acyl groups (formation of conjugated double bonds) was increased. Zommara (16) investigated the oxidation of synthetic liposomes supplemented with plasmenyl ethanolamine phospholipids and measured the appearance of thiobarbituric acid reactive substances as a result of both Cu(II)- and AAPH-induced free-radical oxidation. Only in the case of Cu(II)-induced oxidation were plasmenyl phospholipids inhibitory on thiobarbituric acid reactive substance formation. AAPH-mediated lipid peroxidation was not found to be dependent upon the presence of plasmenyl phospholipids, which led these authors to suggest that the plasmenyl phospholipids

bound to the transition metals by way of the vinyl ether substituent at sn-1 as a mechanism for the antioxidant properties of plasmenyl phospholipids. Reiss and co-workers (17) used NMR spectroscopy and the unique chemical shifts of the vinyl ether methine proton and methine protons present in polyunsaturated fatty acyl groups to measure extent of oxidation of these phospholipid structural units. These investigators found a time-dependent loss of the plasmalogen phospholipids vinyl ether methine proton signal relative to the methine signals from the polyunsaturated fatty acyl groups with a delay of up to 30 min before any loss of signal was observed for the polyunsaturated acyl groups. The data were consistent with plasmenyl phospholipids decreasing the oxidation of polyunsaturated diester phospholipids, leading the authors to conclude that the interaction of peroxy radicals primarily occurred with the vinyl ether double bond. Furthermore, the oxidation of the vinyl ether substituent did not appear to promote decomposition of the polyunsaturated fatty acyl substituents. However, but direct measurement of any modified fatty acyl groups was not attempted. More recently, Sindelar (18) studied the oxidation of phospholipids present in liposomes derived from phospholipids isolated from brain with and without plasmalogens present. The plasmenyl phospholipids were removed from the total brain phospholipid extracts using the unique chemical reactivity of this phospholipid class to mineral acids followed by removal of the resulting aldehydes by normal-phase chromatography. The treatment of total brain liposomes with Fe(III)-ADP and ascorbate resulted in a rapid formation of thiobarbituric acid reactive substances and a rapid decrease in the content of polyunsaturated fatty acid such as docosahexanoic acid and arachidonic acid when plasmalogens were not present. However, when plasmalogens were present in the treated total brain liposomes, there was a substantial decrease in the total production of thiobarbituric acid reactive substances and decrease in the loss of polyunsaturated fatty acids. The authors interpreted their results to suggest that plasmenyl phospholipids inhibited the peroxidation of polyunsaturated fatty acids

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Chem. Res. Toxicol., Vol. 14, No. 5, 2001

present in mixtures of phospholipid liposomes and that the vinyl ether bond was consumed in this process (18). They also carried out studies using AAPH as a freeradical initiator and found similar results which would not be consistent with a major protective effect of plasmalogens due to chelation of transition metals. Overall, the results were consistent with the suggestion that plasmenyl phospholipids interfered with the propagation rather than the initiation of lipid peroxidation. It was suggested that either the vinyl ether radical was somewhat stable and reacted slowly with oxygen or the hemiacetal hydroperoxy radical had a low radical strength (Table 1). No further evidence was available to support this idea of a lower reactivity of the hydroperoxy radical or carbon centered radical derived from the vinyl ether substituent. There has been at least one report to suggest that plasmenyl phospholipids are not antioxidant phospholipids (19). In a study of human skin fibroblasts treated with menadione used to generate reactive oxygen species, there was no change in decomposition of plasmenyl phospholipids in human skin fibroblasts grown from control subjects or individuals with Zellweger’s disease or a deficiency in DHAP-acyl transferase. Plasmenyl phospholipid content was measured as acid-released longchain aldehydes in relationship to the esterified stearic or palmitic acid; however, there was no assessment of the level of polyunsaturated fatty acids such as arachidonic acid or docosahexaenoic acid in these human fibroblasts. There was also no polyunsaturated fatty acyl levels reported in these cell culture studies. Plasmenyl phospholipids have been found to be components within human serum lipoproteins, and there have been reports linking the decrease in plasmalogen phospholipids to their role as antioxidants entities within lipoproteins such as LDLs (9). Detailed studies have been carried out concerning the products obtained from the free-radical decomposition of human LDLs and the formation of R-hydroxy aldehydes. Mass spectrometry was used to structurally characterize the R-hydroxy products, and a relationship was found between the quantity of R-hydroxy aldehydes and the extent of oxidation of LDL (20). Insight into the mechanisms of formation of these R-hydroxy aldehydes has also been obtained through careful studies of the products generated following oxidation of the vinyl ether substituent on plasmenyl phospholipids (21). Additional evidence suggesting that the plasmenyl phospholipids may be primary targets of lipid peroxidation in human LDLs were obtained using fluorescent tagged plasmenyl phospholipids (22) as well as the direct measurement of a decrease in fatty aldehyde dimethylacetals derived from the hydrolysis of plasmenyl phospholipids after oxidative stress (23).

Plasmalogen Biosynthesis The biosynthesis of plasmenyl phospholipids (Figure 3) is quite complex and entirely separate from the phospholipid biosynthetic pathways of phosphatidyl (1,2diacyl) glycerophospholipids taking place in the endoplasmic reticulum (24). The peroxisome is a central and necessary organelle for ether phospholipid generation (25). Human subjects deficient in peroxisomes, such as those with Zellweger cerebrohepatorenal syndrome, lack liver and kidney peroxisomes and are highly deficient in both plasmenyl and plasmanyl phospholipids.

Figure 3. Biochemical pathway proceeding from dihydroxyacetone phosphate (DHAP) and leading to the formation of plamenyl glycerophospholipids. The first several reactions resulting in formation of 1-O-alkyl-glycerophosphate take place exclusively in the peroxisome. Subsequent reactions occur in the mitochondria and endoplasmic reticulum (adapted largely from ref 24).

Two biochemical substrates are required to initiate ether lipid biosynthesis; these are a long chain alcohol (e.g., hexadecanol) and DHAP (dihydroxyacetone phosphate). On the cytosolic-facing peroxisomal membrane, a unique acyl-CoA reductase catalyzes a two-step reduction of fatty acyl-CoA esters to the corresponding long chain alcohol (Figure 3). On the lumenal side of the peroxisome, DHAP is acylated at the sn-1 position of the eventual glycerol backbone by the enzyme dihydroxyacetone phosphate acyl transferase. The lumenal facing side of the peroxisome is also the location of alkyl dihydroxyacetone phosphate synthase (alkyl DHAP synthase) that catalyzes formation of the first ether phospholipid intermediate, alkyl DHAP, by reacting the long chain alcohol synthesized in the peroxisomal membrane with the acyl dihydroxyacetone phosphate. This rather interesting and mechanistically complex reaction is driven in part by the elimination of the long chain acyl substituent as a free carboxylate anion. The cytosolic side of the peroxisome also contains alkyl dihydroxyacetone phosphate dehydrogenase (NADPH: alkyl DHAP oxidoreductase) which catalyzes formation of 1-O-alkyl-2lyso-phosphatidic acid which is passed on to the endoplasmic reticulum or mitochondria for further ether phospholipid biosynthesis. The mitochondria and endoplasmic reticulum are thought to be sites of subsequent processing of 1-O-alkyl-2-lyso-phosphatidic acid. Again,

Invited Review

several steps are involved. First, the sn-2 position is acylated by a fatty acyl-CoA and alkyl-GP acyl transferase. The sn-3 phosphate is then removed by alkylacylGPCho phosphohydrolase and the resultant ether diglyceride transformed to an ether glycerophosphoethanolamine (GPEtn) or glycerophosphocholine (GPCho) lipid by the corresponding CDP-ethanolamine or choline phosphotransferase. The final step of plasmenyl phospholipid biosynthesis is carried out by a microsomal cytochrome b5-dependent electron transport system (∆1-alkyl desaturase) driven by molecular oxygen and NADH. Evidence suggests that both choline and ethanolamine plasmenyl phospholipids are formed by the initial oxidation of only 1-O-alkyl-2acyl-GPEtn and that plasmenyl choline species are formed from subsequent transformations of the plasmenyl GPEtn. In toto, the synthesis of plasmenyl GPEtn phospholipids is quite complex and dependent initially on peroxisomal events followed by an involvement of mitochondrial and endoplasmic reticulum processing to yield the final alk-1′-enyl-GPEtn and -GPCho lipids (24, 25).

Arachidonate Remodeling Experiments carried over the past three decades have clearly established that the acyl substituent at the sn-2 position of plasmenyl GPEtn is not static, but dramatically remodeled during normal cellular activity. Since 1973, arachidonic acid has been known to be an abundant fatty acyl substituent accumulating within the alk-1-enyl subclass of GPEtn (26). For example, in the human polymorphonuclear leukocyte, 70% of total arachidonate is esterified to glycerophosphoethanolamine lipids of which 70-80% are plasmenyl at sn-1 (7, 27, 28). The dynamics of arachidonate remodeling was demonstrated in experiments carried out by incubation of inflammatory cells with low doses of arachidonic acid. Under these circumstances, there was a rapid incorporation of arachidonic acid into 1,2-diacyl phospholipids. Over the next several hours the arachidonate was remodeled into 1-Oalkyl-2-arachidonoyl-GPCho lipids then into 1-alk-1-enyl2-arachidonyl-GPEtn. When these same cells were stimulated during the initial incubation with arachidonic acid, a rapid remodeling into the plasmalogen GPEtn was observed in approximately 4 min (12). Additional experiments revealed that considerable mass of arachidonic acid that appears eventually as lipid mediators, such as leukotrienes following stimulation of mast cells, was derived from plasmenyl GPEtn (12). An interesting aspect of the remodeling of arachidonic acid into alkyl ether GPEtn and plasmenyl GPEtn has been the central involvement of a CoA-independent transacylase and an acyl enzyme intermediate in remodeling arachidonic acid from 1,2-diacyl and 1-O-alkyl phospholipids into the plasmenyl GPEtn during platelet activating factor biosynthesis (29, 30). As a consequence of the extensive enzymatic machinery involved in remodeling arachidonic acid, in many cells this polyunsaturated fatty acid accumulates into phospholipid molecular species with a vinyl ether substituent at sn-1. Docosahexaenoic acid (22:6, n-3) has also been found to be highly abundant in plasmalogen GPEtn molecular species. Interestingly, this phospholipid molecular species does not participate in lipid mediator biosynthesis (PAF or eicosanoids) since it has been found to be a poor


substrate for cPLA2 which forms the plasmenyl lysoGPEtn species thought to drive the remodeling of arachidonic acid required for lipid mediators as well as platelet activating factor biosynthesis (31). However, CoAindependent transacylase readily transfers docosahexaenoic acid into plasmenyl GPEtn (30) leading to the accumulation of DHA into plasmenyl GPEtn.

Mass Spectrometric Analysis of Phospholipids Up until the early 1980s, it was quite difficult to determine the precise structure of phospholipids as individual and intact species because of the requirement of volatility for the analysis of molecules by mass spectrometry and electron ionization. However, with the development of fast-atom bombardment ionization mass spectrometry (32), it became possible to directly generate gas-phase ions of phospholipids as abundant [M + H]+ and [M - H]- pseudo molecular ions. Immediately, the complex mixture of phospholipids present within cellular membranes could be directly observed and studied. Furthermore, the subsequent development of tandem mass spectrometry applied to this ionization technique permitted a direct look of the fatty acyl substituents present within phospholipids of a specific molecular weight (33, 34). Unambiguous assessment of fatty acyl esterification at the carbon atoms of glycerol as well as detailed information concerning the nature and structure of each molecular species of all glycerophospholipid species became possible (35). It was also possible to analyze plasmenyl phospholipids owing to their unique chemical reactivity and susceptibility to acid hydrolysis with mass spectrometry and subtractive analysis (36). The development of electrospray ionization mass spectrometry further advanced phospholipid analysis through direct coupling of powerful chromatographic techniques such as HPLC to the tandem mass spectrometer. The chemical rules developed studying fast-atom bombardment ionization and tandem mass spectrometry of phospholipids were also valid for electrospray ionization and tandem mass spectrometry (37). Thus, it was possible to carry out class separation and structural analysis in the same experiment using normal phase LC/MS/MS. In the past, the analysis of fatty acid composition in phospholipids and the enrichment of arachidonate in plasmalogens specifically required decomposition of the phospholipid to free fatty acids (26), but now molecular species analysis was possible using reversed-phase separation directly coupled as the LC/MS/MS experiment. These techniques in mass spectrometry and chromatography were largely responsible for the recognition of the abovementioned accumulation of arachidonic acid in plasmalogen molecular species as well as the facility of arachidonate remodeling in and through different classes and subclasses of phospholipids (27, 30, 38). It was also possible for the first time to structurally characterize modified phospholipid molecular species, in particular those oxidized in the radyl substituents because of the ability to study rather chemically reactive substituents.

Analysis of Intact Oxidized Plasmalogen Phospholipids A different approach to the study of events taking place within naturally occurring phospholipid mixtures derived

468


Table 2. Relative Abundance of Red Blood Cell Membrane Glycerophosphoethanolamine (GPEtn) and Glycerophosphocholine (GPCho) Plasmenyl and 1,2-Diacyl Phospholipid Molecular Species with Arachidonate or Oxidized Arachidonate at the sn-2 Position before and after Oxidation with tert-Butylhydroperoxidea relative abundance of the sn-2 arachidonate or oxidized arachidonate acyl substituentb GPEtn

plasmalogen 1,2-diacyl GPCho plasmalogen 1,2-diacyl

arachidonate

HETE

HpETE

1.15 1 0.20 1

0.33 1 0c 1

0.33 1 0 1

a Molecular species measured by precursor ion scanning for arachidonate (m/z 303), hydroxyeico-satetraenoic acid (HETE, m/z 319) and hydroperoxyeicosatetraenoic acid (HpETE, m/z 317) in LC/MS/MS experiment (43). b Abundance normalized to 1,2-diacyl arachidonate in each phospholipid class. c Ion transition not detected above background.

from cellular plasma membranes exposed to reactive oxygen species involved the powerful capabilities of tandem mass spectrometry to analyze intact phospholipid oxidation products. Exposure of red blood cell ghosts to tert-butylhydroperoxide has been used as a model of lipid peroxidation in a number of studies (39); furthermore, the red blood cell is a rich source of both plasmenyl phospholipids as well as arachidonic acid which constitutes approximately 15-20% of the entire esterified fatty acid pool (40). Exposure of red blood cell ghosts to tertbutylhydroperoxide for 1.5 h at 37 °C was found to result in abundant formation of oxidized arachidonate as detected following saponification and reverse phase LC/MS/ MS analysis (41, 42). Interestingly, an unexpected high abundance of 5- and 9-hydroperoxyeicosatetraenoic acid (HpETE) were observed as products rather than an even distribution of the six regioisomers of HETE typically seen with direct peroxidation of free arachidonic acid (42). An even more interesting observation was made when the intact phospholipids were directly analyzed. After red blood cell ghost phospholipid classes were separated by normal-phase HPLC followed by tandem mass spectrometric analysis (43), the distribution of arachidonatecontaining molecular species in the 1,2-diacyl and plasmalogen subclasses was determined (Table 2). As expected, a high abundance of arachidonate containing plasmenyl GPEtn phospholipids was observed, equal in abundance to esterified arachidonate in 1,2-diacyl-GPEtn molecular species. However, when analysis of the distribution of molecular species containing oxidized arachidonic acid (either HETE or HpETE using precursor ion scans for m/z 319 and 317, respectively) was carried out with the intact phospholipids after treatment of these cells with tert-butylhydroperoxide, a different distribution was observed. Although the most abundant individual arachidonate containing GPEtn molecular species were plasmenyl GPEtn, the most abundant oxidized arachidonate containing GPEtn molecular species were now 1,2-diacylGPEtn (Table 2). These results were interpreted as representing unique susceptibility of arachidonoyl plasmalogen or plasmenyl phospholipids to oxidation followed by subsequent decomposition, rather than a higher reactivity of 1,2-diacyl-GPEtn to reactive oxygen species generated by decomposition of tert-butylhydroperoxide in the presence of metal ions such as ferrous iron to yield an oxygen-centered alkoxyl radical. Interestingly, several

Table 3. Plasmalogen-Derived Lyso-GPEtn Molecular Species before and after Oxidation Using Cu(II)/H2O2a relative abundance sn-2c

acid hydrolysis before oxidationb

product lyso-GPEtn species after oxidationc

18:0 18:1 20:1 20:4 22:1 22:4 22:6

0.08 1d 0.25 0.76 0.14 0.40 0.60

0.12 1 1.65 0e 0.97 0 0

a Adapted from Khaselev and Murphy (44). b Analysis of lysoGPEtn molecular species obtained after acid hydrolysis of brain GPEtn phospholipids by tandem mass spectrometry. c Radyl substituent at the sn-2 position of the sn-1 lyso-GPEtn species formed after oxidation. d The abundance of each molecular species normalized to the signal for this alk-1-enyl-2-octadecenoyl-GPEtn (18:1 at sn-2) plasmenyl phospholipid. e Ion transition not detected above background.

products of arachidonic acid free-radical oxidation were found to be produced in this model system and several were generated in sufficient quantity to assess biological activity, including 5-oxo-ETE and 5-HpETE (41). The phospholipid mixture isolated from brain was also employed as a model system to study the free-radical oxidation chemistry using Cu(II) and H2O2 (43) to generate a hydroxyl radical by the Fenton reaction (1, 2). The distribution of plasmenyl phospholipids present in brain is somewhat different from that observed for red blood cell membranes with the former having a high abundance of plasmenyl species with 18:1, 20:1, and 22:1 fatty acyl groups at the sn-2 position, which is comparable in abundance to those molecular species with 20:4, 22:4, and 22:6 fatty acyl groups at sn-2 (Table 3). Following oxidation of this mixture of brain phospholipids, sn-1 lyso phospholipids were generated as previously reported for the oxidation of plasmenyl phospholipids. However, the only molecular species observed as lyso phospholipids had monounsaturated fatty acyl group esterified at sn-2 (44). However, none of the sn-1 lyso products retained a polyunsaturated fatty acyl substituent at sn-2 (such as arachidonate or docosahexaenoate) despite the almost equal abundance of these plasmenyl species in the starting lipid mixture (Table 3). These experiments clearly suggested an involvement of the polyunsaturated sn-2 group in the oxidation process once the sn-1 hydroperoxy hemiacetal radical was formed. To more precisely probe the chemical events taking place during free-radical oxidation of plasmenyl phospholipids containing polyunsaturated fatty acyl substituents such as arachidonate, it was necessary to synthesize this molecular species with trace tritium atoms to facilitate isolation and purification of products derived from arachidonate (45). When pure plasmenyl, plasmanyl, and 1-acyl-GPCho phospholipid molecular species (each containing arachidonic acid esterified at the sn-2 position) were oxidized using Cu(II)/H2O2 to generate hydroxyl radical (1, 2), plasmenyl arachidonyl GPCho was destroyed at a considerably more rapid rate than either of the sn-1 variant phospholipid molecular species (45). This was expected based upon the previous reports of the rapid oxidation of plasmenyl phospholipids using other model systems (17, 18, 46, 47). However, completely unexpected was the production of arachidonate metabolites predominantly oxidized to carbon-5. 5-HETE and

Invited Review


Figure 4. Products obtained following free-radical oxidation of 1-hexadec-1′-enyl-2-arachidonoyl-sn-glycero-3-phosphocholine. Products determined by electrospray tandem mass spectrometry as described in ref 47. Abbreviations for the sn-1/sn-2 substituent indicate carbon chain length: degrees of unsaturation: oxygenation state followed by GPCho, the glycerophosphocholine polar headgroup. The substituent at sn-1 is an ester (a), vinyl ether (p), or free hydroxyl (OH) for sn-1 lyso species. The unique sn-2 substituents have a carbonyl (c) or hydroxyeicosatetraenoic acid (HETE). An approximation of the abundance of these products relative to the most abundant (1-lyso-2-arachidonoyl-GPCho) is provided as the integer under each structure. The estimate was based on tandem mass spectrometric analysis and on normalizing the signal for m/z 184 to 100% from 1-lyso-2-arachidonoyl-GPCho and expressing the abundance of each precursor ion forming m/z 184 (phosphocholine ion) on this scale. Since the relative yield of this product ion from each metabolite is unknown, this estimation is not expected to be accurate, but provides an indication of major and minor species.

several 5,12-diHETE were exclusively formed by this free-radical chemistry. Furthermore, several of these 5-oxygenated metabolites of arachidonate were found to possess biological activity in terms of ability to elevate intracellular free calcium ion concentration within the human polymorphonuclear leukocyte. Even when mixed micelles containing the alk-1-enyl-2-arachidonoyl-GPCho were oxidized (45) and other phospholipids such as 1,2diacyl and plasmanyl arachidonoyl GPCho, the predominant oxygenation of the plasmenyl arachidonoyl GPCho phospholipid at the arachidonate carbon-5 was not altered. Detailed analysis of the major phospholipid products derived from the AAPH-mediated free-radical oxidation of alk-1′-enyl-2-arachidonoyl-GPCho by tandem mass spectrometry (47) revealed numerous products (Figure 4A) such as 1-lyso-2-arachidonyl-GPCho, 1-formyl-2arachidonyl-GPCho, and alk-1′-enyl-2-(5′-HpETE)-GPCho, as well as other products corresponding to the oxidation of arachidonate at carbon-5 including chain-shortened products with 5 carbon atoms at the sn-2 position (Figure 4B). Importantly, there were also phospholipids produced which had oxidation reactions taking place at both sn-1 and sn-2 (Figure 4C), but oxidation at sn-2 largely was limited to carbon-5. It is of interest to note that one of the minor sn-1 oxidized products was reported to be an epoxide either product of plasmalogen oxidation consistent with the mechanism suggested by Spiteller and coworkers leading to the formation of R-hydroxy aldehyde

products of plasmalogen oxidation (20, 21, 48).

Proposal A considerable body of data supports the hypothesis that plasmalogens are antioxidant entities and protect cells and cellular membranes from oxidative stress (49). Detailed chemical investigations using NMR spectroscopy clearly suggest that the vinyl ether substituent, unique to plasmenyl phospholipids, is rapidly attacked by radical oxygen species and after their formation, delay subsequent oxidation of polyunsaturated fatty acyl groups. Mass spectrometric analysis has revealed the formation of numerous plasmalogen metabolites derived from the sn-1 position as hydroxy aldehydes and other more complex species (48). Thus, plasmalogen or plasmenyl phospholipids do not appear to inhibit free-radical initiation, but more likely divert the pathway of reactions to the alk-1′-enyl group. Thus, an initial hydroperoxy radical intermediate at the 1′-carbon atom position of plasmenyl phospholipids (Figure 5) likely is a predominate radical product. But how can such a radical intermediate reduce propagation reactions and explain the apparent antioxidant properties of plasmenyl phospholipids? One possible explanation is to consider that this oxygen-centered radical is in quite close proximity to the polar region of the lipid bilayer, somewhat displaced from the hydrophobic bilayer interior where most bisallylic methylene

470


Figure 5. Proposed radical reactions of plasmenyl phospholipids with arachidonate esterified at the sn-2 position responsible for the antioxidant properties of plasmalogens. The initial formation of the plasmalogen hemiacetal hydroperoxy radical would be rapid, but subsequent propagation of the radical reactions would be slowed largely because of the requirement for a bisallylic methylene group to come within reasonable proximity of the hydroperoxy radical for hydrogen abstraction to occur. Favored formation of oxidized products at carbon-5 of arachidonate would result.

Figure 6. Initial hydroperoxy radical formed from a hydrogen atom removed at carbon-7 and formation of the hydroperoxy radical at carbon-5 of an arachidonate-containing phospholipid molecular species in a membrane bilayer would rapidly react with a plasmalogen phospholipid to form hemiacetal hydroperoxy radical after the reaction with molecular oxygen. Formation of this intermediate would cause subsequent slowing of the propagation reaction due to the unavailability of susceptible carbon-hydrogen bonds for subsequent hydrogen abstraction reactions.

groups from the polyunsaturated fatty acyl substituents reside. Thus, there could be a reduced propagation of subsequent radical reactions because of the lack of susceptible C-H bonds capable of being abstracted by the hydroperoxy radical. The alkyl C-H bonds are too strong to be removed by the hemiacetal hydroperoxy radical. Nonetheless, with the high abundance of arachidonic acid esterified at the sn-2 position of plasmenyl phospholipids one bisallylic methylene group at carbon-7 of arachidonic acid could come within a reasonable proximity that would permit radical propagation reaction to proceed (Figure 5). Esterified docosahexaenoate has a bisallylic methylene moiety even closer to the polar region at carbon-6. Propagation eventually does occur with esterified arachidonate and formation of a carbon-7 bisallylic radical that, after reaction with dissolved molecular oxygen, leads to subsequent formation of either

a hydroperoxy radical at carbon-5 or carbon-9 of arachidonyl substituted fatty acyl groups. The most prominent reactions appear to proceed from carbon-5 oxygenation (42). Decomposition reactions of the carbon-5 hydroperoxy radical could undoubtedly take place, including chainshortening reactions prior to eventual termination reactions, likely mediated by other antioxidant species present in cellular membranes such as vitamin E or ascorbate. It is also clear that free-radical events lead to formation of hydroperoxy radicals from polyunsaturated fatty acyl substituents at the sn-2 position without oxidation of the sn-1 vinyl ether group (Figure 6). Likely, such hydroperoxy radicals would favorably propagate the radical reaction to the vinyl ether position of plasmenyl phospholipids because of the bond strength of the 3′-methylene group, again bringing the radical site toward the polar region of the bilayer. In these ways, radical reac-

Invited Review


tions would be slowed by populating radicals in the region of the lipid bilayer close to the hydrophilic polar headgroup. In conclusion, plasmenyl phospholipids appear to be antioxidant species present within cellular membranes of certain cells. The chemical basis of this antioxidant property is not entirely clear, but certainly involves more than the chemical reactions taking place only at the vinyl ether substituent at sn-1 and likely is related to localization of the radical propagation reactions close to the polar region of the bilayer. Structural characterization of the products derived from the free-radical reactions of plasmalogens in lipid bilayers has been critical in expanding our understanding of the antioxidant properties of plasmalogens effect. Polyunsaturated fatty acid such as arachidonic acid and docosahexaenoic acid at the sn-2 position of plasmenyl phospholipids clearly participate in the free-radical propagation reactions because of their high abundance in this subclass of phospholipid and positioning of a susceptible bisallylic methylene group close to the polar region of the phospholipid bilayer. A secondary result of this unusual susceptibility of plasmenyl arachidonoyl phospholipids to free-radical oxidative events is the formation of esterified arachidonate oxidized at carbon-5 in high abundance. Even 5,12dihydroxyeicosatetraenoic acid trienes as esterified species are formed as unique products of free-radical reactions. The biological activity of these later eicosanoids, once liberated as free acids by a phospholipase A2, could mediate and regulate biochemical events in nearby cells because many of these eicosanoids are recognized by specific receptor proteins.

Acknowledgment. The author wishes to thank Drs. Joseph Hankin, Kathleen Harrison, and Keith Clay for critical reading of this manuscript and providing valuable suggestions. This work was supported, in part, by a grant from the National Institutes of Health (HL34303).

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Free-Radical-Induced Oxidation of Arachidonoyl Plasmalogen

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