Identification of Oxidatively Truncated Ethanolamine Phospholipids in

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Chem. Res. Toxicol. 2006, 19, 262-271

Identification of Oxidatively Truncated Ethanolamine Phospholipids in Retina and Their Generation from Polyunsaturated Phosphatidylethanolamines Bogdan G. Gugiu,†,‡ Clementina A. Mesaros,‡ Mingjiang Sun,‡,§ Xiaorong Gu,† John W. Crabb,†,‡,§ and Robert G. Salomon*,‡ Cole Eye Institute and Lerner Research Institute, CleVeland Clinic Foundation, and Department of Chemistry, Case Western ReserVe UniVersity, CleVeland, Ohio 44106 ReceiVed September 8, 2005

Oxidized (ox) phospholipids are receiving growing recognition as important messengers in oxidative stress signaling pathways and as endogenous electrophilic toxins that interfere with protein function through covalent modifications. Phosphatidylcholine lipids predominate in low-density lipoproteins (LDL). Our previous studies of oxLDL identified a family of biologically active oxidatively truncated phosphatidylcholines that are also present in atherosclerotic plaques. In contrast, phosphatidylethanolamine (PE) lipids are extraordinarily abundant in retina. Because photoreceptors contain the most highly unsaturated fatty acids found in vertebrate tissues, these membranes are expected to be especially susceptible to oxidative damage. Here, we report that oxidatively truncated ethanolamine phospholipids (oxPEs) are present in retina. As expected, the most abundant oxPEs, succinyl (2.2 ( 0.8 pmol/retina) and ω-oxobutyryl (1.5 ( 1.0 pmol/retina) esters of 2-lysophosphatidylethanolamine, are derived from the docosahexaenoyl ester, the most abundant polyunsaturated PE in retina. However, a large amount of the ω-oxononanoyl ester (1.3 ( 0.6 pmol/retina), derived from linoleyl-PE, is also present even though linoleate is an order of magnitude less abundant than docosahexenoate in retina. There is a notable trend for the presence in retina of greater amounts, relative to the levels of their precursors, of longer chain homologous aldehydes and alkanedioate monoesters. We considered the possibility that this trend results from differences in the proclivities of various polyunsaturated fatty acyl (PUFA)-PEs to generate these homologous products. Therefore, we examined oxidative cleavage of various PUFA-PEs in small unilamellar vesicles. Alkanedioate monoesters are the major stable end products. Particularly notable is the fact that ω-oxononanoyl-PE levels either do not decline or decline less than those of the analogous aldehydes ω-oxobutyryl-PE or ω-oxovaleryl-PE during autoxidation for 33 h. The resistance of ω-oxononanoylPE, as compared with ω-oxobutyryl-PE and ω-oxovaleryl-PE, to further oxidation may contribute to the greater amount of this oxPE relative to its precursor, linoleyl-PE, in retina. Introduction Previously, malondialdehyde, a hallmark of lipid oxidation, was found in the subretinal fluid from individuals with retinal detachment (1). Lipid oxidation is involved in pathological processes such as age-related macular degeneration (AMD)1 (2), ischemia reperfusion injury associated with heart attack and stroke, Alzheimer’s disease, Batten’s disease (juvenile lipofuscinosis), Parkinson’s disease (3), and amyotrophic lateral sclerosis (4). The involvement of oxidized phospholipids (oxPLs) is suspected in the pathogenesis of diseases such as the antiphospholipid antibody syndrome (5), rheumatoid arthritis (6, 7), inflammatory bowel disease (8), and multiple sclerosis (9, 10). Understanding these processes at the molecular level is an important challenge. Photoreceptor cell membranes in the retina are expected to be highly susceptible to oxidative damage because they contain the most highly unsaturated fatty acids found in vertebrate tissues. Over 50% of the total retinal fatty acids are unsaturated * To whom correspondence should be addressed. Tel: 216-368-2592. Fax: 216-368-3006. E-mail: [email protected]. † Cole Eye Institute, Cleveland Clinic Foundation. ‡ Case Western Reserve University. § Lerner Research Institute, Cleveland Clinic Foundation.

(11). Photoreceptor cells contain a stack of membrane disks that are bathed in oxygen and light (Figure 1). The entire stack of 1 Abbreviations: AA, arachidonic acid; AA-PE, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylethanolamine; AMD, age-related macular degeneration; BHT, butylated hydroxytoluene; DHA, docosahexaenoic acid; DHA-PC, 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphatidylcholine; DHA-PE, 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphatidylethanolamine; EDTA, ethylenediaminotetraacetic acid; EI, electron impact; EP, ethanolamine phospholipid; ESI, electrospray ionization; HDdiA, 4-hydroxydodec-2-enedioic acid; HDdiA-PE, 11-palmitoyl-2-(9-hydroxy-12-carboxydodec-10-enoyl)-sn-glycero-3-phosphatidylethanolamine; HOHA, 4-hydroxy7-oxohept-5-enoic acid; HOOA, 5-hydroxy-8-oxooct-6-enoic acid; HODA, 9-hydroxy-12-oxo-10(E)-dodecenoic acid; HO-PE, 1-palmitoyl-2-hydroxysn-glycero-3-phosphatidyl-ethanolamine; KDdiA-PE, 1-palmitoyl-2-(9-oxo12-carboxydodec-10-enoyl)-sn-glycero-3-phosphatidylethanolamine; KHdiAPE, 1-palmitoyl-2-(7-carboxy-4-oxohept-5-enoyl)-sn-glycero-3-phosphatidylcholine; KOdiA-PE, 1-palmitoyl-2-(8-carboxy-5-oxooct-6-enoyl)-snglycero-3-phosphatidylcholine; LA, linoleic acid; LDL, low-density lipoprotein; Lyso-PE, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphatidylethanolamine; MPO, myeloperoxidase; MS/MS, tandem mass spectrometry; MRM, multiple reaction monitoring; OB-PE, 1-palmitoyl-2-(4-oxobutyroyl)sn-glycero-3-phosphatidylethanolamine; ON-PE, 1-palmitoyl-2-(9-oxononanoyl)-sn-glycero-3-phosphatidyl-ethanolamine; OV-PE, 1-palmitoyl2-(5-oxovaleroyl)-sn-glycero-3-phosphatidylethanolamine; oxLDL, oxidized low-density lipoprotein; oxPE, oxidized phosphatidyl-ethanolamine; oxPC, oxidized phosphatidylcholine; oxPL, oxidized phospholipids; PC, phosphatidylcholine; PE, phosphatidylethanolamine; LA-PE, 1-palmitoyl-2linoleoyl-sn-glycero-3-phosphatidylethanolamine; ROS, rod outer segment; PUFA, polyunsaturated fatty acid; RPE, retinal pigment epithelium; S-PE, 1-palmitoyl-2-(succinoyl)-sn-glycero-3-phosphatidylcholine.

10.1021/tx050247f CCC: $33.50 © 2006 American Chemical Society Published on Web 01/04/2006

OxidatiVely Truncated Ethanolamine Phospholipids in Retina

Chem. Res. Toxicol., Vol. 19, No. 2, 2006 263

burst of retinal ROS disk shedding soon after the onset of extensive light, with the number of large packets of ROS in RPE substantially greater than at any other time of day or night, suggesting that light damage triggered ROS phagocytosis by RPE (12). Phosphatidylethanolamine (PE) lipids account for approximately half of the phospholipids in photoreceptor outer segment membranes. This unusually high PE content is thought to be important for the membrane fusion required to generate new photoreceptor disks and for the shedding of ROS tips (15). In analogy with our previous studies on oxidized choline phospholipids, we predicted that oxidized phosphatidyl-ethanolamine (oxPEs) 2-7 would be generated through autoxidation of docosahexaenoic acid (DHA), arachidonic acid (AA), and linoleic (LA) acid esters of 2-lysophophatidylethanolamine (Scheme 1). We now report the identification of oxPEs in retina. oxPEs from retinal extracts were separated and characterized by LC-tandem mass spectrometry (MS/MS). Their molecular structures were confirmed by derivatizations and comparisons with authentic samples available through our unambiguous total syntheses (16). We also examined the generation of oxPEs through oxidative cleavage of 2-lyso-PE esters of AAs, LAs, and DHAs in unilamellar vesicles and found that the same oxPEs are formed using either UV irradiation, myeloperoxidase (MPO), an enzymatic source of free radicals in vivo, or Cu(II) to initiate oxidation.

Materials and Methods

Figure 1. Rod photoreceptor cell, RPE cell, and choroid.

disks is replaced every 12 days (12, 13). New disks are generated at the nuclear end of the stack, and old disks are shed at the tip of the photoreceptor rod outer segment (ROS) where they are phagocytized by the retinyl-pigmented epithelium (RPE) (14). They are routinely damaged, apparently by free radical-induced oxidative modifications spawned by photogenerated radicals. For example, albino rats reared in cyclic light demonstrate a

1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphatidylethanolamine (Lyso-PE), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphatidylethanolamine (DHA-PE), 1,2-dipalmitoyl-sn-glycero-phosphatidyl3-ethanolamine (DP-DP), 1-palmitoyl-2-arachidonoyl-sn-glycero3-phosphatidylethanolamine (AA-PE), 1-palmitoyl-2-linoleyl-snglycero-3-phosphatidylethanolamine (LA-PE), and 1,2-dimyristoylsn-glycero-phosphatidyl-3-ethanolamine (DM-PE) were obtained from Avanti Polar Lipids (Alabaster, AL), and MPO was purchased from Calbiochem (La Jolla, CA). Dry pyridine and chloroform were supplied by ACROS (Morris Plains, NJ). Extraction of Phospholipids from Retina. Rat retinas were harvested from five normal albino rats. To preclude contamination by blood and to prevent in vitro oxidation, the retinas were rinsed

Scheme 1. Postulated Generation of oxPEs from Polyunsaturated EPs

264 Chem. Res. Toxicol., Vol. 19, No. 2, 2006 with saline antioxidant cocktail [saline PBS, pH 7.4, containing 2 mM ethylenediaminotetraacetic acid (EDTA) and 100 µM butylated hydroxytoluene (BHT)]. The retinas were immediately homogenized manually in a plastic vial using a stainless steel pestle coated with Teflon, and lipid extraction was performed immediately after homogenization using the method of Bligh and Dyer (17). The extract was dried under a stream of nitrogen and stored under argon in an amber vial at -80 °C briefly ( AA > LA, yields of the specific products, e.g., alkanedioate monoesters, might increase from the autoxidations of DHA-PE < AA-PE < LAPE. To test this hypothesis, we examined autoxidations of the PUFA-PEs in vitro. Reaction Profiles for Oxidative Consumption of PUFAPEs. Aerobic oxidation of small unilamellar lipid vesicles (18) containing 50% DHA-PE (AA-PE or LA-PE) and 50% 1,2dipalmitoyl-sn-glycero-phosphatidylcholine (DP-PC) was initiated with Cu2+, or with MPO (19), or with UV light of 350 nm

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Gugiu et al. Table 3. Yields of oxPEs from DHA-PEa product yields (%) initiation

OB-PE

S-PE

KHdiA-PE

MPO max MPO 33 h Cu(II) max Cu(II) 33 h 350 nm max 350 nm 33 h

2.8 ( 0.5 1.1 ( 0.1 1.6 ( 0.1 0.4 ( 0.1 11 ( 1 1.5 ( 0.2

4.0 ( 0.1 4.0 ( 0.1 3.2 ( 0.3 3.0 ( 0.0 17 ( 4 17 ( 4

2.7 ( 0.3 2.1 ( 0.4 1.8 ( 0.7 0.7 ( 0.2 7.5 ( 0.3 1.4 ( 0.3

a Autoxidations of DHA-PE were promoted by various modes of free radical initiation. Yields shown are maximum (shaded) and final after 33 h. See the legend of Figure 4 for details.

Figure 4. Evolution profiles of the oxPEs (OB-PE, S-PE, and KHdiAPE) generated by autoxidation of DHA-PE. Yields were calculated by dividing the amount of each analyte by the amount of starting DHAPE. Data are the average of two sets of independent experiments. MRM chromatograms are given in the appendix. Quantitation of four oxPEs and DHA-PE in the reaction mixtures was achieved by LC-MS/MS in the positive ion mode using the MRM function. An appropriate [M + H]+ and a specific fragment ion were monitored for each analyte. Calibration curves were produced using synthetic authentic samples, for which the accurate amounts were determined by a microphosphorus assay (see Supporting Information). Traces of oxPEs were detected in the commercial DHA-PE. These levels were subtracted from the levels detected in the reaction mixtures. (A) UV-promoted autoxidation, (B) MPO-promoted autoxidation, and (C) Cu(II)-promoted autoxidation.

(19). Aliquots from the reaction mixtures were collected at various times, and oxidation was stopped with catalase and BHT. Lipids were extracted immediately (17), stored at -80 °C, and analyzed by LC-MS/MS within 24 h. Preliminary identification

of products was achieved by comparing retention times with authentic samples of OB-PE, S-PE, KHdiA-PE, OV-PE, G-PE, KOdiA-PE, ON-PE, A-PE, HDdiA-PE, and KDdiA-PE. As expected (Scheme 1), UV irradiation of PE-containing liposomes produced oxPEs. A single peak with a retention time that is identical to the synthetic standard was observed in the MRM chromatograms for OB-PE, S-PE, KHdiA-PE, OV-PE, G-PE, KOdiA-PE, ON-PE, A-PE, HDdiA-PE, and KDdiA-PE (see Supporting Information). DHA-PE irradiated in air with 350 nm light was completely consumed within 33 h. Autoxidation in the presence of Cu2+ consumed about 90% of DHA-PE, while autoxidation in the presence of MPO only consumed about 50% DHA-PE within 33 h (Figure 3A). Similarly, AA-PE irradiated in air with 350 nm light was consumed almost completely after 33 h while Cu(II) promoted about 80% consumption and MPO promoted only about 40% consumption of AA-PE within 33 h (Figure 3B). LA-PE was consumed about 90% upon irradiation in air with 350 nm light, 75% upon Cu(II)-promoted autoxidation, and about 60% upon with MPO-promoted autoxidation within 33 h (Figure 3C). The results for oxidative consumption of the three different PUFA PEs, DHA-PE, AA-PE, and LA-PE, are remarkably consistent. The mildest autoxidation was observed with the enzymatic catalyst, MPO. Cu(II)-promoted oxidation is more extensive, and UV irradiation caused the most oxidation. Evolution of oxPEs during the Autoxidation of DHA-PE. Figure 4 and Table 3 show the time course and maximum and final yields for oxPEs produced upon autoxidation of DHA-PE for three different modes of initiation. In all cases, S-PE accumulates throughout the autoxidation reactions and is the most abundant species at long reaction time. Clearly, S-PE is a stable end product. Upon irradiation with 350 nm UV light, the aldehyde OB-PE is formed more rapidly than S-PE (Figure 4A). The yield of OB-PE reached a maximum (11%) in 4 h and then decreased, falling to 1.5% by 33 h. Thus, OB-PE is not a stable end product. Rather, it is expected to be susceptible to further autoxidation to give S-PE whose yield reached 17% after 33 h. The yield of KHdiA-PE rose to 7.5% after 8 h but eventually declined, reaching 1.4% after 33 h, indicating that it too is not a stable end product. In the oxidations promoted by MPO (Figure 4B) and Cu(II) (Figure 4C), the yields of oxPE are much lower than in the UV-promoted oxidation (Figure 4A). However, as in the UVpromoted autoxidations, the dominant product in both cases is S-PE with a yield of 3-4% after 33 h. The yield of KHdiA-PE reaches 2.7% in the MPO-promoted oxidation and 1.8% in the Cu(II)-promoted oxidation and then declines to 2.1 and 0.7%, respectively, by 33 h. Likewise, the yield of OB-PE reaches 2.8% in the MPO-promoted oxidation and 1.6% in the Cu(II)promoted oxidation and then declines to 1.1 and 0.4%, respectively, by 33 h.


Chem. Res. Toxicol., Vol. 19, No. 2, 2006 267 Table 4. Yields of oxPEs from AA-PEa product yields (%) initiator

OV-PE

G-PE

KOdiA-PE


3.8 ( 0.2 1.4 ( 0.1 1.2 ( 0.2 0.1 ( 0.1 4.8 ( 0.1 0.6 ( 0.1

3.0 ( 0.2 3.0 ( 0.2 3.4 ( 0.3 3.4 ( 0.3 16.1 ( 0.7 16.1 ( 0.7

2.6 ( 0.8 1.2 ( 0.7 0.5 ( 0.1 0.5 ( 0.1 2.4 ( 0.1 1.8 ( 0.1

a Autoxidations of AA-PE were promoted by various modes of free radical initiation. Yields shown are maximum (shaded) and final after 33 h. See the legend of Figure 5 for details.

Figure 5. Evolution profile of the oxPEs generated from AA-PE. Yields were calculated by dividing the amount of each analyte by the amount of starting AA-PE. For details, see the legend of Figure 4. Traces of oxPEs were detected in the commercial AA-PE. These levels were subtracted from the detected levels in the reaction mixtures. (A) UV-promoted autoxidation, (B) MPO-promoted autoxidation, and (C) Cu(II)-promoted autoxidation.

Evolution of oxPEs during the Autoxidation of AA-PE. Figure 5 and Table 4 show the time course and the maximum and final yields for each oxPE produced from autoxidation of AA-PE with three different modes of initiation. In all cases, G-PE accumulates throughout the autoxidation reactions and is the most abundant species at long reaction time. Thus, G-PE is a stable end product from A-PE in analogy with the production of S-PE from DHA-PE. Also, in analogy with the autoxidation of DHA-PE to give unstable products OB-PE and KHdiA-PE,

autoxidation of AA-PE generates OV-PE and KOdiA-PE that are consumed by further oxidation. Comparison of the yields of homologous products in Tables 3 and 4 does not reveal a trend toward substantially higher yields from the less unsaturated precursor. In fact, the maximum and final yields of OV-PE are lower than those for OB-PE except for the mildest oxidation conditions, i.e., MPO, for which modestly higher yields are found for OV-PE. Evolution of oxPEs from Autoxidation of LA-PE. Figure 6 and Table 5 show the time course and the maximum and final yields for each oxPE produced from autoxidation of LA-PE with different oxidation initiation systems. In all cases, A-PE accumulates throughout the autoxidation reactions and is the most abundant species at long reaction time. A-PE is a stable end product from LA-PE in analogy with the production of S-PE from DHA-PE and G-PE from AA-PE. However, there are important contrasts in the product profiles from LA-PE (Figure 6) and those from DHA-PE or AA-PE (Figure 4 or 5). Particularly notable is the fact that ON-PE levels either do not decline or decline less than those of the analogous aldehydes OB-PE or OV-PE during autoxidation for 33 h. ON-PE appears to be relatively resistant to further oxidation as compared with OB-PE and OV-PE. The final yield of ON-PE after a 33 h of autoxidation of LA-PE initiated by Cu(II) or UV irradiation is an order of magnitude greater than that of OV-PE after similar treatment of AA-PE (see Tables 4 and 5). Summary of Results. As expected, the most abundant oxPEs, succinyl (2.2 ( 0.8 pmol/retina) and ω-oxobutyryl (1.5 ( 1.0 pmol/retina) esters of 2-lysophosphatidylethanolamine, are derived from the docosahexaenoyl ester, the most abundant polyunsaturated PE in retina. However, a large amount the ω-oxononanoyl ester (1.3 ( 0.6 pmol/retina) derived from linoleyl PE is also present even though linoleate is an order of magnitude less abundant than docosahexanoate in retina. The oxidation profiles of the three different PUFA PEs, DHA-PE, AA-PE, and LA-PE, are remarkably consistent. The mildest autoxidation was observed with the enzymatic catalyst, MPO. Cu(II)-promoted oxidation is more extensive, and UVA irradiation caused the most oxidation. All three modes of initiation generated the same oxPEs from each PUFA-PE. Thus, autoxidations of PUFA-PEs produce transient levels of simple aldehydes and keto or hydroxy acids, which are further oxidized to the alkanedioate monoesters azeleyl, glutaryl, and succinyl PEs, the major stable end products from linoleyl, arachidonyl, and docosahexaenoyl PEs, respectively. ON-PE appears to be relatively resistant to further oxidation as compared to OB-PE and OV-PE. The resistance of ω-oxononanoyl-PE, as compared with ω-oxobutyryl-PE and ω-oxovaleryl-PE, to further oxidation may contribute to the greater amount of this oxPE relative to its precursor, linoleyl-PE, in retina.


Gugiu et al. Table 5. Yields of oxPEs from LA-PEa product yield (%) initiator

ON-PE

A-PE

HDdiA-PE

KDdiA-PE


3.2 ( 0.4 1.5 ( 0.1 1.0 ( 0.4 1.0 ( 0.4 6.4 ( 1.0 6.4 ( 1.0

1.7 ( 0.2 1.7 ( 0.2 1.2 ( 0.12 1.2 ( 0.12 22.2 ( 1.2 22.2 ( 1.2

0.08 ( 0.01 0.07 ( 0.01 0.04 ( 0.02 0.02 ( 0.02 0.45 ( 0.05 0.45 ( 0.05

1.7 ( 0.3 1.3 ( 0.2 0.35 ( 0.05 0.10 ( 0.01 0.45 ( 0.15 0.45 ( 0.15

a Autoxidations of LA-PE were promoted by various modes of free radical initiation. Yields shown are maximum (shaded) and final after 33 h. See the legend of Figure 6 for details.

Figure 6. Evolution profiles of the oxPEs generated from LA-PE. Yields were calculated by dividing the amount of each analyte by the amount of starting LA-PE. For details, see the legend of Figure 4. Trace levels of oxPEs present in the commercial LA-PE were subtracted from the amounts detected in the reaction product. (A) UV-promoted autoxidation, (B) MPO-promoted autoxidation, and (C) Cu(II)-promoted autoxidation.

Discussion Protein Adducts Derived from 4-Hydroxy-7-oxohept-5enoic Acid (HOHA) Accumulate in AMD Retina. Macular degeneration is characterized by the breakdown of photoreceptor and RPE cells in the small central portion of the retina (∼2 mm in diameter) responsible for high acuity vision. AMD is a slow, progressive disease with multiple environmental risk factors, and oxidative damage has long been suspected of contributing to AMD (24, 25). Indirect evidence that oxidative damage plays a role in AMD comes from studies showing that smoking significantly increases the risk of AMD (26) and that

antioxidant vitamins and zinc can slow the progression of the disease for select individuals (27). We recently obtained presumptive evidence that DHA phospholipids undergo oxidative fragmentation in the retina producing oxidatively truncated phospholipids, e.g., oxPEs, and that this may contribute to retinal pathology. Thus, Western blot analysis of PAGE gels revealed markedly elevated levels of ω-carboxyethylpyrrole (CEP)immunoreactive protein in extracts of retinas from victims of AMD vs normal retina (28). Generation of CEPs (8a) can occur through the adduction with proteins of, e.g., HOHA-PE (4a) derived from DHA-PE (1a) (Figure 7). We also showed that oxPCs are generated upon exposure of DHA-PC liposomes to air in the presence of Cu(II) or the MPO-NO2-glucose/glucose oxidase system (19). Oxidatively Truncated PCs Are Biologically Active Constituents of oxLDL. The central role of oxLDL in atherogenesis prompted investigations of the molecular basis of its pathological effects. The outer phospholipid shell of LDL particles contains mainly PC lipids (29). Previously, we found homologous carboxyalkyl pyrroles 8b and 8c in oxLDL (30). This observation prompted us to postulate that the oxidatively truncated choline phospholipid 5-hydroxy-8-oxooct-6-enoic acid (HOOA)PC is generated upon oxidation of LDL. Our prediction was confirmed, and HOOAPC promotes monocyte binding to endothelial cells, expression of interleukin-8 and monocyte chemotactic protein-1 in endothelial cells, and inhibition of E-selectin expression (31, 32). Binding to endothelial cells may promote infiltration of monocyte macrophages into the subendothelial space where they become foam cells, progenitors of atherosclerotic plaques, through unregulated endocytosis of oxLDL. Our subsequent studies identified a family of oxPCs that are present in oxLDL (33). Only a few molecules of some oxPCs in cholesterol-containing liposomes, prepared from unoxidized LDL lipids, are sufficient to trigger macrophage binding, uptake, metabolism, cholesterol accumulation, and the formation of foam cells (23). The oxPCs apparently promote endocytosis of oxLDL by macrophage cells through binding with the scavenger receptor CD36 (33, 34). γ-Hydroxyalkenal functionality in oxPCs that are present in oxLDL inhibits the activity of the thioprotease cathepsin B through covalent modification of essential nucleophilic functionality (35, 36). Consequently, oxLDL acts like a Trojan horse, delivering toxic oxPC electrophiles into macrophages where they reduce the ability of these cells to degrade protein that has been internalized by receptor-mediated endocytosis. Oxidatively Truncated EPs Are Present in Retina. By analogy, we expected to find oxPEs in retina. Because it is especially abundant in retina, we also expected that oxPEs derived from DHA-PE would predominate. Indeed, OB-PE (1.5 ( 1.0 pmol/retina) and S-PE (2.2 ( 0.8 pmol/retina) are the most abundant oxPEs in retina. Unexpectedly, a high level of ON-PE (1.3 ( 0.6 pmole/retina) was also found even though


Chem. Res. Toxicol., Vol. 19, No. 2, 2006 269

Figure 7. Generation of carboxyalkylpyrroles from PUFA-PE.

the levels of its precursor, LA-PE, are an order of magnitude lower than those of DHA-PE. An investigation of the in vitro oxidation of individual PUFA-PEs in small unilammelar vesicles revealed a possible factor contributing to this paradox. Thus, ON-PE is relatively resistant to further oxidation as compared with OB-PE and OV-PE. Another factor that deserves further scrutiny is the possibility that ON-PE is a poorer substrate for endogenous lipases than OB-PE or OV-PE. Human plasma platelet-activating factor acetylhydrolase is the LDL-associated phospholipase A2 activity that specifically degrades oxPCs (37). It preferentially hydrolyzes short sn-2 alkanoyl groups. However, the presence of an ω-oxo function, e.g., in ON-PC, makes it an excellent substrate (38). It is possible that similar preferential hydrolysis of shorter chain oxPEs, e.g., OB-PE or S-PE, would favor the accumulation of longer chain oxPEs, i.e., ON-PE or A-PE. Thus, slower degradation than for OB-PE or OV-PE might also explain the unexpectedly high levels of ON-PE present in retina. ω-Oxoalkanoyl EPs Are Abundant in Retina. Of the three reaction conditions examined, the in vitro oxidation induced by UV light of 350 nm gives the highest yields of oxPEs. Previously, dose-dependent UV light-induced peroxidation of dry lipid films (39), linolenic acid micelles (40), and PC liposomes (41) was observed by detecting lipid hydroperoxides, conjugated dienes, and thiobarbituric acid reactive substances. Antioxidants inhibit UV light-induced lipid peroxidation up to 90% (42), indicating that free radical mechanisms are involved. The mechanisms of lipid peroxidation promoted by UV light may include catalysis by redox active metal ions (41). Because there are generally traces of hydroperoxides present in polyunsaturated lipids, metal-catalyzed breakdown of hydroperoxides may be a source of hydroxyl and alkoxyl radicals. UV light is known to promote reduction of Fe(III) to Fe(II). Thus, iron may participate in redox cycles that catalyze the peroxidation process (41). This view is supported by the observation that EDTA inhibits UV light-induced peroxidation of unilamellar liposomes (42). Redox cycling Cu(I) with Cu(II) in reactions with hydroperoxides can also account for the promotion of lipid autoxidation by Cu(II). Our in vitro oxidation experiments revealed that autoxidations promoted by UV irradiation or Cu(II) tend to favor the conversion of ω-oxoalkanoyl phospholipids, i.e., OB-PE, OV-PE, and ON-PE, into the corresponding carboxylic acids, i.e., S-PE, G-PE, and A-PE after 33 h. In contrast, autoxidations promoted by MPO and all autoxidations

after short reaction times gave relatively higher ratios of ω-oxoalkanoyl phospholipids vs the corresponding carboxylic acids. Similarly high ratios were found in vivo indicating that autoxidation reaction conditions are relatively mild in vivo. Potential Biological Roles of oxPEs in Retina. The ability of oxPCs to trigger endocytosis of oxidatively damaged LDL particles by macrophage cells through recognition by the scavenger receptor CD36 suggests a potentially important role for oxPEs in retinal homeostasis and AMD. As noted at the outset, photoreceptor outer segment disks are replaced every 12 days (12, 13) because they are routinely damaged, apparently by free radical-induced oxidative modifications spawned by photogenerated radicals. ROS tips are shed and phagocytized by the RPE (14). The scavenger receptor CD36 contributes to the regulated endocytosis of shed ROS by RPE cells. It therefore seems plausible that oxPEs generated in ROS membranes serve as a physiological signal for CD36-mediated phagocytosis by RPE cells. CD36 is suspected to play a role in ROS clearance under conditions of heightened oxidative stress (43). Excessive generation of oxidatively truncated phospholipids associated with the heightened oxidative stress, as evidenced by CEPs that we detected in AMD retina (44), could foster the pathological phagocytosis of photoreceptor cells that is observed in AMD. The most devastating form of AMD is the result of choroidal neovascularization (CNV). It is the chief cause of irreversible loss of vision of elderly individuals in the western hemisphere. CNV involves vessel growth from the choroiocapillaris (see Figure 1). The new capillaries penetrate Bruch’s membrane and the retinal epithelium, resulting in rapid destruction of RPE and photoreceptor cells. The γ-hydroxyalkenal HOHA-PE (Figure 6) may play a central role in promoting CNV because low picomole amounts of the CEP modifications that it forms, through covalent modification of protein lysyl residues, stimulate neovascularization (45). Plausible Mechanisms for Oxidative Truncation of PUFAs. The biological importance of oxidatively truncated PUFA derivatives is indicated by previous findings that biologically active oxPCs are abundant in oxLDL and atherosclerotic plaques (23, 31, 32) and by the current observation that oxPEs are present in retina. Understanding oxidative fragmentation mechanism(s) is important, inter alia, to facilitate the design of therapeutic countermeasures. All three initiation methods, enzymatic (MPO), photochemical (UV irradiation), and redox active metal (Cu), can promote free radical-induced lipid

Scheme 2. Two Consecutive Allylic Hydrogen Abstractions Convert PUFAs into Dihydroperoxydienes


Gugiu et al.

Scheme 3. Postulated Mechanisms for Oxidative Fragmentation of Dihydroperoxydienes

oxidation. MPO catalyzes the generation (from hydrogen peroxide and nitrite) of reactive oxygen (•OH) and reactive nitrogen (•NO2) species (46) that initiate autoxidation through hydrogen atom abstraction. Photolysis of lipid hydroperoxides can generate both peroxy and alkoxy radicals (47). Cu ions promote the generation of peroxyl radicals from traces of PUFA hydroperoxides (48). Preferential abstraction of doubly allylic hydrogen (by •OOR, •OR, •OH, or •NO2) affords pentadienyl radicals that react with oxygen to deliver hydroperoxy dienes, the major primary products of PUFA autoxidation (Scheme 2). A second allylic hydrogen abstraction can then lead to the formation of dihydroperoxy diene intermediates that have been adduced as key intermediates in two different mechanisms postulated to account for oxidative truncation of PUFAs. It has been shown that oxidative fragmentation of optically pure 13(S)hydroperoxy-9,11-octadecadienoate produces optically pure 4(S)-hydroxy-2-nonenal [4(S)-HNE in Scheme 3] (49). This observation was rationalized with a mechanism involving Hock rearrangement of a 10,13(S)-diHPODE intermediate to generate a 4-(S)-hydroperoxy hemiacetal that, through hydrolysis and reduction, affords 4(S)-HNE. An alternative mechanism is also consistent with the production of optically pure 4(S)-HNE from 10,13(S)-diHPODE. Thus, β-scission of the derived doubly allylic alkoxy radical delivers 4(S)-HPNE and a vinyl radical (50). However, the relative instability of vinyl radicals disfavors this β-scission. We now propose a third alternative that provides a more plausible fragmentation pathway for such doubly allylic alkoxy radical intermediates. Thus, cyclization of allylic alkoxy radicals to epoxycarbinyl radicals is kinetically favored (51), and subsequent C-C bond cleavage generating an allylic radical is precedented and favored by conjugation (52, 53). Capture of this radical by oxygen and hydrogen atom transfer would deliver an enol ether whose hydrolysis would produce optically pure 4(S)-HPNE. Pathways for the generation of 10,13-diHPODE are not limited to free radical-induced oxidation. Thus, 10,13diHPODE can be produced through photosensitized singlet oxygenation of LA (54, 55). Furthermore, lipofuscin, a pigment that accumulates in the retina with age, is known to sensitize photogeneration of singlet oxygen (56). To obtain evidence that allows a choice between the alternative oxidative fragmentation mechanisms presented in Scheme 3, we recently prepared the putative intermediate 10,13-diHPODE (57) and are now investigating its oxidative fragmentation. Results of those studies will be reported in due course. Acknowledgment. We thank the NIH for support of this research via Grant GM21249. We thank Dennis Curran for suggesting the involvement of epoxycarbinyl radical rearrangements in oxidative fragmentation reactions.

Supporting Information Available: MRM chromatograms of standards and their derivatives and of the analyses of oxidized lipid samples as well as retinal lipid extract samples. Mass spectra and calibration curves and equations for all lipids analyzed. This material is available free of charge via the Internet at http://pubs.acs.org.

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TX050247F

Identification of Oxidatively Truncated Ethanolamine Phospholipids in

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