Ethanolamine plasmalogens prevent the oxidation of cholesterol by reducing the oxidizability of cholesterol in phospholipid bilayers

The aims of the present study are to establish an appropriate system for assessing the oxidizability of cholesterol (1111156) in phospholipid (PL) bilayers, and to explore the effect of ethanolamine plasmalogens on the oxidizability of CH with the system, through comparing with those of choline plasmalogens, phosphatidylethanolamine, and antioxidant α-tocopherol (Toc). Investigation of the effects of oxidants, vesicle lamellar forms, saturation level, and constituent ratio of PLs in vesicles on CH oxidation revealed the suitability of a system comprising unilamellar vesicles and the water-soluble radical initiator 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH). As CH oxidation in the system was found to follow the rate law for autoxidation without significant interference from oxidizable PLs, the oxidizability of CH in PL bilayers could be experimentally determined from the equation: k p/(2k t)1/2=R p/[LH]Ri1/2 by measuring the rate of CH oxidation. It was found with this system that bovine brain ethanolamine plasmalogen (BBEP), bovine heart choline plasmalogen, and egg yolk phosphatidylethanolamine lower the oxidizability of CH in bilayers. Comparison of the dose-dependent effects of each PL demonstrated the greatest ability of BBEP to reduce the oxidizability.A time course study of CH oxidation suggested a novel mechanism of BBEP for lowering the oxidizability of CH besides the action of scavenging radicals. The aims of the present study are to establish an appropriate system for assessing the oxidizability of cholesterol (1111156) in phospholipid (PL) bilayers, and to explore the effect of ethanolamine plasmalogens on the oxidizability of CH with the system, through comparing with those of choline plasmalogens, phosphatidylethanolamine, and antioxidant α-tocopherol (Toc). Investigation of the effects of oxidants, vesicle lamellar forms, saturation level, and constituent ratio of PLs in vesicles on CH oxidation revealed the suitability of a system comprising unilamellar vesicles and the water-soluble radical initiator 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH). As CH oxidation in the system was found to follow the rate law for autoxidation without significant interference from oxidizable PLs, the oxidizability of CH in PL bilayers could be experimentally determined from the equation: k p/(2k t)1/2=R p/[LH]Ri1/2 by measuring the rate of CH oxidation. It was found with this system that bovine brain ethanolamine plasmalogen (BBEP), bovine heart choline plasmalogen, and egg yolk phosphatidylethanolamine lower the oxidizability of CH in bilayers. Comparison of the dose-dependent effects of each PL demonstrated the greatest ability of BBEP to reduce the oxidizability. A time course study of CH oxidation suggested a novel mechanism of BBEP for lowering the oxidizability of CH besides the action of scavenging radicals. Cholesterol (CH) oxidation caused by free radicals in vivo is of considerable interest, as is intake of CH oxidation products in food, owing to potential pathological application as in the case of atherosclerosis (1Steinbrecher U.P. Zhang H.F. Lougheed M. Role of oxidatively modified LDL in atherosclerosis.Free Radic. Biol. Med. 1990; 9: 155-168Google Scholar, 2Sevanian A. Bittolo-Bon G. Cazzolato G. Hodis H. Hwang J. Zamburlini A. Maiorino M. Ursini F. LDL- is a lipid hydro-peroxide-enriched circulating lipoprotein.J. Lipid Res. 1997; 38: 419-428Google Scholar). CH oxidation is characterized by the following features: CH has a very low ability to propagate radical chain reactions (3Barclay L.R.C. Cameron R.C. Forrest B.J. Locke S.J. Nigam R. Vinqvist M.R. Cholesterol: Free radical peroxidation and transfer into phospholipid membranes.Biochim. Biophys. Acta. 1990; 1047: 255-263Google Scholar), and its oxidation products, i.e., oxysterols, have various biological activities such as cytotoxicity, atherogenicity, mutagenicity, and carcinogenicity (4Smith L.L. Johnson B.H. Biological activities of oxy-sterols.Free Radic. Biol. Med. 1989; 7: 285-332Google Scholar), in addition to their effects on CH metabolism in cells (5Goldstein J.L. Brown M.S. Regulation of the mevalonate pathway.Nature. 1990; 343: 425-430Google Scholar). Considering the characteristics of CH oxidation, in vivo defensive mechanisms appear to differ from those for other oxidizable lipids, such as polyunsaturated fatty acids, possessing a high ability to propagate radical chain reactions. One of the mechanisms of these oxidizable lipids is to suppress the expansion of oxidative injury via active radical species by blocking chain reactions by chain-breaking antioxidants such as α-tocopherol (Toc) (6Niki E. Saito T. Kawakami A. Kamiya Y. Inhibition of oxidation of methyl linoleate in solution by vitamin E and vitamin C.J. Biol. Chem. 1984; 259: 4177-4182Google Scholar). On the other hand, in CH oxidation, the most effective way to avoid the damage seems to be to inhibit the formation of oxysterols as far as possible by reducing the susceptibility of CH to radical attack. Protecting CH in biomembranes from oxidation is especially important, because CH in the phospholipid (PL) bilayer is the form most susceptible to attack by free radicals generated in the water phase (7Lijana R.C. McCracken M.S. Rudolph C.J. The oxidation of cholesterol in vesicles.Biochim. Biophys. Acta. 1986; 879: 247-252Google Scholar). CH content in biomembranes differs noticeably among various species of cells or intracellular organelles, and is abundant in nervous-system myelin and red blood cells at almost the equivalent molar ratio of CH to PLs (8Demel R.A. London Y. Geurts van Kessel W.S.M. Vossenberg F.G.A. van Deenen L.L.M. The specific interaction of myelin basic protein with lipids at the air-water interface.Biochim. Biophys. Acta. 1973; 311: 507-519Google Scholar, 9Nelson G.J. Composition of natural lipids from erythrocytes of common mammals.J. Lipid Res. 1967; 8: 374-379Google Scholar). Nervous-system myelin and red blood cells may readily incur oxidative injury, being the sites of oxygen consumption and exposure to oxygen, but the vivo life spans of these tissues are comparatively long. These biomembranes would appear to possess structures capable of resisting oxidative stress, especially CH oxidation. These biomembranes contain major distribution of 1-O-alk-1 eny-2-acyl-sn-glycero-3-phosphoethanolamine (i.e., ethanolamine plasmalogen) in glycerophosphoethanolamines (10Horrocks L.A. Sharm M. Plasmalogens and O-alkyl glycerophospholipids.in: Hawthorne J.N. Ansell G.B. New Comprehensive Biochemistry. Elsevier Biochemical Press, Amsterdam.1982: 51-95Google Scholar). Plasmalogens are glycerophospholipids with vinyl ether double bonds (-CH2-O-CH=CH-) at the sn-1 position of the glycerol backbone, and are widely distributed in most mammalian cells and tissues (11Snyder F. Metabolism, regulation, and function of ether-linked glycerolipids and their bioactive species.in: Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier Science Publishers B.V., Amsterdam,1991: 241-267Google Scholar). The physiological role of plasmalogens is not fully understood, but recent studies on plasmalogen-deficient mutant cells lead to the proposal that these ether lipids serve to protect cells from oxidative stress as endogenous antioxidants by scavenging radicals at the vinyl ether linkage (12Zoeller R.A. Morand O.H. Raetz C.R.H. A possible role for plasmalogens in protecting animal cells against photosensitized killing.J. Biol. Chem. 1988; 263: 11590-11596Google Scholar, 13Morand O.H. Zoeller R.A. Raetz C.R.H. Disappearance of plasmalogens from membranes of animal cells subjected to photosensitized oxidation.J. Biol. Chem. 1988; 263: 11597-11606Google Scholar, 14Zoeller R.A. Lake A.C. Nagan N. Gaposchkin D.P. Legner M.A. Lieberthal W. Plasmalogens as endogenous antioxidants: somatic cell mutants reveal the importance of the vinyl ether.Biochem. J. 1999; 338: 769-776Google Scholar). However, their ability to scavenge radicals is far less than that of Toc (15Hahnel D. Beyer K. Engelmann B. Inhibition of peroxyl radical-mediated lipid oxidation by plasmalogen phospholipids and α–tocopherol.Free Radic. Biol. Med. 1999; 27: 1087-1094Google Scholar). On the other hand, it is known that ethanolamine plasmalogens have a stronger propensity for hexagonal phase formation than diacyl analog (16Lohner K. Balgavy P. Hermetter A. Paltauf F. Laggner P. Stabilization of non-bilayer structures by the etherlipid ethanolamine plasmalogen.Biochim. Biophys. Acta. 1991; 1061: 132-140Google Scholar), which contributes to membranes fusion (17Glaser P.E. Gross R.W. Plasmenylethanolamine facilitates rapid membrane fusion: a stopped-flow kinetic investigation correlating the propensity of a major plasma membrane constituent to adapt an HII phase with its ability to promote membrane fusion.Biochemistry. 1994; 33: 5805-5812Google Scholar). Such a modification of the physical features of membranes may serve to reduce the oxidizability of membranes. Ethanolamine plasmalogens are considered by the authors to prevent the oxidation of CH by lowering susceptibility to attack by free radicals. To confirm this possibility, in the present study an appropriate system for assessing the oxidizability of CH in PL bilayers has been established, and the effect of bovine brain ethanolamine plasmalogen (BBEP) on the oxidizability of CH in bilayers has been explored with this system by comparing them with those of bovine heart choline plasmalogen (BHCP), egg yolk phosphatidylethanolamine (EYPE), and an antioxidant (Toc). CH, 7-ketocholesterol (7K), 7β-hydroxycholesterol (7βOH), CH 5α,6α-epoxide (α-EPOX), cholestane-3β,5α,6β-triol (α-TRIOL), dioleoyl phosphatidylethanolamine (DOPE), bovine brain phosphatidylserine (BBPS), Toc, and l-ascorbic acid (AsA) were purchased from Sigma Chemicals (St. Louis, MO). 1-Radyl-2-acyl-sn-glycero-3-phosphoethanolamine from bovine brain (BBPE), BBEP, 1-radyl-2-acyl-sn-glycero-3-phosphocholine from bovine heart (BHPC), 1-O-alk-1 eny-2-lyso-sn-glycero-3-phosphoethanolamine from bovine brain (LyEP), and 1-acyl-2-lyso-sn-glycero-3-phosphoethanolamine from porcine liver (LyPE) were obtained from Doosan Serdary Research Laboratories (Englewood Cliffs, NJ). EYPE and dimyristoyl phosphatidylcholine (DMPC) were purchased from Nichiyu Liposome Co., Inc. (Tokyo, Japan). Soybean phosphatidylcholine (SPC) was kindly provided form Tsuji Seiyu Co., Inc. (Mie, Japan) and purified by chromatography prior to use. 2,2′-Azobis (2-amidino-propane) dihydrochloride (AAPH) was obtained from Wako Pure Chemical Industries (Osaka, Japan). 7K was purified by preparative thin layer chromatography prior for use as a standard for analysis. All other chemicals were of the highest purity available from commercial sources. Ethanolamine- and choline-plasmalogens were respectively purified from commercial 1-radyl-2-acyl-sn-glycero-3-phosphoethanolamine from bovine brain (BBPE) and 1-radyl-2-acyl-sn-glycero-3-phos-phocholine from bovine heart (BHPC) with Rhizopus arrhizus lipase (18Paltauf F. Preparation of choline and ehanolamine plasmalogens by enzymatic hydrolysis of the accompanying diacyl analogs.Lipids. 1977; 13: 165-166Google Scholar). The purity was estimated as 82–91% for BBEP and 86–94% for BHCP by acid-catalyzed hydrolytic procedure with HCL fumes followed by thin layer chromatography (19Horrochs L.A. The alk-1-enyl group content of mammalian myelin phosphoglycerides by quantitative two-dimensional thin-layer Lipid Res. 9: and A. E. of lipid in the Scholar). The purified plasmalogens were in at use. CH was with various glycerophospholipids in Toc in at a molar The was in a of The lipids were in at the phase for lipids with or without acid in a for of unilamellar vesicles were by vesicles through of and in that by with an P. of by a rapid Biophys. Acta. 1986; Scholar). The was with a lamellar was by with a Japan) bilayer Biophys. Acta. Scholar). of vesicles were with a B. Press, New Scholar). The constituent molar ratio of CH and PLs in vesicles have been by of enzymatic with a commercial for CH and for PLs of vesicles with The content of Toc in vesicles was by high chromatography Japan) a Japan) K. G. of in by high Scholar). vesicles were for oxidation The oxidation of vesicles was in an and with and acid or water-soluble radical initiator (AAPH) for the at with at CH and oxysterols lipids were from by the of and A rapid of and J. with as an antioxidant and as The lipids were subjected to with J. of in plasma and Scholar), and to CH and oxysterols with as in the R. H. N. of 7-ketocholesterol by a from the of cholesterol Scholar). of PLs fatty and fatty from of plasmalogens were with Pure Japan) to fatty acid methyl and from fatty The PLs in the present are in and almost with those in the Y. L.A. of and diacyl and their species by high Lipid Res. Scholar, A. Y. of on the and of from and Chem. 1988; Scholar, antioxidants for as Chem. bovine brain ethanolamine bovine brain bovine heart choline bovine heart egg yolk acid methyl and from fatty by and as of in a BBEP, bovine brain ethanolamine bovine brain bovine heart choline bovine heart egg yolk acid methyl and from fatty by and as of The of BBEP, ethanolamine plasmalogen (LyEP), and Toc radical were with a with a stopped-flow by following the in of at K. N. E. of action of as antioxidant against lipid peroxidation in solution and Radic. Res. 1999; Scholar). To establish an appropriate system for assessing the oxidizability of CH in PL bilayers, the effects of vesicle lamellar form saturation of phosphatidylcholine oxidizable and constituent molar ratio of CH to PL in vesicles on the of CH oxysterols were oxidation with the of CH was noticeably by the and in the constituent ratio of phosphatidylcholine in or which that CH oxidation by is on the of PLs in On the other hand, in oxidation with the of CH was by the and in the constituent ratio of phosphatidylcholine in it was by the of PL in vesicles oxysterols in the CH oxidation products in vesicles were into and and and and and the oxidation products It is known that the products are by the oxidation of PLs present with CH T. A. M. N. A. A mechanism for of cholesterol by lipid Biophys. Acta. 1984; Scholar). the of of PL oxidation in CH oxidation was estimated by the of to oxidation A was obtained for the oxidation of vesicles with the of PL oxidation in CH oxidation. A obtained for vesicles with the formation ratio in the of of The ratio for with was than that for with the effect of PLs in vesicles with vesicles These that a system comprising vesicles and the water-soluble radical initiator would be most appropriate for assessing the oxidizability of CH in bilayers without significant interference from oxidizable of the of cholesterol (CH) in unilamellar and vesicles comprising or phosphatidylcholine in oxidation CH were in with in the of or with 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH) in the of at for are as or vesicles of phosphatidylcholine and or phosphatidylcholine and (PL) or is the molar ratio of CH to PL in the The CH was obtained from and and standard oxysterols in is the for of CH is the for of CH 2,2'-azobis (2-amidino-propane) or or or cholesterol or the as in the to CH and oxysterols ether were by with as the on as is the for of CH in a 2,2'-azobis (2-amidino-propane) or or or cholesterol or the as in the to CH and oxysterols ether were by with as the on as To the characteristics of CH oxidation in the system, the effects of the of CH and on the rate of CH oxidation were with of CH and dimyristoyl phosphatidylcholine (DMPC) at the equivalent molar ratio CH was with time and the rate of CH oxidation, estimated from the obtained the time a was on the of CH in the and on the of the rate of radical which was is the of initiator in the for is as at E. Free radical as of or peroxyl in Press, Scholar), and the of free radical is as from the by the L.R.C. of biological The autoxidation of a A of the autoxidation of egg phosphatidylcholine in water and in Chem. with a water-soluble L.R.C. Locke S.J. J. of and membranes. kinetic be by water-soluble or with water-soluble or chain-breaking Chem. 1984; of the of CH on the rate of CH oxidation in unilamellar vesicle by of CH and dimyristoyl phosphatidylcholine at the equivalent molar ratio were at and in with in the of at The of CH in each were determined by CH with The of CH oxidation, were estimated from the obtained the time a and are against the of CH in the of the of on the rate of CH oxidation in molar were in with of and in the of at The of CH oxidation, were estimated from the obtained the time a and are against the of the rate of radical which was from the following equation: as in on the of the of on the rate of CH oxidation in molar were in with of and in the of at The of CH oxidation, were estimated from the obtained the time a and are against the of the rate of radical which was from the following equation: as in on the These that the CH oxidation in the system the rate law for autoxidation by rate for reactions of Free Chem. is the rate of the rate of radical the of and k and are the rate for chain and The ratio of these rate k p/(2k is to as the susceptibility of a to that oxidizability. the oxidizability of CH in bilayers be experimentally determined To confirm the suitability of the system, the oxidizability were and in various of CH and dimyristoyl phosphatidylcholine (DMPC) or phosphatidylcholine (SPC) at various constituent of PLs to CH the were which that the system is appropriate for assessing the oxidizability of CH in PL bilayers without significant interference from oxidizable of the saturation and constituent ratio of PLs on the oxidizability of CH in molar oxidizability of CH was determined by measuring the rate of CH oxidation in each with in a The oxidizability of CH was determined by measuring the rate of CH oxidation in each with this system, the effects of various and an antioxidant on the oxidizability of CH in bilayers were explored BBEP, and EYPE the oxidizability of CH in bilayers, ethanolamine plasmalogen from bovine brain and Toc the oxidizability of CH To the ability of BBEP, and EYPE to lower the oxidizability of CH, the dose-dependent effects of each PL were in of CH and each lipid at the equivalent molar ratio of PLs to It was found among BBEP has the greatest ability to reduce the oxidizability of CH in bilayers oxidizability of CH in various as on a by CH with oxidation of with in the or of in at for as standard on by the following equation: is as as in the by the the oxidizability of CH in in the of as by the the oxidizability of CH in in the of as by the the oxidizability of CH in in the of as by the the oxidizability of CH in in the of as by the the oxidizability of CH in in the of as by the the oxidizability of CH in in the of as by the the oxidizability of CH in in the of as by the the oxidizability of CH in in the of as by the the oxidizability of CH in in the of as by the the oxidizability of CH in in the of as by the the oxidizability of CH in in the of as by the the oxidizability of CH in in the of as by the the oxidizability of CH in in the of as by the the oxidizability of CH in in the of as by the the oxidizability of CH in in the of as bovine brain ethanolamine bovine brain bovine brain bovine heart choline bovine heart CH, dimyristoyl dioleoyl egg yolk from bovine 1-acyl-2-lyso-sn-glycero-3-phosphoethanolamine from porcine as on a by CH with oxidation of with in the or of in at for as standard on by the following equation: is as as in the by the the oxidizability of CH in in the of as by the the oxidizability of CH in in the of as by the the oxidizability of CH in in the of as in a BBEP, bovine brain ethanolamine bovine brain bovine brain bovine heart choline bovine heart CH, dimyristoyl dioleoyl egg yolk from bovine 1-acyl-2-lyso-sn-glycero-3-phosphoethanolamine from porcine It is known that plasmalogens an action via scavenging radicals at the vinyl ether linkage (12Zoeller R.A. Morand O.H. Raetz C.R.H. A possible role for plasmalogens in protecting animal cells against photosensitized killing.J. Biol. Chem. 1988; 263: 11590-11596Google Scholar, 13Morand O.H. Zoeller R.A. Raetz C.R.H. Disappearance of plasmalogens from membranes of animal cells subjected to photosensitized oxidation.J. Biol. Chem. 1988; 263: 11597-11606Google Scholar, 14Zoeller R.A. Lake A.C. Nagan N. Gaposchkin D.P. Legner M.A. Lieberthal W. Plasmalogens as endogenous antioxidants: somatic cell mutants reveal the importance of the vinyl ether.Biochem. J. 1999; 338: 769-776Google Scholar, J.E. Lipoproteins by contain the antioxidant Biophys. Acta. 1990; Scholar, D. Beyer K. Engelmann B. oxidative of polyunsaturated diacyl phospholipids in the of plasmalogen phospholipids in J. 1997; Scholar, N. R.C. of phospholipids products from Lipid Res. Scholar). It was the effect of plasmalogens in reducing the oxidizability of CH would be to the ability of scavenging radicals by measuring the which is a radical and has been to the of a antioxidant K. N. E. of action of as antioxidant against lipid peroxidation in solution and Radic. Res. 1999; Scholar). The rate of of BBEP and radical were estimated as and that for Toc was The that the ability of plasmalogens is less than that of Toc in a of the ability of plasmalogens to the in the oxidizability of However, plasmalogens appear to an effect to that of Toc in plasma B. J. phospholipids as potential against lipid peroxidation of low Biophys. Res. 1994; Scholar), plasmalogens may scavenge radicals in membranes. The effects of BBEP, and Toc on the time course of CH oxidation were BBEP and EYPE the rate of CH oxidation their effects in lowering the oxidizability of the radical Toc a time effect on the rate of oxidation ability to reduce the oxidizability. a in the rate of oxidation and of the to the of in vesicles that choline plasmalogens have the ability to reduce the their ability is less than that of addition to the the following the of mechanism for reducing the oxidizability of CH besides the action of scavenging radicals. BBEP has ability to lower the oxidizability of CH than BHCP of vinyl ether double bonds in the EYPE has a reducing ability the action of scavenging radicals of BBEP, and Toc on time course of CH oxidation in by of CH, dimyristoyl phosphatidylcholine or were in with in the of at BBEP at molar to CH EYPE at molar to CH or contain at molar to CH contain Toc at molar to CH of BBEP, and Toc on time course of CH oxidation in by of CH, dimyristoyl phosphatidylcholine or were in with in the of at BBEP at molar to CH EYPE at molar to CH or contain at molar to CH contain Toc at molar to CH of BBEP, and Toc on time course of CH oxidation in by of CH, dimyristoyl phosphatidylcholine or were in with in the of at BBEP at molar to CH EYPE at molar to CH or contain at molar to CH contain Toc at molar to CH It is known that ethanolamine plasmalogens have a stronger propensity for hexagonal phase formation than phosphatidylethanolamine (16Lohner K. Balgavy P. Hermetter A. Paltauf F. Laggner P. Stabilization of non-bilayer structures by the etherlipid ethanolamine plasmalogen.Biochim. Biophys. Acta. 1991; 1061: 132-140Google Scholar), which contributes to membranes fusion (17Glaser P.E. Gross R.W. Plasmenylethanolamine facilitates rapid membrane fusion: a stopped-flow kinetic investigation correlating the propensity of a major plasma membrane constituent to adapt an HII phase with its ability to promote membrane fusion.Biochemistry. 1994; 33: 5805-5812Google Scholar). it has been that the of CH in PL bilayers from group to fatty hexagonal phase formation E. M. K. T. T. of and of cholesterol J. Scholar). Such membrane may serve to CH to a in the bilayers, it to free radical attack in the water Such a modification of the physical features of caused by ethanolamine plasmalogens, may serve to reduce the oxidizability of CH in bilayers. study is to the mechanism by which ethanolamine plasmalogens a lowering of the oxidizability of CH in PL bilayers. The present study that ethanolamine plasmalogens a role in CH oxidation in biomembranes by reducing the oxidizability of CH in bilayers as a physiological antioxidant for The authors for in by at the Research and for in measuring the radical by a stopped-flow at the Research for Science and of The authors of the of for 2,2'-azobis dihydrochloride bovine brain ethanolamine plasmalogen bovine heart choline plasmalogen cholesterol egg yolk phosphatidylethanolamine vesicle phospholipid α-tocopherol