Ethanolamine plasmalogens prevent the oxidation of cholesterol by reducing the oxidizability of cholesterol in phospholipid bilayers
2003; Elsevier BV; Volume: 44; Issue: 1 Linguagem: Inglês
10.1194/jlr.m200340-jlr200
ISSN1539-7262
Autores Tópico(s)Cholesterol and Lipid Metabolism
ResumoThe 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 chromatography.J. Lipid Res. 1968; 9: 469-472Google Scholar) and phosphorus analysis (20Chalvardjian A. Rudnicki E. Determination of lipid phosphorus in the nanomolar range.Anal. Biochem. 1970; 36: 225-226Google Scholar). The purified plasmalogens were stored in chloroform at −80°C under N2 gas atmosphere until use. CH was mixed with various glycerophospholipids and, in some cases, supplemental Toc in chloroform at a specified molar ratio. The solvent was evaporated completely in a stream of nitrogen (N2) gas. The dried lipids were dispersed in phosphate buffered saline (PBS; 10 mM, pH 7.4) kept at 42°C, above the phase transition temperature for all lipids used, with or without 0.1 mM ethylenediamine tetraacetic acid (EDTA) in a vortex mixer for preparation of multilamellar (ML) vesicles. Large unilamellar vesicles (LUV) were prepared by passing ML vesicles through polycarbonate filters (Corning Glass Works, Corning, NY) of pore sizes 3.0 μm, 1.0 μm, 0.4 μm, and 0.1 μm in that order by N2 gas pressure with an extruder (Lipex Biomembranes Inc., Vancouver, Canada) (21Mayer L.D. Hope M.J. R Cullis P. Vesicles of variable sizes produced by a rapid extrusion procedure.Biochim. Biophys. Acta. 1986; 858: 161-168Google Scholar). The final extrusion was recycled five times with a dual 0.1 μm filter. Vesicle lamellar structure was visually confirmed by negative dyeing with uranyl acetate (Merck, Darmstadt, Germany) under a transmission electron microscope (JEM-2000FX; JEOL, Tokyo, Japan) (22Johnson S.M. Bangham A.D. Hill M.W. Korn E.D. Single bilayer liposomes.Biochim. Biophys. Acta. 1971; 233: 820-826Google Scholar). Particle sizes of vesicles were measured with a light scattering instrument (NICOMP 370; Particle Sizing Systems Inc.) (23Chu B. Laser Light Scattering. Academic Press, New York1974Google Scholar). The constituent molar ratio of CH and PLs in vesicles have been checked by means of enzymatic method with a commercial kit for CH (CH C-test; Wako), TLC separation, and phosphorus analysis for PLs after preparation of vesicles with extrusion. The content of Toc in vesicles was checked by high performance liquid chromatography (880-PU; JASCO, Tokyo, Japan) using a Senshupak NH2-1251-N column (4.6 mm × 25 cm; SSC Corporation, Tokyo, Japan) (24Abe K. Katsui G. Determination of tocopherols in serum by high speed liquid chromatography.Vitamins. 1975; 49: 259-263Google Scholar). Freshly prepared vesicles were used for oxidation experiments. The oxidation of vesicles was carried out in an open glass tube, and incubation with 0.4 mM FeSO4 and 4 mM ascorbic acid (Fe/AsA) or 5–100 mM water-soluble azo radical initiator (AAPH) for the designated periods at 37°C with shaking at 150 oscillations per min. For CH and oxysterols analyses, lipids were extracted from incubation mixtures by the method of Bligh and Dyer (25Bligh E.G. Dyer W.J. A rapid method of total extraction and purification.Can. J. Biochem. Physiol. 1959; 31: 911-917Google Scholar) with chloroform-methanol (1:2, v/v) containing butylated hydroxytoluene (50 μg/ml; Sigma) as an antioxidant and 5α-cholestane (100 μg/ml; Sigma) as internal standard. The lipids were subjected to mild saponification with alcoholic KOH (26Brooks C.J.W. McKenna R.M. Cole W.J. MacLachlan J. Lawrie T.D.V. "Profile" analysis of oxygenated sterols in plasma and serum.Biochem. Soc. Trans. 1983; 11: 700-701Google Scholar), and then derived to CH and oxysterols trimethylsilyl ethers (TMSi) with N,O-Bis-TMS-trifluoroacetamide-trimethylchlorosilane (5:1, v/v; Tokyo Kasei Kogyo) as described detail in the literature (27Maeba R. Shimasaki H. Ueta N. Generation of 7-ketocholesterol by a route different from the decomposition of cholesterol 7-hydroperoxide.J. Oleo Sci. 2001; 50: 109-119Google Scholar). For quantification of PLs component, fatty acids and fatty aldehydes derived from alkenyl chains of plasmalogens were transesterified with anhydrous HCl-methanol (5% HCl, w/w; Muto Pure Chemicals, Tokyo, Japan) to yield fatty acid methyl esters and dimethylacetal derivatives from fatty aldehydes. The PLs composition used in the present experiments are shown in Table 1, and almost agree with those previously reported in the literature (28Nakagawa Y. Horrocks L.A. Separation of alkenylacyl, alkylacyl, and diacyl analogues and their molecular species by high performance liquid chromatography.J. Lipid Res. 1983; 24: 1268-1275Google Scholar, 29Smiles A. Kakuda Y. MacDonald B.E. Effect of degumming reagents on the recovery and nature of lecithins from crude canola, soybean and sunflower oils.J. Am. Oil Chem. Soc. 1988; 65: 1151-1155Google Scholar, 30Hudson B.J.F. Lewis J.I. Polyhydroxy flavonoid antioxidants for edible oils. Phospholipids as synergists.Food Chem. 1983; 10: 111-120Google Scholar).TABLE 1PLs compositionComponentSPCBBPEBBEPBHPCBHCPEYPEmol %16:015.9 6.1 2.4 25.6 0.7 19.518:04.0 16.1 8.0 5.1 30.718:111.4 25.4 24.9 20.9 5.7 15.818:264.0 28.8 26.3 8.918:35.7 1.320:1 5.3 6.520:3 1.6 3.420:4 6.0 5.0 6.1 8.1 15.422:4 6.5 5.522:5 4.122:6 7.8 6.3 5.616:0 ald 8.3 12.0 10.5 43.718:0 ald 8.2 13.618:1 ald 10.3 15.8BBEP, bovine brain ethanolamine plasmalogen; BBPE, bovine brain glycero- phosphoethanolamine; BHCP, bovine heart choline plasmalogen; BHPC, bovine heart glycerophosphocholine; EYPE, egg yolk phosphatidylethanolamine; SPC, soybean phosphotidylcholine. Fatty acid methyl esters and dimethylacetal derivatives from fatty aldehydes analyzed by GC/MS, and shown as means of duplicate determinations. Open table in a new tab BBEP, bovine brain ethanolamine plasmalogen; BBPE, bovine brain glycero- phosphoethanolamine; BHCP, bovine heart choline plasmalogen; BHPC, bovine heart glycerophosphocholine; EYPE, egg yolk phosphatidylethanolamine; SPC, soybean phosphotidylcholine. Fatty acid methyl esters and dimethylacetal derivatives from fatty aldehydes analyzed by GC/MS, and shown as means of duplicate determinations. The reactivities of BBEP, lyso ethanolamine plasmalogen (LyEP), and Toc toward galvinoxyl radical were measured with a spectrophotometer equipped with a rapid-mixing stopped-flow apparatus (RX-1000, Applied Photophysics) by following the decrease in maximum absorption of galvinoxyl at 429 nm (31Nishino K. Noguchi N. Niki E. Dynamics of action of bisphenol as radical-scavenging antioxidant against lipid peroxidation in solution and liposomal membranes.Free Radic. Res. 1999; 31: 535-548Google Scholar). To establish an appropriate system for assessing the oxidizability of CH in PL bilayers, the effects of oxidant (Fe/AsA vs. AAPH), vesicle lamellar form [unilamellar (UL) vs. ML], saturation level of phosphatidylcholine [unoxidizable saturated (S) vs. oxidizable unsaturated (U)], and constituent molar ratio of CH to PL in vesicles (CH/PL= 0.2 vs. 1.0) on the percentage of CH oxidized (Fig. 1)and oxysterols formed (Table 2) were investigated. In oxidation with Fe/AsA, the percentage of CH oxidized was noticeably promoted by the unsaturation and increase in the constituent ratio of phosphatidylcholine in either UL or ML vesicles, which indicates that CH oxidation by Fe/AsA is largely dependent on the amount of unsaturated PLs in vesicles. On the other hand, in oxidation with AAPH, the percentage of CH oxidized was only slightly enhanced by the unsaturation and increase in the constituent ratio of phosphatidylcholine in UL vesicles, although it was doubled by the unsaturation of PL in ML vesicles (Fig. 1). Major oxysterols in the CH oxidation products formed in vesicles were classified into C-7 (isomeric 7α- and 7βOH and 7K) and C-5,6 (isomeric α- and β-EPOX and the reduced form, TRIOL) oxidation products (Table 2). It is known that the latter products are enhanced by the oxidation of unsaturated PLs present with CH (32Watabe T. Tsubaki A. Isobe M. Ozawa N. Hiratsuka A. A mechanism for epoxidation of cholesterol by hepatic microsomal lipid hydroperoxides.Biochim. Biophys. Acta. 1984; 795: 60-66Google Scholar). Therefore, the degree of involvement of PL oxidation in CH oxidation was estimated by the product ratios of C-5,6 to C-7 oxidation products. A maximum product ratio, 0.27, was obtained for the oxidation of ML/U vesicles (CH/PL=0.2) with Fe/AsA, indicating the large participation of PL oxidation in CH oxidation. A minimum ratio, 0.08, obtained for UL/S vesicles (CH/PL=1.0) with AAPH represents the formation ratio in the absence of participation of unsaturated PLs. The ratio for UL/U (CH/PL=1.0) with AAPH, 0.14, was significantly smaller than that for ML/U (CH/PL=0.2) with AAPH, 0.22, indicating the reduced effect of unsaturated PLs in UL vesicles compared with ML vesicles (Table 2). These results suggest that a system comprising UL vesicles and the water-soluble radical initiator AAPH would be most appropriate for assessing the oxidizability of CH in bilayers without significant interference from oxidizable PLs.Fig. 1Comparison of the percentage of cholesterol (CH) oxidized in unilamellar (UL) and multilamellar (ML) vesicles comprising saturated or unsaturated phosphatidylcholine in two different oxidation systems. Vesicles containing CH (∼3 mM) were incubated in PBS with (A) Fe/AsA (0.4 mM/4 mM) in the absence of EDTA, or with (B) 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH) (10 mM) in the presence of EDTA, at 37°C for 17 h. Vesicles are indicated as UL or ML vesicles composed of saturated dipalmitoyl phosphatidylcholine (UL/S and ML/S) or unsaturated soybean phosphatidylcholine (UL/U and ML/U). CH/phospholipid (PL) (=0.2 or 1.0) is the molar ratio of CH to PL in the vesicles. The % CH oxidized was obtained from triplicate determinations. Columns and bars represent means and standard deviations, respectively.View Large Image Figure ViewerDownload (PPT)TABLE 2Major oxysterols formed in vesiclesC-7 Oxidation ProductsC-5,6 Oxidation ProductsVesicleCH/PL Molar RatioOxidantCH Oxidized7αOH7βOH7KTotalα-EPOXβ-EPOXTRIOLTotalC-5,6/C-7 Products Ratio%%aPercentage is the value for amount of CH oxidized.%aPercentage is the value for amount of CH oxidized.UL/S1.0 AAPH42.7 ± 4.93.0 ± 0.52.2 ± 0.432.1 ± 2.337.3 ± 2.81.1 ± 0.30.6 ± 0.11.1 ± 0.22.8 ± 0.60.08UL/U1.0 AAPH49.0 ± 3.42.0 ± 0.24.0 ± 0.219.4 ± 1.225.3 ± 1.21.4 ± 01.9 ± 00.2 ± 0.23.5 ± 0.30.14ML/U0.2 AAPH32.9 ± 2.24.7 ± 0.27.6 ± 1.220.9 ± 0.733.2 ± 0.63.1 ± 0.22.4 ± 0.51.9 ± 0.57.3 ± 1.20.22ML/U0.2Fe/AsA38.9 ± 1.43.9 ± 0.35.4 ± 0.218.6 ± 0.228.0 ± 0.73.1 ± 0.21.5 ± 0.22.9 ± 0.37.5 ± 0.40.27AAPH, 2,2'-azobis (2-amidino-propane) dihydrochloride; 7α or 7βOH, 7α- or 7β-hydroxycholesterol; α- or β-EPOX, cholesterol 5α,6α- or 5β, 6β-epoxide; 7K, 7-ketocholesterol; TRIOL, cholestane-3β,5α,6β-triol. Experimental conditions the same as in the legend to Fig. 1. CH and oxysterols trimethylsilyl ether derivatives were quantified by GC with 5α-cholestane as the internal standard. Value determination based on triplicate results, expressed as average ± SD.a Percentage is the value for amount of CH oxidized. Open table in a new tab AAPH, 2,2'-azobis (2-amidino-propane) dihydrochloride; 7α or 7βOH, 7α- or 7β-hydroxycholesterol; α- or β-EPOX, cholesterol 5α,6α- or 5β, 6β-epoxide; 7K, 7-ketocholesterol; TRIOL, cholestane-3β,5α,6β-triol. Experimental conditions the same as in the legend to Fig. 1. CH and oxysterols trimethylsilyl ether derivatives were quantified by GC with 5α-cholestane as the internal standard. Value determination based on triplicate results, expressed as average ± SD. To elucidate the characteristics of CH oxidation in the system, the effects of the concentrations of CH and AAPH on the rate of CH oxidation were examined with LUV composed of CH and dimyristoyl phosphatidylcholine (DMPC) at the equivalent molar ratio (CH/DMPC=1/1) (Figs. 2, 3). CH was linearly oxidized with time until 6–12 h (Figs. 2A, 3A), and the rate of CH oxidation, R p, estimated from the straight line obtained within the time holding a linear correlation, was exactly dependent on the concentration of CH in the reaction mixtures (Fig. 2B) and on the square root of the rate of radical initiation, R i, (Fig. 3B), which was calculated from: [Ri=2ki[AAPH]e](Eq. 1) where [AAPH] is the concentration of initiator in the reaction mixtures, 2ki for AAPH is taken as 1.36 × 10−6 s−1 at 37°C (33Niki E. Free radical initiators as source of water- or lipid-soluble peroxyl radicals.in: Methods in Enzymol. 186. Academic Press, San Diego1990: 100-108Google Scholar), and the efficiency of free radical production, e, is taken as 0.64 from the average value measured 15 times by the induction period method (34Barclay L.R.C. Ingold K.U. Autoxidation of biological molecules. 2. The autoxidation of a model membrane. A comparison of the autoxidation of egg lecithin phosphatidylcholine in water and in chlorobenzene.J. Am. Chem. Soc. 1981; 103: 6478-6485Google Scholar) with a water-soluble antioxidant, Trolox (35Barclay L.R.C. Locke S.J. MacNeil J.M. VanKessel J. Burton G.W. Ingold K.U. Autoxidation of micelles and model membranes. Quantitative kinetic measurements can be made by using either water-soluble or lipid-soluble initiators with water-soluble or lipid-soluble chain-breaking antioxidants.J. Am. Chem. Soc. 1984; 106: 2479-2481Google Scholar).Fig. 2Effect of the concentration of CH on the rate of CH oxidation in large unilamellar vesicle (LUV) by AAPH. A: LUV composed of CH and dimyristoyl phosphatidylcholine at the equivalent molar ratio (CH/DMPC=1/1) were incubated at indicated concentrations (0.225 mM, 0.75 mM, and 2.25 mM CH) in PBS with 25 mM AAPH in the presence of EDTA at 37°C until 24 h. The amounts of oxidized CH (mM) in each LUV were determined by quantifying unoxidized CH with GC. B: The rates of CH oxidation, R p (M/s), were estimated from the straight lines obtained within the time holding a linear correlation, and are plotted against the concentration of CH in the reaction mixtures, [CH] (M).View Large Image Figure ViewerDownload (PPT)Fig. 3Effect of the concentration of AAPH on the rate of CH oxidation in LUV. A: LUV (CH/DMPC=1/1, molar ratio, 0.75 mM CH) were incubated in PBS with indicated concentrations of AAPH (5 mM, 25 mM, and 100 mM) in the presence of EDTA at 37°C until 24 h. B: The rates of CH oxidation, Rp (M/s), were estimated from the straight lines obtained within the time holding a linear correlation, and are plotted against the square root of the rate of radical initiation, Ri, which was calculated from the following equation: R i = 2ki[AAPH]e, as described in detail on the text.View Large Image Figure ViewerDownload (PPT)Fig. 3Effect of the concentration of AAPH on the rate of CH oxidation in LUV. A: LUV (CH/DMPC=1/1, molar ratio, 0.75 mM CH) were incubated in PBS with indicated concentrations of AAPH (5 mM, 25 mM, and 100 mM) in the presence of EDTA at 37°C until 24 h. B: The rates of CH oxidation, Rp (M/s), were estimated from the straight lines obtained within the time holding a linear correlation, and are plotted against the square root of the rate of radical initiation, Ri, which was calculated from the following equation: R i = 2ki[AAPH]e, as described in detail on the text.View Large Image Figure ViewerDownload (PPT) These results indicate that the CH oxidation in the system follows the classical rate law for autoxidation given by (36Howard J.A. Absolute rate constants for reactions of oxylradicals.Adv. Free Radical Chem. 1972; 4: 49-173Google Scholar): [Rp=kp/(2kt)1/2[LH]Ri1/2](Eq. 2) where R p is the rate of oxidation; R i, the rate of radical initiation; [LH], the concentration of substrate; and k p and 2k t are the rate constants for chain propagation and termination, respectively. The ratio of these rate constants, k p/(2k t)1/2, is referred to as the susceptibility of a substrate to oxidation; that is, oxidizability. Accordingly, the oxidizability of CH in bilayers can be experimentally determined from: [kp/(2kt)1/2=Rp/[LH]Ri1/2](Eq. 3) To confirm the suitability of the system, the oxidizability values were measured and compared in various LUVs composed of CH and dimyristoyl phosphatidylcholine (DMPC) or soybean phosphatidylcholine (SPC) at various constituent ratios of PLs to CH (Table 3). Almost the same values, 8.2 × 10−2-11.1 × 10−2 (Ms)−1/2, were obtained, which confirms that the system is appropriate for assessing the oxidizability of CH in PL bilayers without significant interference from oxidizable PLs.TABLE 3Effects of the saturation level and constituent ratio of PLs on the oxidizability of CH in bilayersLUVCH/DMPCCH/SPCConstituent molar ratio1/11/31/101/11/31/10Oxidizability (Ms)−1/2 × 10−29.28.78.29.49.411.1The oxidizability of CH was determined by measuring the rate of CH oxidation in each LUV with 25 mM AAPH. Open table in a new tab The oxidizability of CH was determined by measuring the rate of CH oxidation in each LUV with 25 mM AAPH. Using this system, the effects of various glyceroPLs and an antioxidant on the oxidizability of CH in bilayers were explored (Table 4). BBEP, BHCP, and EYPE significantly lowered the oxidizability of CH in bilayers, whereas lyso ethanolamine plasmalogen from bovine brain (LyEP) and Toc only slightly reduced the oxidizability of CH (Table 4). To compare the ability of BBEP, BHCP, and EYPE to lower the oxidizability of CH, the dose-dependent effects of each PL were examined in LUVs composed of CH (CH), soybean phospatidylcholine (SPC), and each test lipid (BBEP, EYPE, BHCP) at about the equivalent molar ratio of total PLs to CH. It was found that, among them, BBEP has the greatest ability to reduce the oxidizability of CH in bilayers (Fig. 4).TABLE 4The oxidizability values of CH in various LUVsLUVConstituent Molar RatioParticle SizeaExpressed as average ± SD based on particle size distribution, using a light scattering instrument.103 [CH]BufferCH/PLMolar Ratio10 9 R pcMeasured by quantifying unoxidized CH with GC during oxidation o
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