title: Oxidative degradation of cholesteryl esters in low-density lipoproteins: analysis by liquid chromatography-light scattering and protection by a new synthetic antioxidant, S20478.
abstract: Cholesteryl esters in the hydrophobic core of low-density lipoprotein (LDL) particles constitute a major molecular target during copper-mediated oxidation. To facilitate the rapid analysis and quantitation of the oxidative degradation of LDL cholesteryl esters, we describe a new approach based on light scattering detection following separation by HPLC. We have applied this approach to the evaluation of the protective capacity of a new synthetic antioxidant, S20478, during oxidation of LDL in the presence of copper ions. HPLC separation of cholesterol and the four major molecular species of cholesteryl esters (C16:0, C18:1, C18:2 and C20:4) of LDL was achieved in a single run of 20 min with high sensitivity (50 ng) and low background. Time course studies of the oxidative modification of LDL (ratio LDL protein: copper, 100 micrograms/mL: 1 microM) revealed that the content of unsaturated cholesteryl esters (C20:4 and C18:2) decreased (-30% and -15%, respectively) within 90 min of copper-mediated oxidation, while only minor degradation (up to 15%) of monounsaturated (C18:1) and saturated (C16:0) esters occurred. At 24 hours of oxidation, only traces (< 5%) of the C20:4 and C18:2 esters were detectable; whereas 52% of the C18:1 ester remained (P < 0.01). Of the saturated esters, only minor proportions (35% or less) underwent oxidative modification. In addition, some 81% of free cholesterol was conserved as the native sterol. The synthetic antioxidant, S20478 (50 microM) was capable of inhibiting the initiation and the propagation of copper-mediated LDL oxidation as determined by the time- and dose-dependent inhibition of the formation of conjugated dienes and thiobarbituric acid-reactive substances, as well as the conservation of the net electrical charge of LDL; indeed S20478 conserved cholesteryl esters in their native form up to 24 hours. However, after prolonged exposure to copper ions (48 hours), only 47% of the unsaturated esters remained (C18:2, P < 0.05). Nonetheless, S20478 (10 microM) was more efficient in inhibiting copper-mediated LDL oxidation as compared to probucol at the same concentration. These findings suggest that S20478 may be of potential interest in a new antioxidant approach to therapeutic stabilisation and regression of atherosclerotic plaques. Moreover, this method should prove useful in the assessment of the integrity of native LDL, and provides a new chemical marker of the degree of LDL oxidation.
authors.affiliation: Institut National de la Santé et de la Recherche (Inserm), Unité de Recherches sur les Lipoprotéines et l'Athérogénèse, U 321, Pavillon Benjamin Delessert, Hôpital de la Pitié, Paris, France.
authors.forename: M
authors.initials: M
authors.lastname: Arborati
authors.forename: D
authors.initials: D
authors.lastname: Benchorba
authors.forename: I
authors.initials: I
authors.lastname: Lesieur
authors.forename: J G
authors.initials: JG
authors.lastname: Bizot-Espiard
authors.forename: B
authors.initials: B
authors.lastname: Guardiola-Lemaitre
authors.forename: M J
authors.initials: MJ
authors.lastname: Chapman
authors.forename: E
authors.initials: E
authors.lastname: Ninio
mesh_terms: D000975:Antioxidants / Q000494:pharmacology*; D001583:Benzoxazoles / Q000494:pharmacology*; D002784:Cholesterol / Q000032:analysis* / Q000097:blood; D002788:Cholesterol Esters / Q000032:analysis / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D008027:Light; D016014:Linear Models; D008077:Lipoproteins, LDL / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D015203:Reproducibility of Results; D012542:Scattering, Radiation
chemical_list: D000975:Antioxidants; D001583:Benzoxazoles; D002788:Cholesterol Esters; D008077:Lipoproteins, LDL; C105958:S 20478; D002784:Cholesterol