title: Cholesterol as a natural probe for free radical-mediated lipid peroxidation in biological membranes and lipoproteins.
abstract: We describe a relatively convenient and reliable procedure for assessing the magnitude of free radical-mediated (chain) lipid peroxidation in biological systems. The approach is based on use of radiolabeled cholesterol ([(14)C]Ch) as a probe and determination of well-resolved oxidation intermediates/products ([(14)C]ChOX species), using high performance thin layer chromatography with phorphorimaging detection (HPTLC-PI). In a lipid hydroperoxide-primed liposomal test system treated with ascorbate and a lipophilic iron chelate, the following well-resolved [(14)C]ChOX are detected and quantified: 7α/7β-OOH, 7α/7β-OH, and 5,6-epoxide, their levels increasing with incubation time at 37°C. [(14)C]Ch also serves as an excellent probe for lipid peroxidation in lipoproteins and plasma membranes of mammalian cells. Because this approach utilizes Ch as a natural in situ probe, it eliminates potential artifacts associated with artificial probes such as spin traps and fluorophores.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, USA. Electronic address: agirotti@mcw.edu.
authors.forename: Albert W
authors.initials: AW
authors.lastname: Girotti
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, USA; Department of Biophysics, Jagiellonian University, Krakow, Poland.
authors.forename: Witold
authors.initials: W
authors.lastname: Korytowski
mesh_terms: D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D002784:Cholesterol / Q000737:chemistry* / Q000378:metabolism; D002855:Chromatography, Thin Layer / Q000379:methods*; D005609:Free Radicals / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D015227:Lipid Peroxidation; D008074:Lipoproteins / Q000737:chemistry* / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D010777:Photochemistry / Q000379:methods*
chemical_list: D005609:Free Radicals; D008074:Lipoproteins; D008081:Liposomes; D002784:Cholesterol
keywords: Cholesterol; Cholesterol oxides; Free radicals; Lipid peroxidation; Phosphorimaging; Thin layer chromatography