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A. Berra, A. Terrasa, M. Guajardo, G. Zapata; Oxidative Damage of Lipids in Equine Rod Outer Segments (ROS) . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2085.
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The aim of this study was to evaluate: 1) in vitro non–enzymatic lipid peroxidation in equine ROS membranes; 2) the fatty acid composition of total lipids isolated from ROS membranes, and their alterations during the oxidative damage; 3) α–tocopherol (α–toc) protecting action.
equine ROS membranes were obtained from retinal homogenates by sucrose–gradient differential centrifugation. Non–enzymatic lipid peroxidation, induced by Fe2+–ascorbate prooxidizing system, was monitored using chemiluminescence (CL). ROS membranes (0.5 mg protein) were incubated at 37°C in 0.05 M PBS, pH 7.4 with enough iron to start peroxidation (2.15 mM) with or without 0.4 mM ascorbate. Light emission (cpm) was measured every 10 min for 180 min. Similar experiments were performed using ROS membranes treated with α–toc (0.5 and 1 µmol/mg protein). The fatty acid composition of lipids isolated from native ROS membranes with or without oxidative damage were analyzed by gas–liquid chromatography.
CL values were low in controls without ascorbate during the entire experiment. However, in the presence of ascorbate, CL increased, reaching its maximum peak at 221,515 ± 10,719 cpm at 80 min, then, it declined to its initial values after 180 min. When peroxidation was carried out on α–toc–treated membranes (0.5 µmol/mg protein), CL values were decreased (maximum peak at 99,104 ± 5,500 cpm at 60 min). Total CL (sum of records done every 10 min for 180 min) was used to obtain peroxidation percent inhibition. CL inhibition of 80.55 ± 2.76% and 85.55 ± 0.64% was observed in α–toc–treated membranes (0.5 and 1 µmol/mg protein, respectively). The fatty acid composition of total lipids isolated from equine ROS membranes showed a high percentage of PUFAs; docosahexaenoic acid (22:6n–3) was the most abundant (20.65 ± 3.20% total fatty acids). The 94.68 ± 1.24% of PUFAs have disappeared after 3 h peroxidation. When the same oxidative damage was performed on α–toc–treated ROS membranes, we observed a protection in the content of PUFAs of 46.89 ± 2.49 and 61.37 ± 2.95% with 0.5 and 1 µmol/mg protein respectively.
Equine ROS membranes were highly sensitive to oxidative damage since their fatty acid composition was markedly modified during lipid peroxidation process, showing a substantial loss of PUFAs. The protecting role of α–tocopherol as antioxidant was remarkable in that it could be utilized to treat equine ocular diseases in which free radicals are involved
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