The in vitro protective effect of alpha-tocopherol on oxidative injury in the dog retina.

Oxidative stress is a risk factor for eye diseases. Free radicals elicited during the inflammatory process often lead to oxidative damage of lipids (lipid peroxidation). The retina is highly vulnerable because of its high content of polyunsaturated fatty acids (PUFAs). The aim of this study was to investigate in vitro the effect of alpha-tocopherol on the Fe(2+)-ascorbate induced lipid peroxidation in the canine retina. Lipid peroxidation of retinal homogenates was carried out with and without the addition of alpha-tocopherol and monitored both by chemiluminescence and production of thiobarbituric acid reactive substances (TBARS). Total chemiluminescence counts per minute was lower in those homogenates pre-incubated with alpha-tocopherol. Thus, with 1 micromol alpha-tocopherol/mg of protein, 100% inhibition of chemiluminescence and a decrease of TBARS content from 20.46+/-0.85 to 2.62+/-2.77 nmol/mg protein were observed. Simultaneously, changes produced by oxidative stress were noted in the fatty acid composition of retinal lipids. Docosahexaenoic acid was decreased from 14.33+/-2.32% to 1.84+/-0.14% after peroxidation, but this fatty acid remained unaltered in the presence of 1 micromol alpha-tocopherol. These results show that under these experimental conditions, alpha-tocopherol may act as anti-oxidant protecting retinal membranes from deleterious effects. Further studies are required to assess its use in free radical generating conditions affecting the canine retina.

Selective inhibition of the non-enzymatic lipid peroxidation of phosphatidylserine in rod outer segments by alpha-tocopherol.

In the present study it was investigated if alpha-tocopherol shows protection against in vitro lipid peroxidation of phospholipids located in rod outer segment membranes (ROS). After incubation of ROS in an ascorbate-Fe2+ system, at 37 degrees C during 160 min, the total cpm originated from light emission (chemiluminescence) was found to be lower in those membranes incubated in the presence of alpha-tocopherol. The fatty acid composition of total lipids isolated from rod outer segment membranes was substantially modified when subjected to non-enzymatic lipid peroxidation with a considerable decrease of docosahexaenoic acid (22:6 n-3). The incorporation of alpha-tocopherol (0.35 micromol/mg protein) produce a 43.37% inhibition of the lipid peroxidation process evaluated as chemiluminescence (total cpm originated in 160 min). The phospholipid species containing the highest amount of docosahexaenoic acid: phosphatidylethanolamine and phosphatidylserine were more affected than phosphatidylcholine during the lipid peroxidation process. Not all phospholipids, however, were equally protected after the addition of alpha-tocopherol to the incubation medium. Phosphatidylcholine and phosphatidylethanolamine, were not protected by alpha-tocopherol, the vitamin provides selective antioxidant protection only for phosphatidylserine. These results indicate that alpha-tocopherol may act as antioxidant protecting rod outer segment membranes from deleterious effect by a selective mechanism that diminishes the loss of docosahexaenoic acid from phosphatidylserine.

The effect of alpha tocopherol, all-trans retinol and retinyl palmitate on the non enzymatic lipid peroxidation of rod outer segments.

The effect of a tocopherol, all-trans retinol and retinyl palmitate on the non enzymatic lipid peroxidation induced by ascorbate-Fe2+ of rod outer segment membranes isolated from bovine retina was examined. The inhibition of light emission (maximal induced chemiluminescence) by alpha tocopherol, all-trans retinol and retinyl palmitate was concentration dependent. All trans retinol showed a substantial degree of inhibition against ascorbate-Fe2+ induced lipid peroxidation in rod outer segment membranes that was 10 times higher than the observed in the presence of either at tocopherol or retinyl palmitate. Inhibition of lipid peroxidation of rod outer segment membranes by alpha tocopherol and retinyl palmitate was almost linear for up to 0,5 micromol vitamin/mg membrane protein, whereas all-trans retinol showed linearity up to 0,1 micromol vitamin/mg membrane protein. Incubation of rod outer segments with increasing amounts of low molecular weight cytosolic proteins carrying I-[14C] linoleic acid, [3H] retinyl palmitate or [3H] all-trans retinol during the lipid peroxidation process produced a net transfer of ligand from soluble protein to membranes. Linoleic acid was 4 times more effectively transferred to rod outer segment membranes than all-trans retinol or retinyl palmitate. Incubation of rod outer segments with delipidated low molecular weight cytosolic proteins produced inhibition of lipid peroxidation. The inhibitory effect was increased when the soluble retinal protein fraction containing alpha tocopherol was used. These data provide strong support for the role of all-trans retinol as the major retinal antioxidant and open the way for many fruitful studies on the interaction and precise roles of low molecular weight cytosolic retinal proteins involved in the binding of antioxidant hydrophobic compounds with rod outer segments.

Lipoperoxidation of rod outer segments of bovine retina is inhibited by soluble binding proteins for fatty acids.

In the present study it was investigated if soluble-binding proteins for fatty acids (FABPs) present in neural retina show protection from in vitro lipoperoxidation of rod outer segment membranes (ROS). After incubation of ROS in an ascorbate-Fe++ system, at 37 degrees C during 90-120 min, the total cpm originated from light emission (chemiluminescence) was found to be lower in those membranes incubated in the presence of soluble binding proteins for fatty acids. The fatty acid composition of rod outer segment membranes was substantially modified when subjected to non-enzymatic lipoperoxidation with a considerable decrease of docosahexaenoic acid (22:6 n-3) and arachidonic acid (20:4 n-6). As a result of this, the unsaturation index, a parameter based on the maximal rate of oxidation of specific fatty acids was higher in the native and control membranes when compared with peroxidized ones. A similar decrease of chemiluminescence was observed with the addition of increasing concentrations of native or delipidated FABP retinal containing fractions to rod outer segment membranes. These results indicate that soluble proteins with fatty acid binding properties may act as antioxidant protecting rod outer segment membranes from deleterious effect.