RESUMO
PURPOSE: In addition to acting as an optical filter, macular (carotenoid) pigment has been hypothesized to function as an antioxidant in the human retina by inhibiting the peroxidation of long-chain polyunsaturated fatty acids. However, at its location of highest density in the inner (prereceptoral) layers of the foveal retina, a specific requirement for antioxidant protection would not be predicted. The purpose of this study was to determine whether lutein and zeaxanthin, the major carotenoids comprising the macular pigment, are present in rod outer segment (ROS) membranes where the concentration of long-chain polyunsaturated fatty acids, and susceptibility to oxidation, is highest. METHODS: Retinas from human donor eyes were dissected to obtain two regions: an annular ring of 1.5- to 4-mm eccentricity representing the area centralis excluding the fovea (perifoveal retina) and the remaining retina outside this region (peripheral retina). ROS and residual (ROS-depleted) retinal membranes were isolated from these regions by differential centrifugation and their purity checked by polyacrylamide gel electrophoresis and fatty acid analysis. Lutein and zeaxanthin were analyzed by high-performance liquid chromatography and their concentrations expressed relative to membrane protein. Preparation of membranes and analysis of carotenoids were performed in parallel on bovine retinas for comparison to a nonprimate species. Carotenoid concentrations were also determined for retinal pigment epithelium harvested from human eyes. RESULTS: ROS membranes prepared from perifoveal and peripheral regions of human retina were found to be of high purity as indicated by the presence of a dense opsin band on protein gels. Fatty acid analysis of human ROS membranes showed a characteristic enrichment of docosahexaenoic acid relative to residual membranes. Membranes prepared from bovine retinas had protein profiles and fatty acid composition similar to those from human retinas. Carotenoid analysis showed that lutein and zeaxanthin were present in ROS and residual human retinal membranes. The combined concentration of lutein plus zeaxanthin was 70% higher in human ROS than in residual membranes. Lutein plus zeaxanthin in human ROS membranes was 2.7 times more concentrated in the perifoveal than the peripheral retinal region. Lutein and zeaxanthin were consistently detected in human retinal pigment epithelium at relatively low concentrations. CONCLUSIONS: The presence of lutein and zeaxanthin in human ROS membranes raises the possibility that they function as antioxidants in this cell compartment. The finding of a higher concentration of these carotenoids in ROS of the perifoveal retina lends support to their proposed protective role in age-related macular degeneration.
