A NEW METHOD FOR ULTRASTRUCTURAL-LOCALIZA TION OF LIPID PEROXIDES IN THE EYE

Lipid peroxidation is considered a prominent feature of age-related re tinal degeneration. It is known that lipid peroxides can oxidize benzi dine. This property was used to localize lipid peroxides ultrastructur ally in the retina. Methods: (1) Lipid peroxides were formed by incuba tion of linoleic acid with lipoxygenase from soybean, separated by thi n layer chromatography and incubated with tetramethylbenzidine. (2) Li pid peroxides were formed by incubation of porcine retinae with soybea n lipoxygenase in an oxygen-saturated atmosphere. For ultrastructural localization, isolated retinae with and without enzymatically synthesi zed lipid peroxides were fixed with 2 % glutaraldehyde, incubated with 0.5 mg/ml tetramethylbenzidine and embedded for electron microscopy. (3) Eye cups from Syrian golden hamsters were treated in the same way except for incubation with lipoxygenase. The hamsters were kept under constant illumination (1000 lux) for 12 h to enhance lipid peroxidatio n. Results: (1) Tetramethylbenzidine was oxidized by linoleic acid per oxides. (2) In the isolated retinae of pigs lipid peroxides became vis ible as electron-dense structures in the rod outer segments (ROS) afte r treatment with lipoxygenase and were lacking in the other parts of t he retina. Without treatment with lipoxygenase lipid peroxides were on ly infrequently seen in ROS. (3) In the eyes of light-exposed hamsters , electron-dense reaction products of lipid peroxides were particularl y prominent between the basal infoldings of the RPE and within the api cal parts of the ROS. Conclusion: light or enzymatically induced lipid peroxides can be localized ultrastructurally due to their ability to react with tetramethylbenzidine and osmium in the absence of H2O2 to a n electron-dense reaction product. Lipid peroxides seem to be removed from the RPE via Bruch's membrane and blood vessels. Disturbance of th is pathway may enhance lipofuscin or drusen formation.