OXIDATION OF ASCORBIC-ACID AS AN INDICATOR OF PHOTOOXIDATIVE STRESS IN THE EYE

When whole retinal pigmented epithelium (RPE) cells isolated from bovine eyes are incubated with C-14-labeled ascorbic acid and exposed to a visible laser, the ascorbic acid is oxidized to dehydro-L-ascorbic acid (DHA). The amount of ascorbic acid which is oxidized is proportional to the radiant exposure of the sample (i.e. the total amount of radiation per unit area delivered over the exposure time). Blue light is more effective than red light in driving the reaction. The amount of label appearing in the DHA fraction is increased if unlabeled DHA is present in the reaction mixture, indicating that some redox cycling of ascorbate is occurring in the RPE cells. The ascorbic acid oxidizing activity does not depend on intact cells, is not inactivated by heating the cells to 80-degrees-C, and appears to reside mainly in the subcellular fraction which contains melanin pigment granules. The ascorbic acid oxidation may be caused by free radicals formed when melanin is illuminated with light. This reaction appears to be a useful method for quantifying the production of free radicals during photooxidative stress.