Studies of the Photooxidation of A2E and Its Precursor A2–PE

Abstract

Abstract: : Purpose:The lipofuscin fluorophore A2E forms by phosphate hydrolysis of the precursor A2–PE and can undergo further transformation through the light–induced oxidation of its double bonds; singlet oxygen has been implicated in this process. Here we have examined for A2–PE photoxidation, tested the ability of the carotenoids lutein and zeaxanthin to protect against the photoxidation of A2–PE and A2E and we have examined for the involvement of additional oxidizing species Methods:Oxidation of A2E and A2–PE was induced by 430 nm irradiation and by exposure to a singlet oxygen generator (endoperoxide of 1,4 dimethylnaphthalene) in the presence and absence of lutein, zeaxanthin and/or vitamin E. A2E and A2–PE were quantified by HPLC and analyzed by FAB–MS. A2E was also irradiated in the presence of the fluorescent probe HEt. Results: Blue light irradiation of A2E and A2–PE resulted in the generation of additional molecular ion peaks indicative of the addition of oxygens to the molecules. Lutein and zeaxathin inhibited the oxidation of A2E and A2–PE under conditions of both 430 nm irradiation and when the fluorophores were incubated with the singlet oxygen generator. Both lutein and zeaxanthin reduced the oxidation of A2E and A2–PE to an extent greater than vitamin E. HEt fluorescence increased upon blue light irradiation of A2E. Conclusions:The appearance of higher mass peaks upon irradiation of A2–PE reveals that this fluorophore of photoreceptor outer segments can undergo photoxidation as does A2E. Lutein and zeaxanthin may play role in inhibiting these photoxidative events. The increase in HEt fluorescence upon blue light irradiation of A2E was consistent with the generation of reactive oxygen species such as superoxide anion.

Keywords: retinal pigment epithelium • aging