Photogeneration of 3beta-hydroxy-5alpha-cholest-6-ene-5-hydroperoxide in rat skin: evidence for occurrence of singlet oxygen in vivo.

We identified singlet oxygen adduct of cholesterol, 3beta-hydroxy-5alpha-cholest-6-ene-5-hydroperoxide (5alpha-OOH), in skin of rats pretreated with oral doses of pheophorbide a and subsequent visible irradiation, that have been known to induce photosensitive diseases in animals and humans. In a living animal body, this is the first demonstration of presence of 5alpha-OOH, that is known to be formed exclusively by reaction in vitro between singlet oxygen and cholesterol. By the quantitative determination with high performance liquid chromatography equipped with a chemiluminescence detector, we observed time-dependent increase in concentrations of 5alpha-OOH in skin of rats pretreated with oral doses of pheophorbide a and subsequent visible irradiation, suggesting the occurrence of a labile activated oxygen species, singlet oxygen, in this system.