Phthalocyanine-sensitized lipid peroxidation in cell membranes: use of cholesterol and azide as probes of primary photochemistry.

Various phthalacyanine (Pc) derivatives of phototherapeutic interest have been shown to be efficient type II (singlet oxygen, 1O2) sensitizers in aqueous and non-aqueous solutions. However, primary Pc photochemistry in biological environments, e.g. cell membranes, has not been studied in a definitive manner. To address this question, we used endogenous cholesterol in the erythrocyte ghost as a mechanistic reporter lipid Membranes sensitized with chloroaluminum Pc tetrasulfonate (AlPcS) and exposed to white light at 10 degrees C underwent lipid peroxidation, as indicated by the accumulation of hydroperoxides and thiobarbituric acid reactivity. Specific analysis of cholesterol photo-products by thin layer chromatography and high performance liquid chromatography revealed the presence of 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), with much smaller amounts of 3 beta-hydroxycholest-5-ene-7 alpha-hydroperoxide (7 alpha-OOH) and 5 alpha-cholest-6-en-3 beta, 5-diol and cholest-5-en-3 beta, 7 alpha-diol (5 alpha-OH and 7 alpha-OH). Identification of 5 alpha-OOH as a major photoproduct provides unambiguous evidence for large scale 1O2 intermediacy. Azide inhibited lipid peroxidation in a dose-dependent fashion, providing additional support for a type II mechanism. However, the 1O2 quenching constant from Stern-Volmer analysis was approximately 50 times lower than that determined for a non-membrane probe, lactate dehydrogenase. The latter value agreed with literature values. A probable explanation is that membrane-bound dye generates most of the 1O2 involved in lipid peroxidation. Although azide can intercept any 1O2 escaping into (or formed in) the medium, it has limited access to 1O2 generated on the membrane and reacting (or being quenched) near its site of origin.