Photosensitized oxidation reactions damage tissue by catalyzing the formation of oxyradicals and singlet oxygen. β-Carotene is hypothesized to exert photoprotective effects by quenching singlet oxygen formed by Type II reactions and by scavenging free radicals formed by Type I reactions. β- Carotene antioxidant mechanisms were studied in a phospholipid membrane model of photooxidation with a new isotope dilution gas chromatography-mass spectrometry (GC-MS) assay that quantitatively distinguishes singlet oxygen- mediated and radical-mediated lipid peroxidation. This assay measures 9- and 10-hydroxylinoleate methyl esters and was used to generate photooxidation profiles for the photosensitizers methylene blue, Rose Bengal, and tetraphenylporphine. These profiles indicate a shift from Type II to Type I photooxidation mechanisms in later stages of photooxidation. β-Carotene (0.45 mol %) inhibited singlet oxygen-mediated lipid peroxidation at early stages of methylene blue-sensitized photooxidation. Production of radical- mediated products increased faster than singlet oxygen-mediated products at later stages. β-Carotene-5,8-endoperoxide, a specific marker for singlet oxygen oxidation of β-carotene in solution, was unstable under the incubation conditions and was not detected in this system. α-Tocopherol (0.45 mol %) was ineffective in inhibiting photosensitized lipid peroxidation, whereas 4.5 mol % α-tocopherol inhibited almost all radical- mediated lipid peroxidation as well as early-stage singlet oxygen-mediated lipid peroxidation. Cumene hydroperoxide stimulated radical-mediated lipid peroxidation, indicating that accumulation of hydroperoxides from Type II photooxidation may enhance Type I reactions. These data suggest that singlet oxygen quenching, rather than radical scavenging reactions, accounts for the photoprotective actions of β-carotene.
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