Kinetics of photoperoxidation of arachidonic acid: Molecular mechanisms and effects of antioxidants

The kinetics of photoperoxidation of 1⁻¹⁴C]arachidonic acid (20∶4n−6) at 1.32 mM was studied either with the unsaturated fatty acid alone or in the presence of 10μM of antioxidants and/or inhibitors of eicosanoid metabolism. The photosensitizer used wasmeso-tetraphenylporphine. The time-course of the reactions was followed by ultraviolet spectral analysis, thiobarbituric acid reactivity and high-performance liquid chromatographic analysis of aliquots sampled every 15 min during the 4 h of irradiation. The kinetics of photoperoxidation of 20∶4n−6 can be divided into three main successive steps: (i) monohydroperoxidation, characterized by the appearance of conjugated diene patterns and monohydroperoxidized 20∶4n−6; (ii) secondary oxidation characterized by polyoxygenated products such as dihydroperoxidized 20∶4n−6 possessing conjugated triene patterns; and (iii) the disappearance of conjugated patterns and the oxidative cleavage of the products of the two first steps into aldehydic molecules reacting with thiobarbituric acid. During the first 90 min of irradiation, the mechanism of monohydroperoxidation (step one) is purely or predominantly type II photoperoxidation involving only singlet oxygen. This step was inhibited by β-carotene and by BW755C (3-amino-1-3-trifluoromethylphenyl]2-pyrazoline). In contrast, the reactions involved in the second and third steps were predominantly type I photoperoxidation involving radical mechanisms. These latter steps were inhibited by β-carotene, BW755C, vitamin E and probucol. Indomethacin and 5,8,11,14-eicosatetraynoic acid did not alter 20∶4n−6 photoperoxidation. Thisin vitro model of lipid photoperoxidation allows the screening of antioxidants in accordance with their singlet oxygen quenching and/or free radical scavenging properties.