Kinetic analysis of the free-radical-induced lipid peroxidation in human erythrocyte membranes: Evaluation of potential antioxidants using cis-parinaric acid to monitor peroxidation

cis - Parinaric acid (PnA), cis - trans - trans - cis-9, 11, 13, 15-octadecatetraenoic acid, is fluorescent (ϵ = 74,000 at 324 nm) when partitioned into a lipid environment and the fluorescence is destroyed upon reaction with free radiacals. It has been used to monitor semiquantitatively free-radical-induced lipid peroxidation in human erythrocyte membranes. We have applied this assay to the quantitative evaluation of potential antioxidants. The kinetics of the reaction of PnA with free radicals were measured in erythrocyte ghosts. After initiation of free radical generation by cumene hydroperoxide and cupric ion, a steady-state rate of fluorescence decay is rapidly established. In the steady state the oxidation of PnA and, hence, the loss of fluorescence is a first-order process. In the presence of antioxidants, such as vitamin E, the rate constant of fluorescence loss decreases, thereby indicating that the antioxidant decreases the steady-state concentration of free radicals. By adding various concentrations of potential antioxidants, pseudo-first-order rate constants [k1] which measure the reactivity of antioxidants with free radicals were determined. Results show that, when incorporated into erythrocyte membranes, U-78,517f, a vitamin E analog, is a potent free radical scavenger, being approximately 50% as effective as vitamin E and 10–15 times more potent than the aminosteroids evaluated (see Table 1).