Monitoring by cis-parinaric fluorescence of free radical induced lipid peroxidation in aqueous liposome suspensions

Abstract

Cis-parinaric acid is fluorescent when partioned into a lipid environment and its fluorescence is destroyed upon reaction with free radicals. In our study 1-palmitoyl-2-parinoyl-phosphatidylcholine (cis-PnA) has been used to monitor the time-course of liposomal lipid peroxidation, using reverse-phase evaporation vesicles (REV) of different composition exposed to oxidative stress in various conditions. This methodology allowed us to estimate the potential damage produced by two different oxidizing systems, namely hydrogen peroxide (H2O2), a water soluble oxidant, and t-butyl hydroperoxide (t-BHP), a hydrophobic hydroperoxide. Furthermore, we evaluated the protective effects of bilayer-associated antioxidants, namely alpha-tocopherol acetate (alpha-THA), vitamin K1 and beta-carotene, as well as of two antioxidants dissolved in the aqueous bulk solution, that is, biverdin and uric acid. Under our experimental conditions, the results suggest that (i) both oxidizing compounds were able to interact with liposomal PnA leading to decay either of the excitation and of emission spectra of the probe; (ii) hydrogen peroxide seemed to be of most effective among the two stressing agents, when employed at similar concentrations; (iii) the alpha-THA appeared to be a stronger antioxidant than vitamin K1 and beta-carotene, resulting in a decrease of the liposomal membrane stress caused by those two oxidizing agents; (iv) among the water soluble antioxidant compounds, biliverdin displayed a protective effect at least 10 x higher than uric acid; (v) the overall damage, as well as the protection mechanisms, seemed to be dependent either on the lipid composition of the vesicles and on the pH of the liposomal suspension. This relatively easy experimental approach suggests the validity of the use of the bilayer associated fluorescent probe PnA in the monitoring of spontaneous and/or chemically induced liposomal lipid damage.