MONITORING BY CIS-PARINARIC FLUORESCENCE OF FREE-RADICAL INDUCED LIPID-PEROXIDATION IN AQUEOUS LIPOSOME SUSPENSIONS

Cis-parinaric acid is fluorescent when partioned into a lipid environm ent and its fluorescence is destroyed upon reaction with free radicals . In our study 1-palmitoyl-2-parinoyl-phosphatidylcholine (cis-PnA) ha s been used to monitor the time-course of liposomal lipid peroxidation , using reverse-phase evaporation vesicles (REV) of different composit ion exposed to oxidative stress in various conditions. This methodolog y allowed us to estimate the potential damage produced by two differen t oxidizing systems, namely hydrogen peroxide (H2O2), a water soluble oxidant, and t-butyl hydroperoxide (t-BHP), a hydrophobic hydroperoxid e. Furthermore, we evaluated the protective effects of bilayer-associa ted 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 exp erimental conditions, the results suggest that (i) both oxidizing comp ounds 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 agent s, when employed at similar concentrations; (iii) the alpha-THA appear ed to be a stronger antioxidant than vitamin K1 and beta-carotene, res ulting in a decrease of the liposomal membrane stress caused by those two oxidizing agents; (iv) among the water soluble antioxidant compoun ds, biliverdin displayed a protective effect at least 10x higher than uric acid; (v) the overall damage, as well as the protection mechanism s, seemed to be dependent either on the lipid composition of the vesic les and on the pH of the liposomal suspension. This relatively easy ex perimental approach suggests the validity of the use of the bilayer as sociated fluorescent probe PnA in the monitoring of spontaneous and/or chemically induced liposomal lipid damage.