Development and validation of fluorescence spectroscopic assays to evaluate antioxidant efficacy. Application to metal chelators

Two fluorescence-based assays were developed for rapid evaluation of compounds for antioxidant activity. These assays were based on the quenching of intensity of the fluorescent probe and an increase in its fluorescence anisotropy due to the free radicals generated during lipid peroxidation. A large unilamellar vesicle system, containing the fluorescence probe diphenylhexatriene-propionic acid, was used to study the effects of chelators on metal-ion-induced lipid peroxidation. In this paper, the actions of the chelating agents ethylenediaminetetraacetic acid disodium salt (EDTA), nitrilotriacetic acid trisodium salt(NTA), adenosine-5'-diphosphate disodium salt (ADP), and sodium citrate on Fe(ll)- and Fe(lll)-induced peroxidation were compared. The effects of chelators on metal-ion-induced peroxidation depended on the type of metal used to initiate peroxidation and, for citrate, also on the concentration of chelator used. EDTA strongly suppressed both Fe(ll)- and Fe(lll)-induced peroxidation in this system. NTA and ADP inhibited Fe(lll)-induced peroxidation but enhanced Fe(ll)-induced peroxidation at all concentrations tested. Citrate promoted both Fe(ll)- and Fe(III)-induced peroxidations at lower chelator-to-metal ratios; however, at higher ratios, it inhibited both peroxidations. The results of the two fluorescence-based assays agreed well with the quantitation of conjugated dienes and hydroperoxides by high-performance liquid chromatography. The combination of sensitivity, speed, and general utility associated with these methods suggests that these methods will be useful in rapid screening of extracts and purified compounds for antioxidant activity.