A. Arora et Gm. Strasburg, DEVELOPMENT AND VALIDATION OF FLUORESCENCE SPECTROSCOPIC ASSAYS TO EVALUATE ANTIOXIDANT EFFICACY - APPLICATION TO METAL CHELATORS, Journal of the American Oil Chemists' Society, 74(9), 1997, pp. 1031-1040
Two fluorescence-based assays were developed for rapid evaluation of c
ompounds for antioxidant activity. These assays were based on the quen
ching of intensity of the fluorescent probe and an increase in its flu
orescence anisotropy due to the free radicals generated during lipid p
eroxidation. A large unilamellar vesicle system, containing the fluore
scence 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 c
helators on metal-ion-induced peroxidation depended on the type of met
al used to initiate peroxidation and, for citrate, also on the concent
ration 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)-indu
ced peroxidations at lower chelator-to-metal ratios; however, at highe
r ratios, it inhibited both peroxidations. The results of the two fluo
rescence-based assays agreed well with the quantitation of conjugated
dienes and hydroperoxides by high-performance liquid chromatography. T
he combination of sensitivity, speed, and general utility associated w
ith these methods suggests that these methods will be useful in rapid
screening of extracts and purified compounds for antioxidant activity.