A Fluorometric Method for Measurement of Peroxyl Radical Scavenging Activities of Lipophilic Antioxidants

A fluorometric method for evaluation of activities of lipophilic antioxidants toward peroxyl radicals in a lipophilic medium (octane:butyronitrile; 9:1, v/v) and dioleoylphosphatidyl choline (DOPC) liposomal suspension in Tris-HCl buffer (pH 7.4 at 40 degrees C) has been developed. This fluorometric method is based on the use of the fatty acid 4,4-difluoro-5-(4-phenyl-1, 3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undec anoic acid (BODIPY 581/591 C11) as an indicator, 2,2'-azobis-2,4-dimethyl valeronitrile (AMVN) as a peroxyl radical generator, and 6-hydroxy-2, 5,7,8-tetramethyl chroman-2-carboxylic acid (Trolox) as a calibrator. In the absence of antioxidants, a linear correlation was found between the fluorescence decay of BODIPY 581/591 C11 and the concentration of AMVN. Trolox was used as a standard reference material for validating this newly developed method. A linear correlation was found between the net protection of the fluorescence of BODIPY 581/591 C11 and the amounts of added Trolox. Employing this method, the relative peroxyl radical scavenging activities of Trolox, alpha-tocopherol, alpha-carotene, beta-carotene, lutein, and probucol in octane:butyronitrile (9:1, v/v) were determined to be 1. 0:0.7:0.5:0.3:0.4:0.3. In the DOPC system, beta-carotene showed an antioxidant activity which is a factor of two greater than that of alpha-tocopherol. This assay is suitable for measurement of activities of antioxidants in an organic lipid environment and a liposomal medium.