Inhibition of free radical generation by biotin

To assess the inhibitory effect of biotin on free radical generation, we used a spectrophotometric assay of cytochrome c reduction and determined the 2-methyl-6-phenyl-3,7-dihydroimidazo[1,3-a]-pyrazin-3-one (CLA)-dependent chemiluminescence response of human neutrophils or a hypoxanthine-xanthine oxidase (XOD) system. In the spectrophotometric assay of cytochrome c reduction, superoxide (O2-) generation by neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (f-MLP) was reduced significantly when biotin was added. In the CLA-dependent chemiluminescence test of neutrophils stimulated by f-MLP, biotin significantly reduced the generation of free radical species, including O2-, in a concentration-dependent manner, the concentration corresponding to 50% inhibition (IC50) of biotin for free radical generation was 1.12 x 10(-7) mol. However, biotin did not exert an inhibitory effect on oxidative metabolism by directly scavenging superoxide anion, as shown by the study using the hypoxanthine-XOD system.