Superoxide generated by pyrogallol reduces highly water-soluble tetrazolium salt to produce a soluble formazan: a simple assay for measuring superoxide anion radical scavenging activities of biological and abiological samples

Abstract

Superoxide anion radical (O2(˙-)) plays an important role in several human diseases. The xanthine/xanthine oxidase system is frequently utilized to produce O2(˙-). However, false positive results are easily got by using this system. The common spectrophotometric probes for O2(˙-) are nitroblue tetrazolium (NBT) and cytochrome c. Nevertheless, the application of NBT method is limited because of the water-insolubility of NBT formazan and the assay using cytochrome c lacks sensitivity and is not suitable for microplate measurement. We overcome these problems by using 1,2,3-trihydroxybenzene (pyrogallol) as O2(˙-)-generating system and a highly water-soluble tetrazolium salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt (WST-1) which can be reduced by superoxide anion radical to a stable water-soluble formazan with a high absorbance at 450 nm. The method is simple, rapid and sensitive. Moreover, it can be adapted to microplate format. In this study, the O2(˙-) scavenging activities of superoxide dismutase (SOD), L-ascorbic acid, N-acetyl-L-cysteine (NAC), albumin from human serum, flavonoids and herbal extracts were assessed by using this method. Meanwhile, the activities of tissue homogenates and serum were determined by using this validated method. This method, applicable to tissue homogenates, serum and herbal extracts, proved to be efficient for measuring O2(˙-) scavenging activities of biological and abiological samples.

Keywords: (1)O(2); 1,2,3-trihydroxybenzene; 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt; 50% scavenging concentration; CL; EDTA; ESR; H(2)O(2); HO; IC(50); N-acetyl-l-cysteine; NAC; NBT; NH(4)HCO(3); O(2)(−); O(2)(−) scavenging activity; Pyrogallol; ROS; RSD; SOD; Superoxide anion radical; WST-1; ammonium hydrogen carbonate; chemiluminescence; electron spin resonance; ethylene diamine tetraacetic acid; hydrogen peroxide; hydroxyl radicals; m/z; mass to charge ratio; nitroblue tetrazolium; pyrogallol; reactive oxygen species; relative standard deviation; singlet oxygen; superoxide anion radical; superoxide dismutase.