Tert-Butylhydroquinone as a Spectroscopic Probe for the Superoxide Radical Scavenging Activity Assay of Biological Samples

Abstract

As a more convenient and less costly alternative to electron spin resonance (ESR) and nonspecific nitroblue tetrazolium (NBT) and cytochrome c assays of superoxide radical (SR, O(2)(•-)) detection, a novel probe, tert-butylhydroquinone (TBHQ), is introduced for SR nonenzymatically generated in the phenazine methosulfate-β-nicotinamide adenine dinucleotide (PMS-NADH) system. SR attacks both TBHQ and SR scavengers incubated in solution for 30 min where scavengers compete with TBHQ for the O(2)(•-) produced. TBHQ, but not its O(2)(•-) oxidation product, tert-butyl-1,4-benzoquinone (TBBQ), is responsive to the CUPRAC (cupric reducing antioxidant capacity) spectrophotometric assay. The CUPRAC absorbance of the ethyl acetate extract of the incubation solution arising from the reduction of Cu(II)-neocuproine reagent by the remaining TBHQ was higher in the presence of O(2)(•-) scavengers (due to less conversion to TBBQ), the difference being correlated to the SR scavenging activity (SRSA) of the analytes. With the use of this reaction, a kinetic approach was adopted to assess the SRSA of amino acids, vitamins, and plasma and thiol antioxidants. This assay, applicable to small-molecule antioxidants and tissue homogenates, proved to be efficient for cysteine, uric acid, and bilirubin, for which the widely used NBT test is nonresponsive. Thus, conventional problems of NBT assay arising from formazan insolubility and direct reduction of NBT by tested scavengers were overcome.