Resorcinol as a Spectrofluorometric Probe for the Hypochlorous Acid Scavenging Activity Assay of Biological Samples

Abstract

A novel spectrofluorometric method was developed and validated for hypochlorous acid (Hod) scavenging activity estimation using resorcinol, which is a highly sensitive and chemically stable fluorogenic probe. This assay is based on the chlorination of resorcinol to its nonfluorescent products in the presence of HOCl. HOCl reacts with both resorcinol and HOCl scavengers incubated in solution for 10 min, where scavengers compete with resorcinol for the HOCl. Thus, the relative increase in fluorescence intensity of intact resorcinol is proportional to the antioxidative activity of HOCl scavengers. Using this reaction, a kinetic approach was adopted to assess the HOCl scavenging activity of amino acids, vitamins, and plasma and thiol antioxidants. This assay, which is applicable to small molecule antioxidants and tissue homogenates, proved to be efficient for thiol-type antioxidants for which the widely used 5-thio-2-nitrobenzoic acid (TNB) test is not accurately responsive. Thus, conventional problems of the TNB assay arising from the reactivity of thiol-type scavengers to produce extra TNB by direct reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were overcome. Moreover, unlike enzymatic assays (e g, elastase), there is no confusion as to whether the putative scavenger actually reacts with HOCl or inhibits the enzyme.