Novel Colorimetric Assay of 2,3-Dihydroxybenzoate among Other Isomers as a Selective Indicator of Hydroxyl Radical Damage and Related Antioxidant Activity

Abstract

Although reactive oxygen species can regulate intracellular signaling pathways, excessive amounts under oxidative stress conditions may cause damage to biomolecules. Hydroxyl radical ((OH)-O-center dot) is a most reactive oxidant that can harm DNA, lipids, and proteins. As the direct determination of (OH)-O-center dot by highly specialized electron paramagnetic resonance techniques is costly, indirect colorimetric determinations have attracted attention. Salicylic acid has been used both as an in vitro and in vivo probes to detect (OH)-O-center dot, itself being converted to 2,3-,2,4-, and 2,5-dihydroxybenzoic acids (DHBA) and catechol, but since 2,5-DHBA may also be generated enzymatically in the cyctochrome P-450 metabolism, 2,3-DHBA is the real marker of oxidative salicylate damage. This work is focused on the development of a selective hydroxyl radical detection assay by modifying a colorimetric nitrite-molybdate method concerning vic-diol determination of 2,3-DHBA among other DHBA isomers. Salicylic acid was hydroxylated to DHBAs and catechol upon the attack of (OH)-O-center dot produced in a Fenton system. An aliquot from the mixture was oxidized with a nitrite-molybdate(VI) reagent to give an intense red product in alkaline medium with maximal absorbance at 510 nm. The assay was selective to only 2,3-DHBA and catechol among all DHBA isomers. The residual salicylic acid, DHBAs, and catechol were measured using high-performance liquid chromatography (HPLC); the spectrophotometrically measured contents of 2,3-DHBA and catechol were compared with HPLC results. Antioxidants, when present, caused a reduction in the hydroxylation of salicylate probe producing less 2,3-DHBA and catechol, thereby enabling the development of an indirect antioxidant activity assay for colorimetrically measuring (OH)-O-center dot scavenging ability.