Novel spectroscopic sensor for the hydroxyl radical scavenging activity measurement of biological samples

Abstract

A novel spectroscopic sensor was developed and validated for hydroxyl radical scavenging (HRS) activity estimation using terephthalate (TP) as probe. This sensor was designed by electrostatic immobilization of the chromogenic oxidizing agent of the CUPric Reducing Antioxidant Capacity (CUPRAC) method, Cu(II)-Neocuproine (Cu(II)-Nc) complex, on a Nafion cation-exchange membrane, and the spectrophotometric assay developed in aqueous–alcoholic solutions was integrated to the CUPRAC sensor. Hydroxyl radicals (•OH) generated from an equivalent mixture of Fe(II)+EDTA with hydrogen peroxide attacked both the probe and the •OH scavengers in 37°C-incubated solutions for 1/2h. The HRS activity was measured using the decrease in CUPRAC absorbance at 450nm – arising from the reduction of Cu(II)-Nc reagent to the Cu(I)-neocuproine chelate – of the hydroxylated probe (TP) undergoing radical attack in the presence of •OH scavengers. The HRS activity was evaluated as the second-order rate constants of biologically active compounds for •OH scavenging and also as the percentage scavenging of a measured compound or sample relative to a reference compound. Using this reaction, a kinetic approach was adopted to assess the HRS activity of amino acids, plasma- and thiol-antioxidants. This assay, applicable to small molecule antioxidants and tissue homogenates, proved to be efficient for serine and albumin for which the widely used TBARS (thiobarbituric acid-reactive substances) test is nonresponsive. Under optimal conditions, about half of the probe (TP) was converted into 2-hydroxyterephthalate (hTP), and this monohydroxylated derivative, being the only product of hydroxylation, was a more specific marker of •OH than the non-specific malondialdehyde end-product of the TBARS test. The sensor gave a linear response to scavenger concentration in the competition kinetic equation.