Spectrophotometric determination of antioxidant activity

Abstract

The spectrophotometric technique for total antioxidant activity (TAA)(1,2) measures the relative abilities of antioxidants to scavenge the 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) radical cation (ABTS(•+)) in comparison with the antioxidant potency of standard amounts of Trolox, the water-soluble vitamin E analogue. This method is based on the progressive consumption of antioxidant activity by ABTS(•+) as it is generated in the reaction cuvette and can be automated with a spectrophotometric analyzer. Several different analytical strategies are possible using the same reagents, enabling the assay system to be used to determine the antioxidant activity of plasma, saliva, lipoprotein fractions, foods and beverages. To determine the activity of pure antioxidant substances, a hydrogen peroxide concentration of 75 μM is used, together with a 6 min measuring time. For biological samples with endogenous peroxidase activity the hydrogen peroxide concentration is increased fivefold and the measuring time shortened to 3.25 min. Assays with improved sensitivity are described for low-density lipoprotein (LDL) preparations and saliva. Use of a spectrophotometric endpoint makes the assay simple to carry out without special laboratory equipment. Measurement at 734 nm avoids a range of potential interfering factors, such as sample turbidity and non-specific absorbance by sample constituents. Current applications of the ABTS antioxidant assay are described and discussed.