Oxygen-radical absorbance capacity assay for antioxidants

Abstract

A relatively simple but sensitive and reliable method of quantitating the oxygen-radical absorbing capacity (ORAC) of antioxidants in serum using a few microliter is described. In this assay system, beta-phycoerythrin (beta-PE) is used as an indicator protein, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) as a peroxyl radical generator, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, a water-soluble vitamin E analogue) as a control standard. Results are expressed as ORAC units, where 1 ORAC unit equals the net protection produced by 1 microM Trolox. The uniqueness of this assay is that total antioxidant capacity of a sample is estimated by taking the oxidation reaction to completion. At this point all of the nonprotein antioxidants (which include alpha-tocopherol, vitamin C, beta-carotene, uric acid, and bilirubin) and most of the albumin in the sample are oxidized by the peroxyl radical. Results are quantified by measuring the protection produced by antioxidants. This solves many problems associated with kinetics or lag-time measurements. A linear correlation of ORAC value with concentration of serum. Trolox, vitamin C, uric acid, and bovine albumin is demonstrated. The coefficient of variation within a run is found to be about 2% and from run to run about 5%. Trolox, alpha-tocopherol, vitamin C, beta-carotene, uric acid, and bilirubin completely protect beta-PE from oxidation, while bovine albumin protects beta-PE only partially. On a molar basis, the relative peroxyl radical absorbance capacity of Trolox, alpha-tocopherol acid succinate, uric acid, bilirubin, and vitamin C is 1:1:0.92:0.84:0.52. Bovine albumin per unit weight has a lower peroxyl absorbing capacity than these antioxidants.(ABSTRACT TRUNCATED AT 250 WORDS)