Antioxidant and Prooxidant Behavior of Flavonoids: Structure-Activity Relationships

1. A new method has been developed for measuring the total antioxidant capacity of body fluids and drug solutions, based on the absorbance of the ABTS.+ radical cation. 2. An automated method for use on a centrifugal analyser, as well as a manual method, is described. 3. The procedure has been applied to physiological antioxidant compounds and radical-scavenging drugs, and an antioxidant ranking was established based on their reactivity relative to a 1.0 mmol/l Trolox standard. 4. The Trolox equivalent antioxidant capacity of plasma from an adult reference population has been measured, and the method optimized and validated. 5. The method has been applied to investigate the total plasma antioxidant capacity of neonates and how this may be compromised in prematurity.

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)