Free Radical Biology & Medicine, Vol. 14, pp. 303-311, | 993 0891-5849/93 $6.00 + .00 Printed in the USA. All fights reserved. 1993 Pergamon Press Ltd. Original Contribution OXYGEN-RADICAL ABSORBANCE CAPACITY ASSAY FOR ANTIOXIDANTS GUOHUA CAO,* HELAINE M. ALESSIO, t and RICHARD G. CUTLER* *Gerontology Research Center, National Institute on Aging, NIH, 4940 Eastern Avenue, Baltimore, MD 21224, USA; and tDepartment of Physical Education, Health and Sport Studies, Miami University, Oxford, OH 45056, USA (Received 29 June 1992; Revised 16 September 1992; Accepted 6 October 1992) Abstract--A relatively simple but sensitive and reliable method ofquantitating the oxygen-radical absorbing capacity (ORAC) of antioxidants in serum using a few #l is described. In this assay system, ~-phycoerythrin (~-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-tetramethylchro- man-2-carboxylic acid (Trolox, a water-soluble vitamin E analogue) as a control standard. Results are expressed as ORAC units, where 10RAC unit equals the net protection produced by l ~M 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 a-tocopherol, vitamin C, E-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, a-tocopherol, vitamin C,/3-carotene, uric acid, and bilirubin completely protect B-PE from oxidation, while bovine albumin protects/3-PE only partially. On a molar basis, the relative peroxyl radical absorbance capacity of Trolox, a-tocopherol acid succinate, uric acid, bilirubin, and vitamin C is l : 1 : 0.92 : 0.84 : 0.52. Bovine albumin per unit weight has a lower peroxyi absorbing capacity than these antioxidants. However, the serum protein fraction, containing some lipid-soluble antioxidants, represents the major contributor to the ORAC value found in whole serum. The minimum amount of vitamin C and uric acid which could still be detectable when added to a serum supernatant fraction is 1.5/~g and 0.59 #g, respectively, which account for about 1% of the total ORAC value of the serum supernatant fraction. Keywords--Oxygen radicals, Antioxidant, a-tocopherol, E-carotene, Vitamin C, Uric acid, Bilirubin, Free radical INTRODUCTION Reactive oxygen species could be important causative agents of a number of human diseases, including cancer and atherosclerosis, as well as the aging process itself.l-5 Thus, mechanisms such as antioxidants that act to control oxidative stress state represent a major line of defense regulating general health status. 5 Human serum contains many different antioxi- dants that may be important for general health main- tenance. These include vitamin C, a-tocopherol,/3- carotene, uric acid, bilirubin, and albumin. There are other antioxidants that appear to be less important, and perhaps others are not yet identified.6 In addition, trace amounts of antioxidant enzymes such as gluta- thione peroxidase and superoxide dismutase are found in serum to a lesser extent. 6 Because of the many different antioxidant compo- nents in serum and the relative difficulty of measuring Address correspondence to: Richard G. Cutler. each antioxidant separately, a simple method to mea- sure net or total resultant antioxidant capacity of serum would be of considerable value. 7 The available methods for this purpose at present are the fluores- cence-based assay developed in Glazer's laboratory8 and the oxygen electrode method developed by Ingold et al. known as TRAP. 9 However, the assay developed by Glazer et al. is not capable of quantitating results and is limited only to screening the free-radical-sca- venging capacity of a sample/The method developed by Ingold et al. is based on the time taken to prevent maximum oxygen uptake in a system containing a free radical generator, lipid, and serum or a specific antioxidant. The problem with this method is that the time taken to prevent maximum oxygen uptake can- not be measured easily and precisely and the total peroxyl radical trapping capability per mole of some antioxidants (for example, vitamin C) is dependent on their initial concentration, l° Our laboratory has developed a method that mea- 303