Submitted to: Nutritional Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/20/2006
Publication Date: 2/20/2006
Citation: Kwak, H., Blumberg, J., Chen, C., Milbury, P. 2006. Microplate-based oxygen radical absorbance capacity (orac) assay of hydrophilic and lipophilic compartments in plasma. Nutritional Sciences. 9(1):48-54.
Interpretive Summary: Measures of total antioxidant capacity of foods present a simple and way of comparing the sum of the potentially hundreds of individual antioxidant ingredients within different products. The Oxygen Radical Absorbance Capacity (ORAC) assay is one of the most popular tests being used today to rank the antioxidant potential of foods. However, little effort has been directed to applying this summary ORAC score to human blood samples and correlating it to the ORAC value of the foods being consumed. Studies that have approached this issue reveal generally poor correlations between food and plasma ORAC values. Some of these findings may result from the constituents of the assay being largely hydrophilic (water soluble) in nature and missing antioxidant activity present in the lipophilic (fat soluble) parts of blood. Thus, we modified the ORAC assay to be more responsive to lipophilic compartments in plasma and characterized the properties of this assay. Our results indicate the modified assay is responsive to the principal fat-soluble antioxidant, vitamin E, but not to other important fat-soluble antioxidants like carotenoids. Thus, available methods for measuring the total antioxidant capacity of human blood are still limited and unable to reflect the activity of the full range of dietary antioxidants.
Technical Abstract: Methods have been developed to evaluate the total antioxidant capacity of foods and plasma but limitations are associated with their ability to determine precisely the contribution of lipophilic antioxidants in a lipid milieu as well as interactions among them. Thus, we modified the Oxygen Radical Absorbance Capacity (ORAC) assay to determine the peroxyradical scavenging ability of both hydrophilic and lipophilic compartments in plasma. The hydrophilic ORAC assay was performed in a phosphate buffer system utilizing 2,2'-azobis (2-amidinopropane) dihydrochloride as a peroxyradical generator and fluorescein as the target. The lipophilic ORAC assay was carried out in a dimethylsulfoxide:butyronitrile (DMSO/BN, 9:1 v/v) system using 2,2'-azobis(2,4-dimethyl valeronitrile) as a peroxyradical generator and BODIPY C11 581/591 as the target. Analyses were conducted in bovine serum supplemented with water- and lipid-soluble antioxidants and in human plasma. Albumin (0.5-5 g/dL) and uric acid (0.1-0.5 mmol/L) increased hydrophilic ORAC values in a dose-dependent fashion (R2 = 0.97 and 0.98, respectively) but had no impact on lipophilic ORAC values. a-Tocopherol (15-200 mmol/L) increased lipophilic ORAC values in a dose-dependent fashion (R2 = 0.94); neither a-tocopherol nor beta-carotene had an impact on hydrophilic ORAC values. However, addition of beta-carotene at physiological concentrations (0.23-1.86 mmol/L), either alone or in combination with other carotenoids, had no significant impact on lipophilic ORAC values. Thus, while assays of "total antioxidant capacity" in biological matrices would be a useful research and clinical tool, existing methods are limited by the lack of complete responsiveness to the full range of dietary antioxidants.