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KMID : 1012320060090010048
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Nutritional Sciences
2006 Volume.9 No. 1 p.48 ~ p.54
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Microplate-Based Oxygen Radical Absorbance Capacity (ORAC) Assay of Hydrophilic and Lipophilic Compartments in Plasma
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Kwak Ho-Kyung
Blumberg Jeffrey B.
Chen Chung-Yen
Milbury Paul E.
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Abstract
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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 (0.1~0.5μmol/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. α-Tocopherol (15~200μmol/L) increased lipophilic ORAC values in a dose-dependent fashion (R²=0.94); neither -tocopherol nor β-carotene had an impact on hydrophilic ORAC values. However, addition of β-carotene at physiological concentration (0.23~1.86 μmol/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.
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KEYWORD
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Oxygen Radical Absorbance Capacity (ORAC), Lipophilic antioxidants, Hydrophilic antioxidants, Microplate reader
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