METHODS FOR ANTIOXIDANT CAPACITY ESTIMATION

From a biology and nutrition perspective, antioxidants can function in vivo to prevent the oxidation of a variety of biological targets and can include substances which (1) chelate metal ions to prevent the formation of reactive oxygen/nitrogen species, (2) directly react with and scavenge reactive oxygen or nitrogen species, (3) inhibit oxidative enzymes (i.e., cyclooxygenases), or (4) induce antioxidant enzyme activities. This expands the scope of antioxidants to include more complex molecules such as enzymes, and numerous reactive oxygen species (ROS) such as peroxyl (ROO ), hydroxyl ( OH), and superoxide anion (O2 _) radicals, singlet oxygen (1DgO2), and reactive nitrogen species such as nitric oxide ( NO), nitrogen dioxide ( NO2), and peroxynitrite (OONO_).
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It is important to note that the antioxidant activity estimation is highly affected by the reactive oxygen or nitrogen species employed in the assay, although the chemical structure of the selected antioxidant molecule determines its antioxidant capacity. For example, while an antioxidant may be extremely effective in scavenging one type of free radical, it may relatively ineffective towards another type of radical or chelating a metal ion. For both food and biological systems, it has been recognized that in addition to the diverse ways in which antioxidants can function, the properties of the assay system can also greatly influence the effectiveness of an antioxidant (3). Physicochemical properties of the testing system including viscosity, polarity, pH, and presence of organic solvent and other molecules including enzymes and solubilizing agents can alter not only the physical location and concentration of antioxidants in the system but also the chemical mechanisms through which they interact with free radicals or other reactive oxygen and nitrogen species. It has therefore become necessary to employ not one but two or more antioxidant assays to characterize the diverse and multifaceted nature of a selected antioxidant preparation. Potential interferences for antioxidant capacity assays are often overlooked when the methods are initially developed and can lead to errors in the reported results. While each assay described in this chapter will be discussed for potential interferences in detail, some general background should be noted. Antioxidants can scavenge free radicals through several mechanism including hydrogen atom transfer (HAT) single electron transfer (ET or SET), and - the recently described sequential proton loss electron transfer (SPLET) mechanism (4). While details of these mechanisms are presented elsewhere (5), it is important to note that each mechanism involves different kinetics. While in most cases free radical scavenging reactions occur through a combination of mechanisms, factors that influence which mechanism predominates can greatly affect the kinetics and therefore results of antioxidant capacity assays. Such factors can include - solvent properties such as polarity, pH, and hydrogen bond accepting ability (6,7). Another common potential interference for antioxidant assays is the ability of solvent to donate hydrogen atoms to free radicals and themselves become radical species that can alter overall results. Evidence of this has been demonstrated recently by Moore and others (8) using electron spin resonance spin trapping assays. Another recent report by Perez-Jimenez and Saura-Calixto (9) examined the effect of sample solvent on results for several antioxidant capacity assays, and found significant interferences. These and other specific interferences described for individual assays below emphasize the importance of avoiding potential interferences with antioxidant capacity assays to ensure the accuracy of results.

Eleven antioxidant activity estimation methods are presented, each including three sections: (1) the principles, chemistry, and history of the development for the method; (2) the detailed protocol and calculations; and (3) a discussion including reported values for food products and the advantages and limitations for the method.