Oxidative stress is a state that occurs when the generation of reactive oxygen species (ROS) exceeds a system's capacity to neutralize them. Similarly, nitrosative stress is a state that occurs when generation of reactive nitrogen species (RNS) exceeds the rate of neutralization. Oxidative and nitrosative stress are believed to play a role in aging and many diseases, including cardiovascular disease, cancer, inflammatory diseases, metabolic disease (such as diabetes), and neurodegenerative diseases.
Antioxidants are compounds which have the capacity to absorb and neutralize free radicals generated by ROS and RNS. These compounds can play an important role in the prevention or treatment of diseases which include a component of oxidative stress. As such, there is an increased focus on antioxidant levels in the diet and other products, such as nutritional supplements.
Standardized tests have been developed which measure antioxidant levels in nutritional and natural products, as well as in blood samples from a subject before and after consuming such products. One such assay is the non-cell based oxygen radical absorbance capacity assay (ORAC) (see Cao et al., Free Radic. Biol. Med. 14:303-311, 1993). In the ORAC assay, a test sample is incubated with a fluorescent indicator dye which is sensitive to oxidative damage and a free radical generator, and fluorescence intensity is measured. The result is expressed relative to the protection provided by the antioxidant standard TROLOX®. Another commonly used non-cell based method is the TROLOX® equivalent antioxidant capacity (TEAC) assay. In this assay, the formation of ferryl myoglobin radicals from metmyoglobin and hydrogen peroxide oxides 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ATBS) to produce a radical cation which is chromogenic and can be detected at 405 nm (Miller et al., Biochem. Soc. Trans. 21:95 S, 1993). As with ORAC, the antioxidant activity of the test sample is expressed relative to the antioxidant activity of the standard TROLOX®. A drawback of these assays is that they do not provide information on the ability of the products tested to penetrate the cell membrane.