Oxygen radical absorbance capacity

Oxygen radical absorbance capacity (ORAC) is a method of measuring antioxidant capacities in biological samples in vitro.

A wide variety of foods have been tested using this method, with certain spices, berries and legumes rated highly in extensive tables once published by the United States Department of Agriculture (USDA), but withdrawn in 2012 since no correlation between test results and biological activity could be determined, stating that no physiological proof in vivo existed in support of the free-radical theory.

Although not all have been unilaterally dismissive, the majority position supports the USDA decision. Alternative measurements include the Folin-Ciocalteu reagent, and the Trolox equivalent antioxidant capacity assay.

The assay measures the oxidative degradation of the fluorescent molecule (either beta-phycoerythrin or fluorescein) after being mixed with free radical generators such as azo-initiator compounds. Azo-initiators are considered to produce the peroxyl radical by heating, which damages the fluorescent molecule, resulting in the loss of fluorescence. Antioxidants are considered to protect the fluorescent molecule from the oxidative degeneration. The degree of protection is quantified using a fluorometer. Fluorescein is currently used most as a fluorescent probe. Equipment that can automatically measure and calculate the capacity is commercially available (Biotek, Roche Diagnostics).

The fluorescent intensity decreases as the oxidative degeneration proceeds, and this intensity is typically recorded for 35 minutes after the addition of the azo-initiator (free radical generator). So far, AAPH (2,2’-azobis(2-amidino-propane) dihydrochloride) is the sole free-radical generator used. The degeneration (or decomposition) of fluorescein is measured as the presence of the antioxidant slows the fluorescence decay. Decay curves (fluorescence intensity vs. time) are recorded and the area between the two decay curves (with or without antioxidant) is calculated. Subsequently, the degree of antioxidant-mediated protection is quantified using the antioxidant trolox (a vitamin E analogue) as a standard. Different concentrations of trolox are used to make a standard curve, and test samples are compared to this. Results for test samples (foods) have been published as "trolox equivalents" or TEs.

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