Oxygen Radical Absorbance Capacity - Method

Method

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.

One benefit of using the ORAC method to evaluate substances' antioxidant capacities is that it takes into account samples with and without lag phases of their antioxidant capacities. This is especially beneficial when measuring foods and supplements that contain complex ingredients with various slow- and fast-acting antioxidants, as well as ingredients with combined effects that cannot be precalculated.

Drawbacks of this method are: 1) only antioxidant activity against particular (probably mainly peroxyl) radicals is measured; however, peroxyl radical formation has never been proven; 2) the nature of the damaging reaction is not characterized; 3) there is no evidence that free radicals are involved in this reaction; and 4) there is no evidence that ORAC values have any biological significance following consumption of any food. Moreover, the relationship between ORAC values and a health benefit has not been established.

Resulting from scientific refutation of the physiological significance of ORAC, the USDA, which had been collating and publishing ORAC data for more than a decade, withdrew its web publication of ORAC values for common American foods in May 2012.

Several modified ORAC methods have been proposed. Most of them employ the same principle (i.e. measurement of AAPH-radical mediated damage of fluorescein); however, ORAC-EPR, electron paramagnetic resonance-based ORAC method directly measures the decrease of AAPH-radical level by the scavenging action of the antioxidant substance.