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Basic Principles of the ORAC Assay
 
The ORAC assay measures the time-dependent decrease in the fluorescence  intensity of the b-PE marker protein, resulting from oxygen radical damage. On a molar basis, the b-PE protein reacts with oxygen radicals over 100 times slower than most biological antioxidants such as thiols, uric acid, bilirubin and ascorbate. However, b-PE is over 60 times more reactive than other non-antioxidant proteins.2 Therefore, all other active antioxidants are completely oxidized before the b-PE protein gets oxidized, thus facilitating the measurement of the total antioxidant capacity of the sample being tested.

Quantitation of Fast and Slow Antioxidant Activity

Each point on the whole serum ORAC curve reflects the level of antioxidant protection at that time, which is made up of many different antioxidants. Genox has developed a computer macro which measures the amount of fast acting antioxidants, such as ascorbic acid, phenolic acids etc. that protect the b-PE protein 100% by completely inhibiting free radical propagation. The slow acting antioxidants consist of the tocopherols, polyphenols, flavonoids, and, in addition, certain proteins and lipids, that are found in the blood stream. This separation technique increases the sensitivity of the assay in showing differences among different groups of individuals.
Contribution of known serum antioxidants that make up the total ORAC value.

Quantitation of the Antioxidant Activity of the Aqueous Soluble Fraction – AQUEOUS ORAC Value

The fast acting ORAC only constitutes a population CV = 9.4% of the total ORAC value - where as over 60% of the total ORAC is from the high concentration of slow acting antioxidants. In order to focus in on the active antioxidants Genox has developed methods to remove the proteins/lipids from a serum sample that results in an aqueous antioxidant fraction of human serum. This increases the sensitivity of the ORAC assay to measure the water-soluble antioxidants like vitamin C by over 200%.
Aqueous Soluble ORAC curve.

Contribution of known serum antioxidants that make up the aqueous soluble fraction ORAC value.

Quantitation of the Antioxidant Activity of the Lipid Soluble Fraction – LIPID ORAC Value

A protocol to quantify the antioxidant activity of lipid soluble fraction of serum and other biological samples is currently being developed.  Until the new protocol is tested and offered as a part of our in-house testing service, the Lipid ORAC value will be calculated and expressed as the difference between the WHOLE ORAC and AQUEOUS ORAC values.