|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.
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%.
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.