Oxidative stress is a physiological condition where there is an imbalance between concentrations of reactive oxygen species (ROS) and antioxidants.  However, excessive ROS accumulation will lead to cellular injury, such as damage to DNA, proteins, and lipid membranes.  The cellular damage caused by ROS has been implicated in the development of many disease states, such as cancer, diabetes, cardiovascular disease, atherosclerosis, and neurodegenerative diseases.  Under normal physiological conditions, cellular ROS generation is counterbalanced by the action of cellular antioxidant enzymes and other redox molecules.  Because of their potential harmful effects, excessive ROS must be promptly eliminated from the cells by this variety of antioxidant defense mechanisms.  Antioxidants include both hydrophilic and lipophilic molecules for metabolizing ROS.

Although the products of ROS-induced oxidative stress are extensively used to monitor the effects of oxidative stress, it is also important to evaluate the antioxidant capacity of biological fluids, cells, and extracts.  The Oxygen Radical Antioxidant Capacity (ORAC) Assay is a classic tool for measuring the antioxidant capacity of biomolecules from a variety of samples.  The ORAC Assay is based on the oxidation of a fluorescent probe by peroxyl radicals by way of a hydrogen atom transfer (HAT) process.  Peroxyl radicals are produced by a free radical initiator, which quenches the fluorescent probe over time.  Antioxidants present in the assay work to block the peroxyl radical oxidation of the fluorescent probe until the antioxidant activity in the sample is depleted.  The remaining peroxyl radicals destroy the fluorescence of the fluorescent probe.  This assay continues until completion, which means both the antioxidant’s inhibition time and inhibition percentage of free radical damage is a single value.  The sample antioxidant capacity correlates to the fluorescence decay curve, which is usually represented as the area under the curve (AUC).  The AUC is used to quantify the total peroxyl radical antioxidant activity in a sample and is compared to an antioxidant standard curve of the water soluble vitamin E analog Trolox™.