Determination of Antioxidant potential using an Oxygen Radical Absorbance Capacity (ORAC) Assay with Synergy™ H4

Oxidative damage to living organisms has been associated with several disease states as well as aging. The sources of reactive oxygen species (ROS) are varied and can lead to the formation of toxic compounds within organisms. ROS sources such as cellular respiration, interaction of biomolecules with ionizing radiation, and dedicated cellular pathways for ROS formation as a protective mechanism ensure chronic exposure of living organisms to ROS species.

A proper balance must be maintained between oxidants and antioxidants to ensure the ubiquitous ROS species do not become deleterious to the organism. Disruption of any one of these mechanisms can result in an imbalance and ensuing damage to a number of critical components required to maintain cellular homeostasis. Thus, there is interest in the ability to accurately determine the antioxidant capabilities of foods, cosmetics, dietary supplements and pharmaceutical agents.

While several methods exist to measure total antioxidant capacity, ORAC has emerged as a low cost method suitable for high throughput automation in a microplate format. The ORAC assay relies on free radical damage to a fluorescent probe, most commonly fluorescein, caused by an oxidizing reagent resulting in a loss of fluorescent intensity over time.The resultant damage can then be correlated with the amount of oxidant present. Conversely, inhibition of oxidative damage to the fluorescent probe can be correlated with the antioxidant capacity of a compound acting as a free radical scavenger.

This application note demonstrates the ability to perform the assay in a 96-well microplate format amenable to higher throughput platforms.