HORAC Assay

Background

It is now well established that oxidative stress is a major risk factor for the development of several diseases including atherosclerosis, cardiovascular disease, and cancer. Oxidative stress is the condition in which there is an imbalance between the concentrations of reactive oxygen species (ROS) and physiological antioxidants, resulting in oxidative damage to many biomolecules within the cell. Products of ROS-mediated oxidation are widely used to monitor oxidative stress. However, it is also important to assess the antioxidant capacity of cells and biological fluids, as well as putative “functional foods” to assess their antioxidant capacity. Organisms possess multiple antioxidant systems to help regulate ROS and prevent oxidative stress. In vertebrates, these include enzymes that metabolize ROS, antioxidant proteins, and smaller molecules that are important antioxidants. These antioxidants include hydrophilic as well as lipid-soluble molecules that are localized throughout various tissues and cell types. Given the multiplicity of antioxidant pathways, their centrality in the prevention of oxidative stress, and the influences of lifestyle and nutritional supplements on an individual’s antioxidant capacity, it is important to be able to quantitatively measure the total antioxidant capacity or antioxidant power in a biological specimen or in nutrients.

Assay Principle

The HORAC assay is based on the oxidation of fluorescein by hydroxyl radicals via a classic hydrogen atom transfer (HAT) mechanism. Free radicals are generated by hydrogen peroxide (H2O2). The hydroxyl radicals thus generated quench the fluorescence of fluorescein over time. The antioxidants block the hydroxyl radical mediated oxidation of fluorescein until all of the antioxidant activity in the sample is exhausted, after which the H2O2 radicals react with and quench the fluorescence of fluorescein. The area under the fluorescence decay curve (AUC) is used to quantify the total hydroxyl radical antioxidant activity in a sample and is compared to a standard curve obtained using various concentrations of gallic acid. Unlike other antioxidant activity assays, the fluorescent HORAC assay provides a direct measurement of antioxidant capacity against hydrophilic chain-breaking hydroxyl radicals.

Sample Standard Curve

Note:

This is a sample standard curve and should not be
used to determine the HORAC value of samples.

Reference:

1. Ou, B., Hampsch-Woddill, M., Flanagan, J., Deemer, EK., Prior, RL., Huang, D. J Agric Food Chem. 50, 2772 (2002).