Introduction
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 Hydroxyl Radical Antioxidant Capacity (HORAC) Assay is a classic tool for measuring
the antioxidant capacity of biomolecules from a variety of samples. The HORAC Activity Assay is
based on the oxidation of a fluorescent probe by hydroxyl radicals by way of a hydrogen atom transfer
(HAT) process. Hydroxyl radicals are produced by hydroxyl radical initiator and fenton reagent, which
quenches the fluorescent probe over time. Antioxidants present in the assay work to block the radical
hydroxyl oxidation of the fluorescent probe until the antioxidant activity in the sample is depleted. The
remaining hydroxyl 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 hydroxyl radical antioxidant activity in a sample and is compared to a gallic acid antioxidant
standard curve.
Cell Biolabs’ OxiSelect™ HORAC Activity Assay is a fast and reliable kit for the direct measurement
of HORAC antioxidant capacity from cell lysate, plasma, serum, tissue homogenates, and food extracts.
Each kit provides sufficient reagents to perform up to 192 assays, including blanks, antioxidant
standards and unknown samples. The assay is designed for use in single plate microplate readers as
well as readers with high-throughput capabilities. Please read the complete kit insert prior to
performing the assay.