Oxidative stress can lead to diverse pathophysiological changes in the body. Neurodegenerative diseases such as Parkinson and Alzheimer are linked to oxidative stress, as well as several cancers and diseases such as chronic fatigue syndrome, fragile X syndrome, heart and blood vessel disorders, heart attack, heart failure, and atherosclerosis (for an overview see Fig 1).
Oxidative stress is based on the imbalance between the production of free radicals and the ability of the body to detoxify their hazardous effects through neutralization by antioxidants. Free radicals and reactive oxygen species (ROS) are highly reactive molecules that are generated by normal cellular processes, environmental stress, and UV irradiation. ROS react with cellular components, damaging DNA, carbohydrates, proteins, and lipids causing cellular and tissue injury. Organisms have developed complex antioxidant systems to protect themselves from oxidative stress, however, excess ROS can overwhelm the systems and cause severe damage.
2 versatile methods to measure the anti-oxidative potential of your sample
Two convenient kits to measure the total antioxidant capacity of biological fluids, cells, and tissue (developed by ZenBio). Both can also be used to assay the antioxidant activity of naturally occurring or synthetic compounds for use as dietary supplements, topical protection, and therapeutics. ORAC is a fluorescence based assay, while the ABTS assay is based on a colorimetric measurement. Let’s take a look at both.
The ORAC assay measures the loss of fluorescein fluorescence over time due to peroxyl-radical formation by the breakdown of AAPH (2,2′-azobis-2-methyl-propanimidamide, dihydrochloride) (Fig 2). Trolox [6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid], a water soluble vitamin E analog, serves as a positive control inhibiting fluorescein decay in a dose dependent manner. The ORAC assay is a kinetic assay measuring fluroescein decay and antioxidant protection over time. The antioxidant activity in biological fluids, cells, tissues, and natural extracts can be normalized to equivalent Trolox units to quantify the composite antioxidant activity present (see Fig.3).
The assay measures ABTS + radical cation formation induced by metmyoglobin and hydrogen peroxide. Trolox [6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid], a water soluble vitamin E analog, serves as a positive control inhibiting the formation of the radical cation in a dose dependent manner. As with the ORAC assay the antioxidant activity in biological fluids, cells, tissues, and natural extracts can be normalized to equivalent Trolox units to quantify the composite antioxidant activity present (see Fig 5).
You can also choose to outsource your oxidative activity measurement, to labs such as tebu-bio who perform this service (saving time and resources for other activities!).
Interested in measuring the antioxidative potential of your samples? Leave your questions and comments below, I’ll be pleased to get in touch with you!