Easy and sensitive methods to measure antioxidative potential

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. Excess production of reactive oxygen species can also lead to inflammation, premature aging disorders  and several disease states including cancer, diabetes, and atherosclerosis. Organisms have developed complex antioxidant systems to protect themselves from oxidative stress, however, excess ROS can overwhelm the systems and cause severe damage.

ZenBio offers two convenient kits to measure the total antioxidant capacity of biological fluids, cells, and tissue. 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.

ORAC assay

ORAC curve

Fig. 1: Effects of antioxidants in ORAC assay Trolox, Epogallocatechin gallate (EGCG), and Gallic acid were tested for their antioxidant activity in the ORAC assay

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). 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.1).

ABTS assay

ABTS curve

Fig. 2: Effects of antioxidants in ABTS assay Trolox, Sodium L-ascorbate (L-Asc), Epigallocatechin gallate (EGCG), and Gallic acid were tested for their antioxidant activity in the ABTS assay.

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.

In combination, both the ABTS and the ORAC assay provide a comprehensive analysis of a test sample’s antioxidant activity.

 

Interested in either of these kits? Leave your questions or comments below to get in touch.

Written by Ali El Baya, PhD
Ali el Bayâ is the Sales Manager at tebu-bio for the North of Europe.