ORAC Assay Measures Antioxidant Capacity
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Everyday metabolism, as well as stress and environmental pollutants, cause cells in the human body to produce molecules that are collectively known as Reactive Oxygen Species (ROS). ROS can be either free radicals or can form free radicals through their interaction with biological molecules (i.e. proteins, DNA/RNA, and lipids).
ROS and free radicals are necessary intermediates in the human body; however, too many free radicals are thought to play a role in cancer, aging, and other degenerative diseases, such as cardiovascular, Alzheimer’s and Parkinson’s (1-5).
Free radicals andROS are eliminated from the human body through enzymes (catalase, glutathione peroxidase, etc.) and through their interaction with biological antioxidants (transferrin, ceruloplasmin, urate, etc.). Nevertheless, these methods are not completely effective at eliminating ALL of these reactive intermediates from the body (6).
One way to help eliminate excess free radicals and ROS from the body is through the consumption of antioxidant laden foods. In recent years, an inverse relation has been shown between the consumption of fruits and vegetables (antioxidant rich foods) and diseases (7-9). A drawback to these studies, though, was determining the exact antioxidant capacity of the food and what in the food (flavenoids, vitamins C, D, E, etc.) was acting as an antioxidant (10-12).
Therefore a method was needed that could measure the antioxidant capacity of a substance, either directly from the food or indirectly in the blood after being consumed. The Oxygen Radical Absorbance Capacity (ORAC) assay is such a method. The ORAC assay was first developed by Dr. Guohua Cao in 1993 (13) and was based upon the work of Glazer (14). Since then, Dr. Ron Prior, currently at the Arkansas Children’s Nutrition Center, has modified and optimized the assay on BMG LABTECH’s FLUOstar OPTIMA (15-17)
Basically, the ORAC assay measures a fluorescent signal from a probe that decreases or is ‘quenched’ in the presence of an ROS generator. Addition of an antioxidant absorbs the generated ROS, thereby allowing the fluorescent signal to persist. Trolox® (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), a vitamin E analogue and a known antioxidant, is used as a standard by which all unknown antioxidants are compared. Subsequent modifications to the ORAC assay include the use of fluorescein instead of β-phycoerythrin as the fluorescent probe (known as ORACFL) (15), the separation of hydrophilic and lipophilic antioxidants to obtain total antioxidant capacity (16, 18), and the adaptation to a high-throughput platform (17).
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Several antioxidant assays have been developed over the years and they all use an ROSgenerator. The ORAC assay is unique in that its ROS generator, AAPH ((2,2’-azobis(2-methylpropionamidine) dihydrochloride)), produces a peroxyl free radical upon thermal decomposition that is commonly found in the body, making the reaction biological relevant. Furthermore, since AAPH is reactive with both water and lipid soluble substances it can be used to measure the total antioxidant potential. The ORAC assay is quickly becoming a standard method by which to measure a substance’s antioxidant capacity. The ORAC scores for some common foods are shown in Table 1. Note in some foods the difference between the lipophilic (L-ORAC) and hydrophilic (H-ORAC) ORAC scores. The total antioxidant capacity ORAC (TAC-ORAC) score is the sum of these two numbers |
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aSource: Wu, et al. 2004 (19). b Samples were taken from two seasons and averaged; each season had duplicates from 4 different U.S. regions c Lipophilic (L-ORAC), Hydrophilic (H-ORAC), and Total Antioxidant Capacity (TAC-ORAC); units are micromoles of Trolox Equivalents per gram |
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FLUOstar OPTIMA was used by Dr. Ron Prior to perform the ORAC assay (15-17), thereby making it one of the first microplate readers to be adapted to the ORAC assay. The FLUOstar OPTIMA is a multifunctional microplate reader that can read in four modes (fluorescence, time-resolved fluorescence, luminescence, and absorbance) and up to 1536 well plates. It can be outfitted with incubation (45°C or 60°C), orbital shaking, top/bottom reading, and two onboard reagent injectors. Incubation and onboard injectors are needed to adequately perform the ORAC assay. |
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To learn more about the ORAC assay performed on a FLUOstar OPTIMA, please read BMG LABTECH’s ORAC assay application note. To learn more about purchasing a FLUOstar OPTIMA, please email or contact your country’s BMG office. |
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