Determining Antioxidant Potential : Using an Oxygen Absorbance Capacity (ORAC) Assay

The oxygen radical absorbance capacity (ORAC) assay is an analytical method to determine the antioxidant potential of nutraceutical, pharmaceutical and food ingredients. The ORAC assay relies on the fluorescent probe to monitor antioxidant activity, which can be read in 96-well microplates using a fluorescence-capable reader.

INTRODUCTION:

Oxidative damage to living organisms is associated with several disease states and aging¹. Sources of reactive oxygen species (ROS) are both endogenous and exogenous, and can lead to toxic compound formation within organisms. A proper balance must be maintained between oxidants and antioxidants to ensure that the ubiquitous ROS species are not deleterious to the organism. Disrupting these mechanisms can result in an imbalance and ensuing damage to critical components required to maintain cells. Thus, there is interest in accurately determining the antioxidant capabilities of foods, cosmetics, dietary supplements and pharmaceutical agents.

While several methods exist to measure total antioxidant capacity, ORAC is a low cost method suitable for microplate-based high-throughput automation²,³. The assay relies on free radical damage to a fluorescent probe resulting in a loss of fluorescent intensity⁴. Inhibition of oxidative damage to the fluorescent probe is correlated with a compound’s antioxidant capacity acting as a free radical scavenger. Kinetic reactions containing antioxidants and blanks are run in parallel with integration of the resultant curve to calculate the area under the curve (AUC). Antioxidant protection is then quantified by the difference between the AUC of the blank reaction and reactions containing antioxidant.

EXPERIMENTAL CONDITIONS:

The ORAC assay was performed as described by Huang et al.² with modifications described by Held⁵. Reactions were initiated by adding 25 μL of AAPH solution using the SynergyTM H4 Hybrid Multi-mode Microplate Reader injectors (BioTek Instruments, Inc., Winooski, VT) in a 200 μL final reaction volume. The fluorescence was monitored kinetically with data taken every minute for two hours.

RESULTS:

Several compounds with known antioxidant properties were assayed by the ORAC method (Figure 1). To determine each compound’s antioxidant capacity, the net AUC for each sample was calculated and Trolox® equivalents calculated using the ratio of the compound’s slope of the linear regression analysis to the Trolox® standard’s slope⁶.

CONCLUSIONS:

High throughput antioxidant determination requires a low-cost assay with high precision and accuracy that is amenable to microplates. Here we show that the ORAC assay provided antioxidant determination of several known antioxidants and subsequent conversion to the commonly accepted Trolox® equivalents for quantitative analysis. The assay used common reagents, a standard 96-well microplate and a fluorescence-capable microplate reader. The ORAC assay provided excellent precision across all microplate wells tested and performed the assay in approximately 60 minutes. Results correlated well with those presented in the literature².

REFERENCES

Figure 1. Trolox® kinetic curves and resultant Trolox® equivalents. (A) ORAC assay kinetic curves of Trolox® antioxidant concentrations ranging from 0-100 µM. (B) Calculated Trolox® equivalents were then used for sample antioxidant comparative analysis.

1. Ames, B.N.; Shigenaga, M.K.; Hagen, T.M. Oxidants, antioxidants and the degenerative diseases of aging. Proc Natl Acad Sci USA. 1993, 90(17), 7915-7922.

2. Huang, D.; Ou, B.; Hampsch-Woodill, M.; Flanagan, J.A.; Prior, R.L. High-throughput assay of oxygen radical absorbance capacity (ORAC) using a multichannel liquid handling system coupled with a microplate fluorescence reader in 96-well format. J Agric Food Chem. 2002, 50(16), 4437-4444.

3. Cao, G.; Prior, R.L. Measurement of oxygen radical absorbance capacity in biological samples. Oxidants and Antioxidants. Methods Enzymol. 1999, 299, 50-62.

4. Ou, B.; Hampsch-Woodill, M.; Prior, R.L. Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. J. Agric Food Chem. 2001, 49(10), 4619-4626.

5. Held, P. Performing Oxygen Radical Absorbance Capacity (ORAC) Assays with Synergy™ HT Multi- Mode Microplate Reader – ORAC Antioxidant Tests. BioTek Instruments, Inc. http://www.biotek.com/ resources/articles/performing-orac-assays.html (accessed Jul 16, 2012).

6. Sun, T.; Powers, J.R.; Tang, J. Evaluation of the antioxidant activity of asparagus, broccoli and their juices. Food Chem. 2007, 105, 101-106.