Wednesday, November 25, 2009

Oxygen Radical Absorbance Capacitance Assays (ORAC) in Microplates

The antioxidant capabilities of foods, cosmetics, supplements and pharmaceutical agents have become of particular interest. This interest is the result of the evidence demonstrating the relationship of reactive oxygen/nitrogen species with aging and pathogenesis. [1,2]. Living organisms use of a fuel (ATP) whose production results in the formation of toxic compounds, requires that a balance be maintained between the oxidants and the antioxidants. ROS species are generally held in check by a combination of antioxidant enzymes, proteins, and antioxidants provided by the diet. A breakdown or reduction in anti-oxidant capability has been associated with a number of chronic diseases. Conceivably, the ingestion of foods or supplements that contain antioxidant activity could provide some protection towards this affect.


Illustration of Antioxidant activity determination expressed as the net area under the curve (AUC).


Figure 1. Illustration of Antioxidant activity determination expressed as the net area under the curve (AUC).


There are several different methods to measure total antioxidant capacity described in the literature (3-7). The oxygen radical absorbance capacity (ORAC) assay is the latest in a lineage of assays that attempt to measure the antioxidant capabilities of compounds and foods [7]. This assay has been automated [8] and over time converted to a microplate format [9]. The ORAC assay depends on the free radical damage to a fluorescent probe, such as fluorescein, to result in a downward change of fluorescent intensity [10]. The assumption of course is that the degree of change is indicative of the amount of radical damage. The presence of antioxidants results in an inhibition in the free radical damage to the fluorescent compound. This inhibition is observed as a preservation of the fluorescent signal. Reactions containing antioxidants and or blanks are run in parallel using equivalent amounts of a ROS generator and fluorescent probe. Because the reaction is driven to completion, one can quantitate the protection by calculating the area under the curve (AUC) from the experimental sample. After subtracting the AUC for the blank, the resultant difference would be the protection conferred by the antioxidant compound (Figure 1).

Comparison to a set of known standards allows one to calculate equivalents and compare results from different samples and experiments. Typically Trolox, (6-hydroxy-2,5,7,8-tetrametmethylchroman-2-carboxylic acid) a water soluble vitamin E analog, is used as the calibration standard and ORAC results are expressed as Trolox equivalents. The ORAC assay is unique in that because the assay is driven to completion the AUC calculation combines both the inhibition time as well as inhibition percentage of free radical damage by the antioxidant into a single quantity. Standardization of the assay with the use of a common calibrator in conjunction with an assay that can be performed easily on many different compounds, foods, and materials has allowed for an easy comparison of antioxidant capabilities of many different materials and the formation of a database [11].



Figure 2. Typical Antioxidant Standard Curve.
When net AUC are calculated from these kinetic curves and plotted against Trolox Concentration a linear relationship is observed (Figure 2). The standard curve can then be interpolated to quantitate unknown samples. The resultant determinations are expressed as Trolox equivalents. Several compounds known to have antioxidant properties were assayed using the ORAC assay as previously described. As depicted in Figure 3, all of these compounds show a significant linear concentration dependent antioxidant activity. The specificity of the assay is demonstrated by the lack of any response from Tris buffer.



Figure 3. Antioxidant Dose Response Curves. The ORAC of several different known antioxidant compounds, as well as Tris buffer were measured as described previously and their Net AUC plotted against their concentration.

The ORAC assay in our hands is very sensitive to slight temperature gradations. Despite extreme efforts, an edge effect was observed when the outside wells were used. Edge effects have been reported in any number of different types of microplate-based assays. Because were used the reader’s reagent injectors to initiate the assay, plate lids were not used. In these experiments, the tighter control between reading the wells and the initiation of the assay by the addition of AAPH was of greater value than the throughput. We found that filling the outer wells with 300 ┬ÁL of water resulted in more consistent data from the interior wells. Besides avoiding the use of the outer wells, where most of the discrepancies were found, the filled wells served as a significant heat mass that eliminated any temperature fluctuations. In addition, we found that reading the plate from the bottom resulted in more consistent results.

References
1. Halliwell, B., Aruoma, O. (1991) DNA Damage by Oxygen Derived Species. Its Mechanisms and Measurement in Mammalian Systems. FEBS Lett. 281:9-19.
2. Ames, B.N., Shigenaga, M.K., and Hagen, T.M. (1993) Oxidants, Antioxidants and the Degenerative Diseases of Aging. Proc. Natl. Acad. Sci. USA 90:7915-7922.
3. Benzie, IFF, Strain, JJ. (1996) The Ferric Reducing Ability of Plasma (FRAP) as a Measure of “Antioxidant Power”: The FRAP Assay. Anal. Biochem. 238:70-76.
4. Rice-Evans, C. and Miller, NJ. (1994) Total Antioxidant Status in Plasma and Body Fluids. Methods Enzymol. 234:279-293.
5. Wayner, DDM, Burton, Gw., Ingold, KU., and Locke, S. (1985) Quantitative Measurement of the Total, Peroxyl Radical-trapping Antioxidant Capacity of Human Blood Plasma by Controlled Peroxidation. FEBS Lett. 187:33-37.
6. Glazer, AN., (1990) Phycoerythrin Fluorescence-based Assay for Reactive Oxygen Species. Methods Enzymol 186:161-168.
7. Cao, GH., Alessio, HM.and Cutler, RG., (1993) Oxygen-radical Absorbance Capacity Assay for Antioxidants. Free Radical Biol. Med. 14:303-311.
8. Cao, G., Verdon, C., Wu, A., Wang, H., and Prior, R. (1995) Automated Assay of Oxygen Radical Absorbance Capacity with the COBRAS FARA II. Clin. Chem. 41:1738-1744.
9. Huang, D., Ou, B., Hampsch-Woodill, M., Flanagan, J., and Prior, R. (2002) 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. 50:4437-4444.
10. Ou, B., Hampsch-Woodill, and Prior, R. (2001) Development and Validation of an Improved Oxygen Radical Absorbance Capacity Assay Using Fluorescein as the Fluorescent Probe. J. Agric Food Chem. 49:4619-4626.
11. Wu, X., Gu, L., Holden, J., Haytowitz, D., Gebhardt, S., Beecher, G., and Prior, R. (2004) Development of a Database for Total Antioxidant Capacity in Foods: a Preliminary Study. J. Food Composition and Analysis. 17:407-422.
12. Cao, G., and Prior, R. (1999) Measurement of Oxygen Radical Absorbance Capacity in Biological samples. Oxidants and Antioxidants. Methods Enzymol. 299:50-62.



By: BioTek Instruments

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