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ORAC Assay Measures Antioxidant Capacity

ROS and Free Radicals

Free RadicalsEveryday 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 and ROS 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)

The ORAC Assay

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).

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.

Table: ORAC Scores for Some Common Foods a
Sample L-ORAC
(µmol TE/g)b,c		 H-ORAC
(µmol TE/g)b,c		 TAC-ORAC
(µmol TE/g)c
Watermelon 0.19 ± 0.04 1.23 ± 0.17 1.4
Cantaloupe 0.15 ± 0.08 2.97 ± 0.62 3.1
Grapefruit 0.35 ± 0.10 15.13 ± 3.36 15.5
Blueberry 0.36 ± 0.18 61.84 ± 7.75 62.2
Strawberry 0.36 ± 0.25 35.41 ± 4.24 35.8
Raspberry 1.62 0.66 47.65 ± 7.18 49.3
Navel Orange 0.29 ± 0.13 17.85 ± 3.79 18.1
Plum 0.17 ± 0.10 62.22 ± 20.22 62.4
Tomato 0.22  ± 0.07 3.13 ± 0.69 3.4
Lettuce, Iceberg 0.33  ± 0.10 4.18 ± 2.80 4.5
Potato, Russet 0.51 ± 0.14 12.72 ± 2.28 13.2
Broccoli 1.72 ± 0.24 14.18 ± 2.04 15.9
Almond 1.72 ± 0.50 42.82 ± 8.71 44.5
Cashew 4.74 ± 1.38 15.23 ± 2.04 20.0
Pecan 4.16 ± 0.98 175.24 ±10.36 179.0
Pistachio 4.25 ± 1.46 75.57 ± 10.50 79.8
Date 0.32 ± 0.16 38.63 ± 3.21 39.0
Raisin 0.35 ± 0.13 30.02 ± 5.23 30.4
Prune 1.79 ± 0.56 83.99 ± 16.56 85.8

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

ORAC Assay Application Notes

BMG LABTECH has published application notes (AN) ORAC using various BMG LABTECH microplate readers:

ORAC Assay on the FLUOstar Optima to Determine Antioxidant Capacity (AN 148)
ORAC Assay Performed on the POLARstar Omega and PHERAstar FS microplate reader (AN 197)

ORAC Assay Compatible Microplate Readers

The following microplate readers can be configured to preform the ORAC assay:

PHERAstar FS HTS Microplate Reader FLUOstar Omega Microplate Reader POLARstar Omega Microplate Reader
FLUOstar OPTIMA POLARstar OPTIMA NOVOstar

 

ORAC References

  1. Ames, B. N.; Shigenaga, M. K.; Hagen, T. M. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl.Acad. Sci. U.S.A. 1993, 90, 7915-7922.
  2. Ames, B. N.; Gold, L. S.; Willet, W. C. The causes and prevention of cancer. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 5258-5265.
  3. Christen, Y. Oxidative stress and Alzheimer’s disease. Am. J. Clin. Nutr. 2000, 71, 621S-629S.
  4. Diaz, M. N.; Frei, B.; Keaney, J. F., Jr. Antioxidants and atherosclerotic heart disease. New Engl. J. Med. 1997, 337, 408-416.
  5. Lang, A. E.; Lozano, A. M. Parkinson’s disease. First of two parts. N. Engl. J. Med. 1998, 339, 111-114.
  6. Davies, K. J. A. Oxidative stress, antioxidant defenses, and damage removal, repair, and replacement systems. IUBMB Life 2000, 50, 279-289.
  7. Block, G.; Patterson, B.; Suber, A. Fruits, vegetables and cancer pervention: a review of the epidemiological evidence. Nutr. Cancer 1992, 18, 1-29.
  8. World Cancer Research Fund, American Institute for Cancer Research. Food, Nutrition and the PreVention of Cancer: A Global PerspectiVe; American Institute for Cancer Research: Washington, DC, 1997.
  9. Joshipura, K.J., Ascherio, A., Manson, J.E., Stampfer, M.J., Rimm, E.B., Speizer, F.E., Hennekens, C.H., Spiegelman, D., Willett, W. Fruit and vegetable intake in relation to risk of ischemic stroke. Journal of American Medical Association 1999,282, 1233–1239.
  10. Cooper, D. A.; Eldridge A. L.; Peters, J. C. Dietary carotenoids and certain cancer, heart diseases, and age-related macular degeneration: a review of recent research. Nutr. ReV. 1999, 57, 210-214.
  11. Emmert, D.H., Kirchner, J.T. The role of vitamin E in the prevention of heart disease. Archives of Family Medicine 1999, 8, 537–542.
  12. Hercberg, S., Galan, P., Preziosi, P. Antioxidant vitamins and cardiovascular disease: Dr. Jekyll or Mr. Hyde? American Journal of Public Health 1999, 89, 289–291.
  13. Cao, G.; Alessio, H. M.; Cutler, R. G. Oxygen-radical absorbance capacity assay for antioxidants. Free Radical Biol. Med. 1993, 14, 303-311.
  14. Glazer, A. N. Phycoerythrin Flurorescence-Based Assay for Reactive Oxygen Species. Methods Enzymol. 1990, 186, 161-168.
  15. 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, 4619-4926.
  16. Prior, R. L.; Hoang, H.; Gu, L.; Wu, X.; Bacchiocca, M.; Howard, L.; Hampsch-Woodill, M.; Huang, D.; Ou, B.; Jacob, R. Assays for hydrophilic and lipophilic antioxidant capacity (oxygen radical absorbance capacity (ORACFL)) of plasma and other biological and food samples. J. Agric. Food Chem. 2003, 51, 3273-3279.
  17. Huang, D.; Ou, B.; Hampsch-Woodill, M.; Flanagan, J.; 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, 4437-4444.
  18. Huang, D.; Ou, B.; Hampsch-Woodill, M.; Flanagan, J. A.; Deemer, E. K. Development and validation of oxygen radical absorbance capacity assay for lipophilic antioxidants using randomly methylated â-cyclodextrin as the solubility enhancer. J. Agric. Food Chem. 2002, 50, 1815-1821.
  19. Xianli Wu, Liwei Gu, Joanne Holden, David B. Haytowitz, Susan E. Gebhardt, Gary Beecher and R.L.Ronald L. Prior. Development of a database for total antioxidant capacity in foods: a preliminary study J. Food Compost. Anal. 2004, 17, 407-422.