Oxygen radical absorbance capacity

Oxygen Radical Absorbance Capacity (ORAC) is a method of measuring antioxidant capacities in biological samples in vitro.^[1][2]

A wide variety of foods has been tested using this method, with certain spices, berries and legumes rated highly.^[3] There exists no physiological proof in vivo that free-radical theory is valid. Consequently, the ORAC method, derived only in test tube experiments, cannot currently be interpreted as relevant to human diets or biology.

Method

The assay measures the oxidative degradation of the fluorescent molecule (either beta-phycoerythrin or fluorescein) after being mixed with free radical generators such as azo-initiator compounds. Azo-initiators are considered to produce the peroxyl radical by heating, which damages the fluorescent molecule, resulting in the loss of fluorescence. Antioxidants are considered to protect the fluorescent molecule from the oxidative degeneration. The degree of protection is quantified using a fluorometer. Fluorescein is currently used most as a fluorescent probe. Equipment that can automatically measure and calculate the capacity is commercially available (Biotek, Roche Diagnostics).

The fluorescent intensity decreases as the oxidative degeneration proceeds, and this intensity is typically recorded for 35 minutes after the addition of the azo-initiator (free radical generator). So far, AAPH (2,2’-azobis(2-amidino-propane) dihydrochloride) is the sole free-radical generator used. The degeneration (or decomposition) of fluorescein is measured as the presence of the antioxidant slows the fluorescence decay. Decay curves (fluorescence intensity vs. time) are recorded and the area between the two decay curves (with or without antioxidant) is calculated. Subsequently, the degree of antioxidant-mediated protection is quantified using the antioxidant trolox (a vitamin E analogue) as a standard. Different concentrations of trolox are used to make a standard curve, and test samples are compared to this. Results for test samples (foods) have been published as "trolox equivalents" or TE.^[4]

One benefit of using the ORAC method to evaluate substances' antioxidant capacity is that it takes into account samples with and without lag phases of their antioxidant capacities. This is especially beneficial when measuring foods and supplements that contain complex ingredients with various slow and fast acting antioxidants, as well as ingredients with combined effects that cannot be pre-calculated.

Drawbacks of this method are: 1) only antioxidant activity against particular (probably mainly peroxyl) radicals is measured; however, peroxyl radical formation has never been proven; 2) the nature of the damaging reaction is not characterized; 3) there is no evidence that free radicals are involved in this reaction; and 4) there is no evidence that ORAC values have any biological significance following consumption of any food. Moreover, the relationship between ORAC values and a health benefit has not been established.

Several modified ORAC methods have been proposed. Most of them employ the same principle (i.e. measurement of AAPH-radical mediated damage of fuorescein); however, ORAC-EPR, electron paramagnetic resonance (EPR) based ORAC method directly measures the decrease of AAPH-radical level by the scavenging action of the antioxidant substance.^[5]

Regulatory guidance

In the following discussion, the term "antioxidant" refers mainly to non-nutrient compounds in foods, such as polyphenols, which have antioxidant capacity in vitro and so provide an artificial index of antioxidant strength—the ORAC measurement.

Other than for dietary antioxidant vitamins -- vitamin A, vitamin C and vitamin E -- no food compounds have been proved with antioxidant efficacy in vivo. Accordingly, regulatory agencies like the Food and Drug Administration of the United States and the European Food Safety Authority (EFSA) have published guidance disallowing food product labels to claim or imply an antioxidant benefit when no such physiological evidence exists.^[6][7] This guidance for the United States and European Union establishes that it is illegal to imply potential health benefits on package labels of products with high ORAC.

Physiological context

Although research in vitro indicates that polyphenols are good antioxidants and probably influence the ORAC value, antioxidant effects in vivo are probably negligible or absent.^[8][9] By non-antioxidant mechanisms still undefined, flavonoids and other polyphenols may reduce the risk of cardiovascular disease and cancer.^[10]

As interpreted by the Linus Pauling Institute and EFSA, dietary polyphenols have little or no direct antioxidant food value following digestion.^[7][8][11] Not like controlled test tube conditions, the fate of polyphenols in vivo shows they are poorly conserved (less than 5%), with most of what is absorbed existing as chemically-modified metabolites destined for rapid excretion.^[12]

The increase in antioxidant capacity of blood seen after the consumption of polyphenol-rich (ORAC-rich) foods is not caused directly by the polyphenols, but most likely results from increased uric acid levels derived from metabolism of flavonoids.^[11][12] According to Frei, "we can now follow the activity of flavonoids in the body, and one thing that is clear is that the body sees them as foreign compounds and is trying to get rid of them."^[12]

Food sources

Scientists with the United States Department of Agriculture have voluntarily published (i.e. USDA does not authorize these values) lists of ORAC values for plant foods commonly consumed by the U.S. population (fruits, vegetables, nuts, seeds, spices, grains, etc.).^[3] Values are expressed as the sum of the lipid soluble (e.g. carotenoid) and water-soluble (e.g. phenolic) antioxidant fractions (i.e., “total ORAC”) reported as in micromoles Trolox equivalents (TE) per 100 gram sample, and are compared to assessments of total polyphenol content in the samples.

USDA data on foods with high ORAC scores

Food	Serving size	Antioxidant capacity per serving size. Units are Total Antioxidant Capacity per serving in units of micromoles of Trolox equivalents.
- Raw unprocessed Cocoa bean	100 grams	28,000
Small Red Bean	½ cup dried beans	13,727
Wild blueberry	1 cup	13,427
Red kidney bean	½ cup dried beans	13,259
Pinto bean	½ cup	11,864
Blueberry	1 cup (cultivated berries)	9,019
Cranberry	1 cup (whole berries)	8,983
Artichoke hearts	1 cup, cooked	7,904
Blackberry	1 cup (cultivated berries)	7,701
Prune	½ cup	7,291
Raspberry	1 cup	6,058
Strawberry	1 cup	5,938
Red Delicious apple	1 apple	5,900
Granny Smith apple	1 apple	5,381
Pecan	1 oz	5,095
Sweet cherry	1 cup	4,873
Black plum	1 plum	4,844
Russet potato	1, cooked	4,649
Black bean[disambiguation needed ]	½ cup dried beans	4,181
Plum	1 plum	4,118
Gala apple	1 apple	3,903

With nearly all vegetables, conventional boiling can reduce the ORAC value by up to 90%, while steaming retains more of the antioxidants.^[13]

Comparisons of ORAC values

When comparing ORAC data, care must be taken to ensure that the units and food being compared are similar. Some evaluations will compare ORAC units per gram of dry weight, others will evaluate ORAC units in wet weight and still others will look at ORAC units per serving. Under each evaluation, different foods can appear to have higher ORAC values. For example, although a raisin has no more antioxidant potential than the grape from which it was dried, raisins will appear to have a higher ORAC value per gram of wet weight than grapes due to their reduced water content. Likewise, large water content in watermelon can make it appear as though this fruit is low in ORAC. Similarly, the typical quantity of food used should be considered; herbs and spices may be high in ORAC, but are applied in much smaller quantities than for other foods.^[14]

Numerous health food and beverage companies and marketers have capitalized on the ORAC rating by promoting products claimed to be "high in ORAC". As most of these ORAC values have not been independently validated or subjected to peer review for publication in scientific literature, they remain unconfirmed, are not scientifically credible, and may mislead consumers.

See also

• Polyphenol antioxidants
• Reduction potential
• List of antioxidants in food
• Trolox (TEAC)

References

1. ^ Cao G, Alessio H, Cutler R (1993). "Oxygen-radical absorbance capacity assay for antioxidants". Free Radic Biol Med 14 (3): 303–11. doi:10.1016/0891-5849(93)90027-R. PMID 8458588. 
2. ^ Ou B, Hampsch-Woodill M, 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 (10): 4619–26. doi:10.1021/jf010586o. PMID 11599998. 
3. ^ ^{a b} Nutrient Data Laboratory, Agriculture Research Service, US Department of Agriculture (May 2010). "USDA Database for the Oxygen Radical Absorbance Capacity (ORAC) of Selected Foods, Release 2 - May 2010". http://www.ars.usda.gov/SP2UserFiles/Place/12354500/Data/ORAC/ORAC_R2.pdf. 
4. ^ Huang D, Ou B, Prior R (2005). "The chemistry behind antioxidant capacity assays". J. Agric. Food Chem. 53 (6): 1841–56. doi:10.1021/jf030723c. PMID 15769103. 
5. ^ Kohri S, Fujii H, Oowada S, Endoh N, Sueishi Y, Kusakabe M, Shimmei M, Kotake Y (2009). "An oxygen radical absorbance capacity-like assay that directly quantifies the antioxidant's scavenging capacity against AAPH-derived free radicals". Anal. Biochem. 386 (2): 167–71. doi:10.1016/j.ab.2008.12.022. PMID 19150323. 
6. ^ Guidance for Industry, Food Labeling; Nutrient Content Claims; Definition for "High Potency" and Definition for "Antioxidant" for Use in Nutrient Content Claims for Dietary Supplements and Conventional Foods U.S. Department of Health and Human Services, Food and Drug Administration, Center for Food Safety and Applied Nutrition, June 2008
7. ^ ^{a b} Scientific Opinion on the substantiation of health claims related to various food(s)/food constituent(s) and protection of cells from premature aging, antioxidant activity, antioxidant content and antioxidant properties, and protection of DNA, proteins and lipids from oxidative damage pursuant to Article 13(1) of Regulation (EC) No 1924/20061, EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA)2, 3 European Food Safety Authority (EFSA), Parma, Italy, EFSA Journal 2010; 8(2):1489
8. ^ ^{a b} Williams RJ, Spencer JP, Rice-Evans C (April 2004). "Flavonoids: antioxidants or signalling molecules?". Free Radical Biology & Medicine 36 (7): 838–49. doi:10.1016/j.freeradbiomed.2004.01.001. PMID 15019969. 
9. ^ Frei, B. (April 10, 2009). "Controversy: What are the true biological functions of superfruit antioxidants?". http://www.npicenter.com/anm/templates/newsATemp.aspx?articleid=23667&zoneid=273. Retrieved April 10, 2010. ^{[dead link]}
10. ^ Arts, I.C. and P.C. Hollman, "Polyphenols and disease risk in epidemiologic studies." American Journal Clinical Nutrition, 2005. 81(1 Suppl): p. 317S-325S.
11. ^ ^{a b} Lotito SB, Frei B (2006). "Consumption of flavonoid-rich foods and increased plasma antioxidant capacity in humans: cause, consequence, or epiphenomenon?". Free Radic. Biol. Med. 41 (12): 1727–46. doi:10.1016/j.freeradbiomed.2006.04.033. PMID 17157175. 
12. ^ ^{a b c} "Studies force new view on biology of flavonoids", by David Stauth, EurekAlert!. Adapted from a news release issued by Oregon State University
13. ^ Ninfali, Paolino; Gloria Mea, Samantha Giorgini, Marco Rocchi, Mara Bacchiocca (2005). "Antioxidant capacity of vegetables, spices and dressings relevant to nutrition". British Journal of Nutrition (Cambridge University Press) 2005 (93): 257–266. doi:10.1079/BJN20041327. ISSN 0007-1145. PMID 15788119. 
14. ^ Tapsell, Linda C; Ian Hemphill, Lynne Cobiac, Craig S Patch, David R Sullivan, Michael Fenech, Steven Roodenrys, Jennifer B Keogh, Peter M Clifton, Peter G Williams, Virginia A Fazio, Karen E Inge (2006-08-21). "Health benefits of herbs and spices: the past, the present, the future". The Medical Journal of Australia 185 (4 Suppl): S4–24. ISSN 0025-729X. PMID 17022438. http://www.ncbi.nlm.nih.gov/pubmed/17022438. Retrieved 2009-09-23. 

External links

• ORAC levels of various foods These are "old" data from the ORAC assay used before 2004 and should be viewed only as qualitative rankings. A revised ORAC assay used since 2004 provides quantitative values several times higher than these measurements. See an abstract of the reference report at this link from PubMed.
• Database of ORAC values based on 2007 USDA study, sortable alphabetically or by ORAC level
• Effect of soaking, boiling, and steaming on total phenolic content and antioxidant activities of cool season food legumes, Feb 2, 2008.