Antioxidant testing: an ORAC update; Evaluating antioxidant capacity is challenging but ORAC and other associated methods may be the best solution amidst all of the confusion.

Over a timeline that spans 138 years of research, the U.S. Agricultural Research Service (the in-house research arm of the U.S. Department of Agriculture) lists the automation of the Oxygen Radical Absorbance Capacity assay (commonly referred to as the ORAC assay) method as one of its major research accomplishments (1). (A detailed description of the ORAC assay was discussed previously in NUTRACEUTICALS WORLD in September 2002 (2).) The original method was developed by Cutler and Cao (3) at the National Institutes on Aging in Baltimore, MD, but this method lacked automation and was cumbersome when measuring the ORAC value of many samples side-by-side. Eventually, Dr. Cao moved to Dr. Ronald Prior's laboratory at the USDA Human Nutrition Research Center on Aging at Tufts University in Boston, MA, taking the method with him. The original objective was to determine if nitric oxide production would alter in vivo antioxidant activity. But before they continued their research they wanted to know if the method could be used to evaluate foods. According to Dr. Prior, who is still involved in ORAC/antioxidant research, the results "really changed the direction of our research program. At that point, we had no idea that the method would have such widespread use."

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Although the fundamental chemical principles and mathematical model behind the ORAC method have remained the same since 1994, it has undergone some significant changes and improvements. In 2001, scientists at Brunswick Laboratories, Inc., Wareham, MA, working in collaboration with Dr. Prior, replaced the original fluorescent probe, b-phycoerythrins (PEs), with fluorescein (FL) (4). FL as compared to PEs does not interact with antioxidants, shows excellent photostability and reduces the cost of experiments. Soon afterward, additional progress was made when a method was developed and validated to measure the ORAC of lipophilic antioxidants using randomly methylated beta cyclodextrin to enhance solubility (5).

In addition to the ORAC assay, which determines free radical scavenging activity against the peroxyl radical for both water-soluble and lipid-soluble substances, Brunswick Laboratories has also developed assays to determine free radical scavenging activity against other free radicals, including the peroxynitrite and hydroxyl radicals (known as NORAC and HORAC).

The collaboration between Brunswick Laboratories and Dr. Prior continues. In 2003, Brunswick Laboratories received a Small Business Innovation Research (SBIR) grant from the USDA to develop ORAC assays for singlet oxygen, hydroperoxide and superoxide anion. Given the method's inherent scientific substantiation and continued USDA support, the ORAC assay has all the makings of the method-of-choice for in vitro and in vivo antioxidant testing of bioactive markers. When complete, this panel of ORAC tests will provide a comprehensive antioxidant profile with important applications across a broad range of nutritional and human health markets.

ORAC: A Critical Review

These timely developments could not have come soon enough for several reasons. First, some critics point out that ORAC does not measure the ability to prevent the formation of oxidants. Under most living conditions, humans are subjected to physiological and environmental stresses that overwhelm their bodies' natural defenses. Prevention of free radical formation is the role of endogenous antioxidants produced by our body. It is commonly understood that in order to combat harmful oxidation in our bodies we must supplement our natural defenses with antioxidants from other sources. The measurement of the antioxidant capacity of various sources, then, becomes important.

Antioxidant capacity is defined as the ability of a compound to reduce pro-oxidants. Free radicals are produced in situ (in the body) and serve important biological functions. Ingested nutrients provide a diverse and complex supply of antioxidants to combat excessive free radical activity. There are seemingly limitless combinations of internal and external antioxidants and the radicals they combat. This might explain why no single measure of antioxidant status is going to provide a sufficient amount of data to evaluate in one assay the free radical scavenging activity of a food in vitro or its potential antioxidant activity in vitro. Then what does the ORAC measure?

The most popular ORAC assays measure the scavenging capacity of antioxidants in nutrients or in vivo against the peroxyl radical, which is one of the most common reactive oxygen species (ROS) found in the body. The ORAC-hydro assay reflects water-soluble antioxidant capacity, while the ORAC-lipo assay measures lipid-soluble antioxidant capacity. The values of these two assays are additive. Trolox, a water-soluble vitamin E analog, is used as the calibration standard and the ORAC result is expressed as micromole Trolox equivalent (TE) per gram. These popular ORAC assays do not measure the scavenging capacity against the larger body of free radicals. However, it is a valid and meaningful representation of antioxidant capacity.

And it is becoming an increasingly useful tool for consumers. It is estimated that only 10% of the U.S. population consumes five servings of fruits and vegetables per day. As consumers become more familiar with ORAC as a measure of antioxidant intake, it will become a standard bearer that can help nutraceutical companies educate consumers on the comparative antioxidant benefits of products.

Hence, it is welcome news to the nutraceutical industry that Brunswick Laboratories recently developed and brought into commercial use additional assays to measure free radical scavenging activity in vitro, namely HORAC and NORAC. They measure quenching capability against the peroxynitrite and hydroxyl radicals, respectively. (Boxin, O. et al. J Agric Food Chem, 2002; 50: 2772-2777). Several studies have found that in Alzheimer's disease, Parkinson's disease and other degenerative neurological diseases, peroxynitrite levels rise as evidenced by the accumulation of nitrotyrosine. Yet, relatively few products are being measured for NORAC, despite fears by millions of the epidemic increase in neuro-degenerative diseases.

The hydroxyl radical is a third generation species of radicals derived from hydrogen peroxide, which in turn is derived from the superoxide radical though the action of the enzyme superoxide dismutase (SOD). These radicals in excess can be extremely harmful in vivo. The need to round out the panel of assays with the SOD would provide a more complete picture of the total antioxidant activity of a product.

Without such a convenient suite of assays (ORAC, HORAC and NORAC), assessing the level of the hydroxyl, peroxyl and peroxynitrite radical activity would require use of a variety of competing methodologies offered around the world, including the trolox equivalent antioxidant capacity (TEAC) method, the 2,2-azinobis-3-ethylbenzothiazoline-6-sulphonate (ABTS) method, the total radical-trapping antioxidant (TRAP) method, the 2,2-diphenyyl-1-picrylhydrazl (DPPH) method, the dichlorofluorescin-diacetate (DCFHDA) method and the N,N-dimethyl-p-phenylenediamine (DMPD) method, to name just a few.

The problem created by so many methods, some of which lack the desired degree of validity, robustness, specificity, and reliability desired by analytical chemists, or suffer from interference confounders, is the difficulty of correlating one method against another. For this reason, the more familiar ORAC assay, now beginning its second decade of use, allows companies to compare their product against other products amongst more than 8000 assays performed on hundreds of food and supplement products. That is not only a considerable data bank, but invaluable to companies wishing to substantiate antioxidant claims against valid and robust standards.

Second, critics also charge that using an in vitro assay to imply in situ in vivo benefits is invalid. A corresponding ORAC value cannot be assumed, however, researchers have made significant gains in being able to compare in vivo and in vitro outcomes. Comparative studies in vitro and in vivo have included investigations of metabolizing enzymes and ORAC in the liver of animals. A good example is a paper by Marnewick and colleagues that examined the modulation of hepatic metabolizing enzymes and oxidative status by rooibos tea (Aspalathus linearis), green and black teas (Camellia sinensis) and honeybush tea (Cyclopia intermedia) in rats (Marnewick JL, Joubert E, Swart P et al. "Modulation of hepatic drug metabolizing enzymes and oxidative status by rooibos (Aspatlathus linearis) and honeybush (Cyclopia intermedia), green and black (Camellia sinensis) teas in rats." J Agricul Food Chem, 2003;51:8113-8119). In this study, antioxidant activity was reflected by the redox state of glutathione and the ORAC assay in liver of rats exposed to the various tea preparations. The enzyme assays included glutathione stransferase assay, UDP-glucuronosyltransferase assay, total glutathione (GSH and GSSH) analysis, and ORAC.

As these kinds of studies begin to extend to human investigations, invaluable supportive data may emerge to substantiate claims of antioxidant activity in vivo for products based on inferential data from in vitro results. Given all the attention to the "French Paradox" for over 10 years, and evidence that serum antioxidant activity is higher in healthy volunteers who drink red wine than those who do not (Lancet, 1994;344:193), there is no question ORAC will begin to appear more often in reference to in vivo antioxidant activity rather than just as in vitro value.

Third, critics have complained that ORAC has received too much attention compared to other procedures. Since its original claim in 1993 that the ORAC method represents a "relatively simple but sensitive and reliable method of quantifying the oxygen-radical absorbing capacity of antioxidants," the method has lived up to its original billing (Cao G, Alessio HM, Cutler RG. "Oxygen radical absorbance capacity assay for antioxidants." Free Radic Biol Med, 1993;14:303-311). There is no question that ORAC has gained considerable brand recognition as a standard for antioxidant capacity, especially for suppliers and manufacturers. But the future of ORAC or any antioxidant assay will depend upon the responsible use of results when making comparisons.

There is a reason why a red grape's ORAC value would be lower than a dehydrated red grape powder. Knowledgeable individuals will know that this is due to the difference in moisture content. Care must be shown by the nutraceuticals industry to compare apples to apples. Red grape extract would very likely have a much higher ORAC value, but this does not mean that from a cost, dose and/or serving size comparison standpoint one product is superior to the other. Manufacturers and suppliers should take note: DHSEA clearly states that in advertising and marketing the "truthful and misleading" yardstick should rule the day when making antioxidant unit comparisons. If comparisons are made let them be made on the basis of sound analyses, otherwise the nutraceutical industry risks destroying the currency of ORAC in the minds of consumers as its value in product comparisons grows.

ORAC's Influence in the Market

Who is using ORAC to market their products and services? A quick "google" search of "ORAC and antioxidants" provided 3880 hits, going fa. beyond the expected dietary supplement company's websites. The search also brought up research articles, laboratories, nutrition and health education sites for the consumer, food companies (two major dried plum manufacturers have ORAC information on their website) and food commissions, boards and associations, such as the Washington Red Raspberry Commission and the Wild Blueberry Association of North America. It appears everyone is jumping on the antioxidant bandwagon, and incorporating ORAC into their marketing efforts seems to be the most concrete means of explaining antioxidants to their audience.

Supplement products have been competing against each other for the title of "Highest ORAC Value." However, with competitive use of ORAC values comes misconceptions and misuse. Are all these references to ORAC values comparable to each other? The translation to consumer vocabulary can be quite confusing. The ORAC value of foods is often reported per 100 grams, while ORAC value for supplements is reported per gram and often the units are completely left out. Many companies are comparing ORAC values of their products to equal antioxidant activity of servings of fruits and vegetables, but which fruits or vegetables and what is the serving size? For example, one company's website claimed its product contained equal antioxidant activity as compared to 10 servings of fruits and vegetables, then further defines the equivalence by listing a head of lettuce and other low ORAC vegetables as part of the 10 servings.

Even some early articles published by the USDA can be misleading. An article published by USDA in 1999 shows a chart comparing ORAC values of a number of fruits, including their dried counterparts (6). According to this article, prunes have an ORAC value six times greater than fresh plums, but in actuality their ORAC values should be very close based on dry weight or calories. In addition, there's still the same old issue of label claim versus actual content, which the supplement industry has been struggling with since its inception. Just because a label claims the product has 5000 ORAC units per serving, are we sure it really does?

Resolving these issues won't be easy. It will take a concerted effort on the part of the manufacturers to promote ORAC values using the same language; confusing the consumer will not help sell antioxidants. As for validation of ORAC value in final products, it is the responsibility of the manufacturer to have a quality control (QC) mandate to guarantee ORAC values in the same way they guarantee other content claims. As more ORAC data on fruits and vegetables become available based on the new fluorescein method (most data available on fruits and vegetables is using data from old method), the issues of recommended ORAC dosages and ORAC values per serving will be solved.

Dr. Prior's lab at the USDA has been working diligently in this area. They have completed analyses of over 100 samples, the majority being fresh fruits and vegetables. Based on their work, which will be published some time this year, Dr. Prior says that seven to 10 servings of fruit and vegetables can contain anywhere from 8900 to 17,500 ORAC units depending on the mix of foods. This may seem like a pretty broad range, but it takes into account the many combinations of fruits and vegetables that can make up seven servings and that fresh fruits have a higher average ORAC value--almost four times the average ORAC value for vegetables. Again, it will still be up to the industry to use this data without misleading the consumer. Perhaps making ORAC comparisons to specific fruits or vegetables can help eliminate the ambiguity.

The challenge, then, is how do we make ORAC a successful tool for making antioxidants more consumer-friendly? The equation for this success must include three major elements. First, we must use similar language in comparing ORAC values to real foods. Second, although the USDA does plan to release ORAC information on its food database website in the future, it does not have any specific plans for further educating the consumer on ORAC. This leaves the education component of the ORAC equation to the supplement, functional food and conventional food industry. Finally, we need to focus on the third piece of the equation: the product label. How ORAC can be used on the product label leads to a discussion and clarification of the regulatory issues, which is an issue that should be part of future conversations.

This article in a nutshell:

* ORAC: a critical review

* ORAC's influence in the market

References

1. Cao G.; Verdon C.P.; Wu A.H.B.; Wang H.; Prior R.L. "Automated Assay of Oxygen Radical Absorbance Capacity with the COBAS FARA II." Clinical Chemistry 1995;41(12):1738-1745.

2. Bank, G.; Lenoble, R. "Oxygen Radical Absorbance Capacity, Standardizing the Way We Look at Antioxidants." NUTRACEUTICALS WORLD September 2002;42-45.

3. Cao, G.H.; Alessio, H.M.; Cutler, R.G. "Oxygen Radical Absorbency Capacity Assay for Antioxidants." Free Radical Biol Med 1993;14(3):303-311.

4. Ou B.; Hampsch-Woodill M.; Prior R.L. "Development and Validation of Oxygen Radical Absorbance Activity using Fluorescein as the Fluorescent Probe." Journal of Agricultural and Food Chemistry 2001;49:4619-4626.

5. Huang, D.; Ou, B.; Hampsch-Woodill, M.; Flanagan, J.A.; Deemer, E.K. "Development and Validation of Oyxgen Radical Absorbance Capacity Assay for Lipophilic Antioxidants using Randomly Methylated Cyclodextrin as the Solubility Enhancer." Journal of Agricultural and Food Chemistry 2002;50(7):1815-1821.

6. "Can Foods Forestall Aging." Agricultural Research Feb 1999.

About the authors: Alexander Schauss, PhD, FACN, CEDS, is the director of natural and medicinal products research, Life Sciences Division, American Institute for Biosocial and Medical Research, Inc., Puyallup, WA. He has studied nutrition and botanical medicine for over 30 years and has authored over 100 papers for various prominent publications spanning a wide range of topics. He can be reached at 253-286-2888; E-mail: [e-mail]. Ginny Bank is technical director at RFI Ingredients, Blauvelt, NY. Ms. Bank has been researching and developing antioxidant products for 10 years and holds four U.S. patents pertaining to antioxidants and their applications. She can be reached at 720-304-7335; E-mail: [e-mail].

By Ginny Bank, MA Technical Director, RFI Ingredients, Blauvelt, NY & Alex Schauss, PhD, FACN, CEDS, Director of Natural and Medicinal, Products Research, Life Sciences Division, American Institute for Biosocial and Medical Research, Inc., Puyallup, WA