Dealing with Antioxidant Assay Issues

Numerous tests have been developed to screen substances for their antioxidant activity for potential use in foods, nutraceuticals, and supplements, and companies utilize the tests to promote the antioxidant capacity of their products. But Karen Schaich ([email protected]), Professor in the Dept. of Food Science at Rutgers University, said that there are drawbacks to these tests and no standardization of procedures and reporting. In addition, she said that results are often used inappropriately.

Schaich is participating in a task force formed in February 2009 by the Analytical Chemistry Division of the International Union of Pure and Applied Chemistry (www.iupac.org) to deal with these issues. The task force expects to complete its evaluation and submit recommendations for standardization to IUPAC in early 2011.

The task force is meeting in Istanbul,Turkey, as this issue goes to press, and an Antioxidants 2010 conference is being held in Brussels, Belgium (www.ni-antioxidants.com) the following week. Schaich will be presenting the same message at both events, namely, that there must be standardization of methodologies, more basic research on the actual assay chemistry, and abandonment of in-vitro assays for predicting in-vivo activity and making dietary choices.

Currently, there is no single antioxidant assay for food labeling, she said, for several reasons:
• The international scientific community has not yet agreed on which methods and procedures should be standardized.

• Antioxidant activity is not a single reaction but encompasses multiple mechanisms—hydrogen atom or electron transfers to quench radicals, metal chelation, singlet oxygen quenching, and others—in both aqueous and lipid phases. No single assay detects them all.

• Specific antioxidant actions needed vary with the system.

Drawbacks of Current Methods
Current methods of antioxidant analysis are briefly described in the sidebar at the end of this article. The most widely used method is the Oxygen Radical Absorbance Capacity (ORAC) assay developed by researchers at the U.S. Dept. of Agriculture’s Agricultural Research Service and used to determine the ORAC values for foods and beverages that appear in the USDA National Nutrient Database for Standard Reference (www.ars.usda.gov/Services/docs.htm?docid=8964). Schaich has concerns about the method.

She said that it is usually conducted using a single concentration of antioxidant or extract but should be conducted at a range of concentrations, and the reaction conditions should be carefully controlled. For example, some test substances are sensitive to light and temperature, so samples stored in brown bottles under refrigeration give different results than those left on the benchtop in clear glass. And commercial autosamplers provide imperfect temperature control. Since the heating tray moves in and out, thermal equilibrium at the 37°C required to decompose azide initiators is never reached. As a result, researchers can’t rely on the oven temperature settings but should measure the temperature in the microwells instead.

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Another problem is interaction of the sample with the fluorescein reagent currently used in the test. Some phenols bind to it, probably through hydrogen bonding or pi interactions, preventing decay of fluorescein fluorescence. When this happens, the test overestimates antioxidant activity, giving extraordinarily high ORAC values that are unrelated to actual radical scavenging activity.

In addition, ORAC values are often reported without accompanying units. They should be reported on a standard basis with units, such as mg of Trolox equivalent per fixed weight or volume. Otherwise, the results are meaningless and can be easily manipulated to make a product look good, she said.

In her opinion, industry should not be selling products on the basis of ORAC test results, anyway. USDA’s recommended consumption of 5,000 ORAC units/day can be provided by eating one apple or 1 oz of pecans, so why suggest eating large amounts of high-antioxidant foods and fortifying many foods with antioxidants? Looking at ORAC values another way, she said that if Trolox is comparable in antioxidant activity to α-tocopherol, then 5,000 ORAC units is equivalent to 3,230 international units of α-tocopherol. However, only 30 IU of α-tocopherol is needed to prevent nutritional deficiency, and 400 IU is the maximum recommended supplement level. In this context, 5,000 ORAC units is way too much. So choosing what to eat on the basis of ORAC values doesn’t make sense, she concluded.

The other two most common antioxidant assays, TEAC and DPPH, have their own problems, Schaich said. The ABTS^+• and DPPH^• radicals used in the assays are nitrogen-centered and sterically hindered. Nitrogen radicals are not good models for antioxidants, since radicals involved in oxidative deterioration are oxygen-centered and phenols react preferentially with oxygen radicals. Also, the steric hindrance means that molecular size controls reaction more than chemistry.

TEAC should be abandoned, she said, because ABTS^+• reactions reflect almost entirely steric hindrance rather than specific reactivity. Flavonoids, for example, are large molecules, so they always react more slowly than single phenols such as gallic acid or small reducing agents such as ascorbic acid. In addition, ABTS^+• and DPPH^• radicals can be quenched by both hydrogen and electron transfer, and the dominant reaction varies with the antioxidant, solvent, and pH, complicating comparison of results in different systems.

She added that every in-vitro assay needs to be studied from a basic chemical standpoint, using optimum reaction conditions, to learn more about what drives antioxidant reactions rather than using them merely as screening assays.

It’s becoming increasingly clear, Schaich said, that in-vitro assays are not good models for what happens in humans. To be active in vivo, antioxidants must be absorbed from the gastrointestinal tract, and this is not modeled in current assays. Actual absorption of polyphenols is extremely low, and there is little evidence that consuming large quantities of polyphenols can increase it. Most absorbed phenols are rapidly conjugated and eliminated, in-vivo pharmacokinetics and tissue distribution are unknown, and endogenous antioxidants that form the main line of physiological defense are largely unreactive in in-vitro assays. Cell-culture assays purport to measure some cell response, she said, but the assays are irrelevant if the test cells never actually see antioxidants in vivo or are exposed to antioxidant levels many orders of magnitude lower than assay concentrations commonly used.

A further problem, she said, is that responses are almost always interpreted as the antioxidant acting by free-radical scavenging without considering or measuring other effects such as protein binding and signal transduction. That is not to say that antioxidants do not have biological activity, but their direct effects may be limited to the GI tract, where they may quench radicals but also bind to toxic dietary components and prevent their absorption, mediate redox tone, interact with intestinal flora, or be metabolized into small active compounds.

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There is increasing recognition that the most important action of antioxidants in vivo may be indirect, mainly through signal transduction, she said. One possible mechanism is that phenolic antioxidants bind to epithelial proteins throughout the GI tract, causing changes that induce proteases to degrade the proteins. The released peptides then bind to genes and set off a cascade of events that lead to systemic responses and mobilization of endogenous defenses. For example, studies have shown that consumption of apples increased the total antioxidant capacity of blood plasma but that the activity resulted from increased endogenous uric acid rather than absorbed phenols. Levels of endogenous tocopherol, glutathione, and antioxidant enzymes may be similarly increased when phenols are consumed in other foods.

Given all these limitations, Schaich concluded, there is no basis or rationale for developing or endorsing a single assay for antioxidant activity of foods, food ingredients, or supplements. Besides, she added, foods have other nutritional value besides antioxidant activity, and these need to be considered along with antioxidant activity when selecting a balanced diet. (See sidebar on flavanols in cocoa products.)

Opportunities in Formulation and Waste Utilization
Schaich said that even if antioxidant assays are inappropriate models for health effects, the chemistry they test does occur in foods, so they can be refocused to identify natural antioxidants or combinations potentially useful as replacements for synthetic antioxidants such as BHA and BHT. If used more definitively, current antioxidant assays can provide important information about how antioxidants act under different conditions, data that will be critical for developing new applications in foods.

She suggested that once we know more about the specific actions and interactions of natural antioxidants, we may be able to combine foods to increase product stability without use of synthetic antioxidants. Research at Brigham Young University on synergisms and antagonisms of phenol mixtures suggests possibilities such as adding strawberry juice to lemonade to improve the stability, flavor, and color, since polyphenols in strawberry juice can inhibit oxidation of unstable citrus oils. Similarly, since cooked meat undergoes autoxidation, combining a food such as fruit or an herb that has high antioxidant activity with meat can provide oxygen radical scavenging and prevent or delay development of undesirable warmed-over flavor. Such combinations are common in culinology for flavor and color reasons, she said, but now have a scientific basis, as well.

Schaich’s group is also working to identify high antioxidant activity in waste or leftover materials, such as coffee grounds, potato peels, apple peels, or the outer leaves of cabbage. Instead of extracting antioxidants from edible materials, she said, we can use the antioxidant assays to identify active components in materials that would normally be discarded. This would add value and increase waste utilization, while providing new natural ingredients. The group routinely uses a battery of antioxidant tests, including at least DPPH, ORAC, FRAP, and EPR, along with composition analysis to quantify the antioxidant activity of these materials and learn more about their reaction mechanisms and specificities.

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Flavanols More Than Just Antioxidants
Mars Inc. and Barry Callebaut AG announced in February 2010 an agreement to cooperate in producing flavanol-rich chocolate and cocoa products containing guaranteed levels of flavanols.

Flavanols, natural compounds found in cocoa, have been linked to important circulatory and other health benefits, according to Hugo Pérez ([email protected]), spokesperson at Mars Botanical, a unit of Mars Inc., but manufacturers have struggled with consistency, reliable measurement methods, and communication of the benefits and level of flavanols in foods. The collaboration will address these issues.

Carefully handling the product throughout the manufacturing process is the first step toward guaranteeing a standard level of active flavanols, Pérez said. Flavanols are naturally abundant in cocoa, but unless you are specifically measuring them and carefully handling a product throughout the manufacturing process, there is no guarantee that final products contain meaningful flavanol levels.

While flavanols may act as antioxidants in a test tube, he said, their cardiovascular benefits have little to do with their antioxidant properties. The actual flavanol content of a food may be a better indicator than antioxidant activity when evaluating the potential value for health and nutrition in this context, he said, and the companies are therefore emphasizing flavanol content rather than antioxidant activity of their products.

Today, there are multiple ways manufacturers describe the flavanol content in their products, Perez said, some based on active flavanols, some based on simply the physical amounts or percentages of cacao powder in the product. Mars and Barry Callebaut will develop a standard measurement method and standard language for communicating amounts of active flavanol compounds in products, including use of a “bean-in-hand” logo on packages as a guarantee of flavanol content.

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Antioxidant Assays
The following are brief descriptions of antioxidant assays:
• Folin-Ciocalteu Total Phenolic Assay. This assay determines total phenolics in foods by measuring the change in color when metal oxides are reduced by polyphenolic antioxidants. The resulting blue solution has maximal absorption at 765 nm. Standard curves are prepared using gallic acid, and results are reported as gallic acid equivalents.

• ORAC. The Oxygen Radical Absorbance Capacity assay measures the fluorescence of a target attacked by free radicals. When a free-radical generator such as an azo compound is added to a fluorescent target such as fluorescein and heated in the presence of oxygen, it produces peroxyl free radicals that damage the target molecule, resulting in the loss of fluorescence. Antioxidants intercept the peroxyl radicals and prevent the loss of fluorescence. Areas under curves of target fluorescence intensity vs time with and without addition of an antioxidant are calculated and compared to a standard curve generated using Trolox™, a water-soluble vitamin E analog trademarked by Hoffman-LaRoche.

• PSC. The Peroxyl Radical Scavenging Capacity assay is a variant of ORAC that uses dichlorofluorescein as the radical target.

• TRAP. The Total Radical-Trapping Antioxidant Parameter assay, similar to ORAC, measures the decay of a fluorescent target during a controlled peroxidation reaction initiated by radical generators such as azides or Fenton reactions (H₂O₂ + Fe²⁺). TRAP values are usually calculated from the length of the lag-phase caused by the antioxidant compared to that of Trolox.

• DPPH. This assay uses a UV-visible spectrophotometer (515 nm) or electron paramagnetic resonance (EPR) spectrometer to measure the ability of antioxidants to reduce the stable deep-purple 2,2-diphenylpicrylhydrazyl (DPPH) radical.

• TEAC. The Trolox Equivalent Antioxidant Capacity assay measures the ability of antioxidants to reduce the blue-green radical 2,2’-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid), ABTS^{+ •}, decolorizing it. The reaction is followed at 734 nm using a spectrophotometer.

• TOSC. The Total Oxyradical Scavenging Capacity assay measures production of ethylene when peroxyl radicals react with α-keto-γ-methiolbutyric acid (KMBA). Antioxidants intercept the peroxyl radicals, decreasing production of ethylene, which is measured by gas chromatography.

• SIFT-MS. The Selected Ion Flow Tube Mass Spectrometric assay developed by Syft Technologies Ltd. (www.syft.com) is based on the TOSC assay.

• FRAP. The Ferric Reducing/Antioxidant Power assay measures the ability of antioxidants to reduce the complex of Fe(III)- 2,3,5-triphenyl-1,3,4-triaza-2-azoniacyclopenta-1,4-diene chloride (TPTZ) to Fe(II) at low pH. The reduction is quantitated by change in absorption at 593 nm.

• CuPRAC. The Cuprıc Ion Reducıng Antıoxıdant Capacıty assay measures the ability of antioxidants to reduce neocuproine-copper(II) complexes to copper(I) at pH 7. Change in absorbance at 450 nm is monitored for 30 min to calculate rate and extent of reaction.

• Crocin CL. The Crocin Chemiluminescence assay measures the ability of antioxidants to prevent bleaching of the yellow-orange color in water-soluble carotenoid crocin by radicals generated by thermal decomposition of azo-initiators in the presence of oxygen. Color loss is monitored at 440 nm for 10 min, and results are expressed as relative bleaching rate.

• TAC. Total Antioxidant Capacity refers to any of the above assays applied to plasma. It measures effects of all antioxidants present, both endogenous (including proteins) and dietary.

• Chronocoulometric Assay. This single-step electrochemical assay—developed at the University of Maryland and reported on at the 2009 IFT Annual Meeting & Food Expo (paper 025-43)—uses chitosan-coated electrodes to estimate polyphenolic contents.

• CAA. The Cellular Antioxidant Activity assay—developed at Cornell University—is a cell-culture method designed to register bioavailability, uptake, and metabolism of antioxidants. It measures the ability of antioxidants to prevent oxidation of dichlorofluorescin by azide-generated peroxyl radicals in human hepatocarcinoma HepG2 cells. The decrease in cellular fluorescence compared to the control cells indicates the antioxidant capacity of the compounds.

by Neil H. Mermelstein, a Fellow of IFT, is Editor Emeritus of Food Technology
[email protected]