Determining ORAC

Unraveling the science of food is a multidisciplinary effort involving a diverse range of chemists, life-sciences experts and physicians. As a result, many of the milestones in food and nutrition have been an outcome of combined biological (in vivo) and analytical (in vitro) assays.

For example, Harry Steenbock's pioneering work with vitamin D was based in large part on work with animals, and a bioassay was the primary method for measuring that compound for decades. More recently, there has been an almost continuous debate over the use of the biological protein efficiency ratio (PER) and the protein digestibility corrected amino acid score (PDCAAS) assays for determining the quality of protein. Now, as the potential therapeutic value of antioxidants becomes further elucidated, a sophisticated approach to determine the efficacy of these substances is being developed.

Antioxidants help break down or "quench" the cell-damaging compounds known as free radicals that are thought to contribute to cancer, cardiovascular disease and cognitive decline. The antioxidants include a broad range of food components, such as vitamins E and C, carotenoids and polyphenols.

The quantitative measurement of vitamins is a routine procedure, and assays to determine levels of other antioxidants are quickly advancing. However, progress in determining the link between dietary content and physiological benefit relies on an effective measurement of biological potential. The oxygen radical absorbance capacity (ORAC) test is an in vitro or in vivo assay designed to measure the total antioxidant power of foods and other chemical substances by tracking chemical biomarkers.

Understanding ORAC

In 1993, physician and chemist Dr. Guohua Cao and his colleagues at the National Institute on Aging, Bethesda, MD, first published the principles of the ORAC assay. The assay combines a sample with a chemical marker that fluoresces and an oxidizing agent. Because the sample can be biological (e.g., blood or tissue) or chemical (e.g., a food extract) the potential applications were theoretically endless. When Cao joined forces with nutritionist Ronald Prior, Ph.D., at USDA's Human Nutrition Research Center on Aging at Tufts University, Boston, the impact of the assay for the evaluation of food became apparent.

Although other methods had been developed to measure antioxidant potential, the ORAC assay had several significant advantages. According to Prior, one key to the success of the ORAC assay is the choice of the oxidizing agent. "Other methods are easier to perform, and thus, many take the easy way out," he says. "However, ORAC still has the most relevance to biology since it utilizes a peroxyl radical generator in the assay."

The peroxyl radical is not only one of the most common reactive oxygen species (ROS), but is also reactive with both water- and lipid-soluble substances, which is key when determining total antioxidant potential.

For food analysis, a freeze-dried sample undergoes an extraction and drying process. The extract, along with a peroxyl radical, such as 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH), and a fluorescent probe are mixed for injection into an analyzer. In Prior and Cao's original research a ß-phycoerythrin probe was utilized. In 2001, this was replaced with a fluoresein (FL) probe. This provided some chemical benefits and was less costly. The modified test method using the FL probe is sometimes denoted as "ORACFL." At the same time, the monumental breakthrough of measuring lipophilic radicals was validated. When combined with the hydrophilic assay, ORAC became the first tool for measurement of "total antioxidant capacity."

The extract, radical and probe mixture is added to the wells of a microplate reader, which contains a detector to measure the strength of the light emitted from the marker. Reference standards and blanks are also included in the sequence. Readings begin 0.5 seconds after the start of the sequence and continue at regular intervals (e.g., two minutes). As the battle between the free radicals and antioxidants progresses, it uses up more of the fluorescing marker until none is left and the level of fluorescence is zero. The measurement and inclusion of all inhibition from the beginning to the end of fluorescence represents another significant advancement.

During development of the assay, researchers were hamstrung by a lack of suitable instrumentation with much of the work being done on an analyzer that had already been discontinued by the manufacturer. "I think a major roadblock in more-widespread use of the method has been the availability and expense of instrumentation to perform the procedure," says Prior.

In addition, the manual process of sample preparation was both time-consuming and labor-intensive. The ORACFL assay was developed on a high-throughput platform that consists of a state-of-the-art robotic eight-channel liquid handling system with attached centrifuge for mixing of samples and a microplate fluorescence reader. This system significantly improved the assay and allowed the analysis of up to 30 samples in one sequence. Validation of the method and increased availability of instrumentation should accelerate the availability and acceptance of the assay.

Translation of the data

A key to all methods is the selection and use of an appropriate reference standard to which sample data can be compared and applied within a mathematical model. In the case of ORAC, it is an analogue of vitamin E known as Trolox® (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). Trolox is a widely accepted standard for antioxidant activity, and ORAC results are expressed as micromole (µM) Trolox equivalents (TE) per gram.

The value is attained by determining the area under the curve of the sample (i.e., the quenching curve) and subtracting the area under the blank curve. The same calculation is done with the Trolox standard curve and corresponding blanks. The resulting net protection area of the sample is then divided by the Trolox net protection area. One ORAC unit is equivalent to the net protection area provided by 1 µM of Trolox. Adding the results of the hydro- and lipophilic assays provides a total ORAC score.

Prior's research on 40 food matrices showed that the water-soluble ORAC values ranged from 7.34 ±0.85 to 568.0 ±14.2 µM of Trolox equivalents per gram. The fat-soluble ORAC values ranged from 1.00 ±0.06 to 31.23 ±0.22 µM and accounted for approximately 0.59% to 39.51% of the total ORAC value. These results demonstrate that measures of total antioxidant capacity of foods need to include both the water- and lipid-soluble antioxidant in order to have an accurate total for a given food sample.

A clinical connection

While chemists and manufacturers wrestle with the validation and value of the ORAC assay in product development, the use and influence of the assay in biological research is expanding. After all, knowing the ORAC value of a compound is only part of the puzzle. Linking consumption of products with high ORAC scores to a corresponding value in human health is another story. By using the ORAC assay with plasma and other biological samples, researchers can measure the antioxidant levels in plasma and tissues from animals and humans fed diets fortified with certain foods or phytochemicals.

Although the studies are far from conclusive, results seem to show a definite connection. For example, studies on middle-aged rats fed diets with ORAC-standardized antioxidants showed evidence of an effect on the loss of long-term memory and learning capacity. Additionally, human-plasma samples from volunteers fed fortified diets of certain antioxidant foods/substances indicate an increase in the antioxidant power of the human body.

However, the interpretation and application of ORAC scores for comparative use still requires some work. For example, the origin and manufacturing of ingredients can have a tremendous effect on ORAC values. "I believe the ultimate impact on food manufacturers and an area that we have limited data on is the effect of processing on many of these antioxidant compounds," says Prior. "Processing, in some cases, seems to have very devastating effects on some of these antioxidants."

One of the goals of the USDA project is to develop a database containing ORAC values for a widespread range of representative matrices. This would allow consumers and scientists alike to make more-accurate comparisons between products and significantly enhance ongoing research.

Only the beginning

Although not yet universally accepted as a definitive method, ORAC is becoming one industry standard for measuring antioxidants. However, due to the complexity of the factors involved, no single assay will be sufficient for assessing the power of these compounds. The recent development of the NORAC and HORAC assays, to measure capability against the peroxynitrate and hydroxyl radicals respectively, represent another addition to the battery of tools at our disposal. Through an ongoing collaboration of disciplines, scientists will be able to expand our understanding of the effect of the diet on the prevention of maladies such as cancer and cardiovascular disease.

Richard Crowley is the editor of the Covance Food Science newsletter and the author of numerous articles on food analysis. He has a B.S. in Agricultural Journalism from the University of Wisconsin–Madison and is a member of the National Association of Science Writers.

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