Determining ORAC

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.