What is the ORAC Assay?
The Oxygen Radical Absorbance Capacity (ORAC) assay is a laboratory-based method designed to measure the total antioxidant power of a substance, such as a food, beverage, or supplement, against certain free radicals. The test is conducted entirely in vitro, or in a test tube, and provides a quantitative score of a substance's ability to neutralize these damaging molecules. The ORAC score is expressed in units known as Trolox Equivalents (TE), referencing a water-soluble vitamin E analogue used as a standard. While once popular, the scientific community now views ORAC scores with significant caveats, primarily due to the disconnection between laboratory conditions and the complex biological processes within the human body.
The Core Chemical Components
For the ORAC assay to work, a precise combination of chemical components is necessary. Each plays a distinct role in the reaction and measurement process:
- Free Radical Initiator: A compound known as 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH) is heated to thermally decompose and generate a steady stream of peroxyl radicals. These are the damaging free radicals the assay is designed to measure.
- Fluorescent Probe: A fluorescent molecule, most commonly fluorescein, is included in the reaction mixture. When exposed to the free radicals, the probe is oxidized, causing its fluorescence intensity to fade over time. This decay is the measurable signal.
- Test Sample: The substance being tested for its antioxidant capacity is added to the mixture. If it contains antioxidants, they will react with and absorb the free radicals, protecting the fluorescent probe and inhibiting its decay.
- Trolox Standard: A series of known concentrations of Trolox, the vitamin E analogue, are also run in the assay to create a standard curve. This curve allows researchers to quantify the antioxidant activity of the unknown sample by comparing its protective effect to that of the Trolox standards.
The ORAC Assay Procedure
The ORAC value is typically measured in a microplate reader, a device that can process many samples simultaneously. The procedure follows several key steps:
- Sample Preparation: The test sample (e.g., a food extract) is prepared, and various dilutions are made. It might be separated into water-soluble and lipid-soluble fractions to determine total antioxidant capacity.
- Plate Setup: The microplate wells are loaded with the test sample, the Trolox standards at different concentrations, and a blank control well containing only the buffer and fluorescent probe.
- Incubation: A solution of the fluorescent probe, fluorescein, is added to all wells. The plate is then incubated in the microplate reader at a controlled temperature, typically 37°C.
- Initiation: The AAPH radical generator solution is rapidly added to each well, and the fluorescence measurement begins immediately.
- Data Collection: The fluorescence intensity of each well is continuously and automatically recorded by the microplate reader over a specific period, often around two hours. In the blank control well, fluorescence will decay rapidly. In wells with antioxidants, the decay will be slower and produce a higher area under the curve (AUC).
- Data Analysis: The data is analyzed to calculate the net Area Under the Curve (AUC) for each sample and standard. The AUC of the sample is compared to the Trolox standard curve to determine its antioxidant capacity in Trolox Equivalents (TE).
Traditional vs. Kinetic Modeling ORAC
While the Area Under the Curve (AUC) method is the traditional way to calculate ORAC, it has limitations, including potentially inaccurate rankings of antioxidants. Newer kinetic modeling offers a more detailed understanding of the chemical reactions.
| Feature | Traditional ORAC (AUC Method) | Kinetic Modeling ORAC |
|---|---|---|
| Core Metric | Integrated Area Under the Curve (AUC) over time. | Absolute rate constants ($k_5$, $k_6$) of reaction. |
| Measurement Focus | Overall extent of protection of the fluorescent probe. | Reactivity of antioxidants towards radicals and ability to repair oxidized fluorescein. |
| Interpretation | A higher AUC value is generally interpreted as stronger antioxidant capacity. | Provides deeper mechanistic insights into how antioxidants act. |
| Example Case | Lemon juice, rich in fast-acting ascorbic acid, shows a lower AUC score than apple juice. | Reveals lemon juice has higher reactivity (higher $k_5$) than apple juice, confirming its stronger antioxidant effect. |
| Scientific Nuance | Can lead to misleading conclusions as it doesn't differentiate between rapid and slow-acting antioxidants. | Offers a more robust and verifiable measure of antioxidant activity. |
The Key Limitations and USDA Stance
It is critical to understand the limitations of the ORAC value. The USDA's decision to remove its ORAC database was based on significant scientific concerns.
- In Vitro vs. In Vivo: The ORAC assay is an artificial, test-tube environment. It does not account for the complexities of human digestion, absorption, and metabolism, which all affect how antioxidants actually behave in the body.
- Bioavailability: A food with a high ORAC value in the lab may have low bioavailability in the body, meaning the antioxidants are not effectively absorbed into the bloodstream.
- Radical Specificity: The assay only measures scavenging against peroxyl radicals, ignoring the diverse range of other reactive oxygen species that exist in the body.
- External Factors: A substance's ORAC value can be significantly altered by simple factors like water removal. Dried fruits will often have a higher ORAC per gram than their fresh counterparts simply because the water has been concentrated. Growing conditions and harvest time can also affect results.
Conclusion: A Tool with Context
To understand how is the ORAC value measured is to understand that it is a specific, in-vitro laboratory procedure, not a holistic indicator of health benefits. The assay uses a fluorescent probe, a free radical generator like AAPH, and a standardized antioxidant, Trolox, to quantify a substance's capacity to absorb certain oxygen radicals. While the test is still used for certain research and comparative purposes, its results must be interpreted with caution. The USDA's 2012 withdrawal of its ORAC database underscores the scientific consensus that these values do not reliably predict a food's health effects within the human body. For more accurate insights, advanced kinetic modeling provides a deeper understanding of antioxidant reactions, but the most reliable health advice remains focusing on a balanced, whole-food diet.
For a deeper dive into the kinetic modeling of ORAC, refer to the research published by the National Institutes of Health (NIH): A Kinetic Approach to Oxygen Radical Absorbance Capacity (ORAC).
