ORAC Method First Action Paper Published


Frequently Asked Questions

  1. What are Free Radicals?

    A free radical is any species containing an unpaired electron. With an unpaired electron in its atomic orbital, the free radical is highly chemically reactive. These species can play a dual role having both deleterious and/or beneficial agents. Free radicals are necessary for life and play an important role in a number of biological processes. For instance, free radicals can kill bacteria by phagocytic cells involved in the immune system. However, excess free radicals can damage cellular lipids, proteins or DNA and impair their normal function. The most important oxygen-centered free radicals are superoxide, peroxyl and hydroxyl radicals. Free radicals in cigarette smoke are implicated in the development of emphysema in the lung and lung cancer.

  2. What is Oxidative Stress?

    Maintaining the balance or homeostatsis of oxidation and antioxidants is a fundamental theme of aerobic life. However, if this balance shifts in favor of the oxidants, a status call oxidative stress is generated. Oxidative stress was often defined as “a serious imbalance between the generation of reactive oxygen species and antioxidant protection in favor of the former, causing excessive oxidative damage”. Oxidative stress can occur in cells as a result of a low level of dietary antioxidants, or through inhibition of the complex antioxidant mechanisms within the cells of the body. These include antioxidants such as glutathione, vitamin C and vitamin E as well as enzymes such as catalase, superoxide dismutase and various peroxidases. Oxidative stress may be both the cause and/or the consequence of disease processes, and is involved with infectection, inflammation, UV radiation, pollution, excess alcohol consumption, cigarette smoking, etc. Evidence indicates that oxidative stress plays a major role in the initiation and progression of cardiovascular dysfunction associated with diseases such as hyperlipidemia, diabetes mellitus, hypertension, ischemic heart disease, and chronic heart failure (Taniyama and Griendling 2003). Some scientists suggest that oxidative stress is involved in the early signs of aging.

  3. What are reactive oxygen species (ROS)?

    There are many types of free radicals and non-radical reactive species in living systems; nonetheless, reactive oxygen species (ROS) are primary reactive species in human body. ROS form as a natural by-product of the normal metabolism of oxygen and have important roles in cell signaling. However, because of their reactivity, they can participate in unwanted side reactions resulting in cell damage. Excessive amounts of ROS can lead to cell injury and death, which may be an initiating factor for many diseases. Diseases that have been shown to have ROS include cancer, stroke, atherosclerosis, coronary heart disease, diabetes, Parkinson’s disease and perhaps others.

  4. What is an Antioxidant?

    An antioxidant is a chemical substance that in small quantities inhibits the oxidation of other molecules. Oxidation is a chemical reaction that in the body induces oxidative stress, usually through either creating reactive oxygen species (ROS) or inhibiting antioxidant defense systems. Oxidation reactions can produce free radicals or non-radical reactive species. Common free radicals are peroxyl radical, hydroxyl superoxide anion, etc. These radicals can start chain reactions. When the chain reaction occurs in a cell, it can cause damage or death to the cell. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions. Antioxidants help prevent or slow down the oxidation process in the body.

  5. What is a Pro oxidant?

    Substances or chemicals that produce cellular oxidation or induce oxidative stress, usually through either creating free radicals or inhibiting antioxidant systems. Some compounds can act as an antioxidant or pro oxidant depending upon concentrations and/or presence of other reactive species.

  6. What are common antioxidants in foods?

    Vitamins A, C, and E; Vitamin cofactors (i.e. Coenzyme Q10, Manganese); Melatonin; Polyphenolic compounds such as flavonoids (i.e quercetin, catechin, epicataechin, proanthocyanidins, anthocyanidins, etc); Phenolic acids (chlorogenic acid, ellagic acid, gallic acid, etc.)

  7. How does variety or genetics affect the antioxidant capacity of a food?

    ORAC Deviation in Southern Highbush Blueberry VarietiesGenetics or variety of a food can be a significant factor accounting for variability in the antioxidant content of foods. This point is illustrated in the deviation from the mean in ORAC values of different varieties of Highbush (Fig. 1) and Southern Highbush (Fig. 2) blueberries. As a deviation from the mean, it can be increased or decreased by as much as ~60-100% (Ehlenfeldt and Prior 2001).

    ORAC Deviation in Highbush Blueberry VarietiesBlueberries represent just one example of the effects of variety on antioxidant content. for The antioxidant capacity and the content of total phenolics can vary up to 3.3-fold in strawberries of differing genetic background. Other fruits and vegetables may reflect similar variations in antioxidant capacity with different varieties (Cho et al. 2005, Cho et al. 2008).

  8. How can processing alter the antioxidant capacity of foods?

    Processing, especially cooking of food, is a factor that can impact antioxidant capacity (Papas 1996). Some vegetables are commonly eaten in cooked form (i.e., potatoes and asparagus), while others are consumed in either raw or cooked forms (i.e., broccoli, carrots, and tomatoes). Thus, cooking is an important issue that needs to be considered in estimating the daily total antioxidant capacity intake. However, few studies have considered this relative to antioxidants. Cooking is generally regarded as being destructive to antioxidant compounds (Krishnaswamy and Raghuramulu 1998). In studies by Wu et al., (Wu et al. 2004b), ORAC of raw broccoli and carrots were significantly higher than that of their cooked forms. However, baked Russet potatoes showed an increase in measured water soluble antioxidant but a reduced amount of lipid soluble antioxidants compared to the raw forms. For tomatoes, both lipophilic and hydrophilic ORAC values of the cooked forms were higher than those of their raw forms, which has also been observed in other studies (Takeoka et al. 2001). An increased bioavailability of carotenoids from processed compared to raw tomatoes ahs also been observed (Shi and Le Maguer 2000). Although it is clear that processing and cooking can alter antioxidant content, it is not possible to predict the effect, which may vary from one food to another.

  9. What are the effects of growing conditions on the antioxidant capacity of food?

    Factors such as temperature, precipitation, fertilization, insects, diseases etc. could affect the quality of plants and may also affect antioxidant contents of the plant foods. Because of these factors, which can vary from year to year, and from region to region, the antioxidant content of foods will also vary from year to year and region to region. For these reasons, most of the foods sampled originally for the ORAC database, were collected in 2 different growing seasons and in 12 different cities in the United States (Wu et al. 2004a). Thus the composite ORAC value will reflect a better estimate of what is available in the U.S. market.

  10. Why does extraction solvent matter when measuring antioxidant capacity of a food?

    Although not often noted, the solvents used in the extraction can affect the amounts of antioxidants being extracted and thus the final value measured in a food. In developing a large database of different foods, a decision has to be made as to what is a reasonable solvent or mixed solvent that will work reasonably well for most foods though may not be optimum for some foods. Acetone/water/acetic acid (70:29.5:0.5) was shown to be an ideal mixed solvent for extraction of water soluble antioxidants. It has been used for extracting the water soluble components from a large part of the ORAC database samples (Wu et al. 2004a), realizing that for foods particularly high in anthocyanins, acidic methanol might be a slightly better extraction solvent for the anthocyanins, but in foods particularly high in proanthocyanins, acidified acetone might be a better solvent. Solvent effects might introduce a small difference usually less than 10-15%.

  11. Why do we need a “total” antioxidant capacity assay?

    Several thousand polyphenolic compounds exist in plants and many of these have antioxidant capacity. Because of the difficulty in quantifying the individual antioxidant compounds, a method which provides a “sum” of the antioxidant components in plants and biological samples is useful. These types of assays are often referred to as “total” antioxidant capacity assays. However, there is in actuality no single assay which provides a “total” measurement of antioxidant capacity. In thinking about antioxidant capacity methods, one has to consider the oxidant source and the mechanism of reaction with potential antioxidants. Experimental evidence has shown that there are six major reactive oxygen species (ROS) causing oxidative damage in the human body. These species include: superoxide anion (O2-); hydrogen peroxide (H2O2); peroxyl radicals (ROO); hydroxyl radicals (HO); singlet oxygen (1O2); and peroxynitrite (ONOO-). The peroxyl radical is the most abundant free radical in the human body. To comprehensively evaluate the oxidant-scavenging capacity of a food sample, assays have to be designed to include these reactive oxygen species. However, so far the majority of assays are designed to measure a sample’s capacity to react with one oxidant (either an organic radical or redox active metal complex).

  12. What are the advantages of the ORAC assay?

    The peroxyl radical has been the most frequently used reactive oxygen species in antioxidant capacity assays because it is the most relevant radical in lipid autoxidation and can be generated conveniently in the laboratory. The peroxyl radical has been used as a radical source in the Oxygen Radical Absorbance Capacity (ORAC). Although there are other antioxidant assays, many of them use an oxidant or radical source which is not present in human biology, so they become less relevant if the end objective is to find antioxidant sources that may impact human biology. Recently, assays have been developed based on the other major radical sources in the body. Radical source and Antioxidant Capacity in VegetablesFigure 3 illustrates the differences measured in some common vegetables using 5 different free radical sources (Peroxyl; Hydroxyl, .OH; Peroxynitrite, ONOO; Superoxide anion, O2- ; and Singlet oxygen, 1O2). This limited data indicates clearly is that one cannot expect a good correlation across foods using different radical sources. Thus direct comparison of antioxidant capacity data from different assays is not feasible. All of the antioxidant assay methods are in vitro (‘test tube’) methods as are most of the other nutrient analyses of food ingredients and are subject to the disadvantages that are inherent in any in vitro method. ORAC using the peroxyl radical provides a measure of the antioxidant capacity primarily of a group of compounds called flavonoids. In the plant kingdom, there are several thousand individual flavonoid compounds. Although we have greatly improved analytical techniques for detecting and quantitating some of these compounds, we still do not have methods or standards to quantitate all of these compounds. ORAC has been an extremely useful analytical tool to compare the relative total quantities of these bioactive compounds plus other nonflavonoid compounds such as vitamins C and E. However, the fact that a food or compound has measured antioxidant capacity in the test tube does not mean that they will have antioxidant effects in vivo or will act through an antioxidant mechanism? The components may not be efficiently absorbed into the body and/or the bioactive compound may not be identical to the parent compound(s) found in the food due to metabolism during the digestion/absorption process. Answers to many of these questions are the domain of ongoing or future in vivo clinical investigation.

  13. Why do we need dietary antioxidants?

    We consume approximately 3.5 kg oxygen/day. In this process of energy metabolism about 2.8% of the oxygen is not completely reduced and forms free radicals which results in the production in our body of several kilograms of the peroxide oxidant in a years’ time. Antioxidants are important in preventing this oxidation process. Past research has shown that dietary antioxidants can prevent oxidative damage to cells and in some cases also help repair damage. Vitamin C and E are the common dietary antioxidants that have a defined amount needed in the diet. Fruits and vegetables contain numerous other phytochemicals that have antioxidant capacity (AOC) (Wu et al. 2004a). Numerous epidemiology studies have indicated that increased consumption of fruits and vegetables is associated with a decreased risk for a number of diseases associated with aging (Joshipura et al. 1999, De Stefani et al. 2000, Hirvonen et al. 2001, Joshipura et al. 2001, Serafini et al. 2002, Rissanen et al. 2003). Increased dietary intake of selected classes of flavonoids (isoflavone, anthocyanidins, flavones and flavonols) was associated with a reduced risk of colorectal cancer (Rossi et al. 2006).

    Recent research has shown that increased consumption of dietary antioxidants resulted in: 1. Increased circulating antioxidants, increased plasma antioxidant capacity and reduced inflammation (Del Rio et al. 2011); 2. Higher levels of plasma antioxidant capacity and a reduced body weight after 3 years of intervention in a high cardiovascular risk population (Razquin et al. 2009); 3. A decrease in LDL and an increase in HDL, decreased plasma lipid peroxidation, and an increase in antioxidant defenses (total plasma glutathione and antioxidant capacity) (Fenercioglu et al. 2009); 4. Decreased risk of cancer of the stomach (Serafini et al. 2002); 5. Increased serum adiponectin concentrations with a decrease in inflammatory markers (Detopoulou et al. 2009); 6. A reduction in incidence of ischemic stroke and to a lesser extend in all types of stroke in a cross-sectional and randomized intervention study of 41,620 men and women (Del Rio et al. 2011); 7. Increased insulin sensitivity in insulin-resistant obese adults and enhanced effects of the insulin-sensitizing drug metformin (2010, Endocrine Society) and 8. Increased total antoxidant capacity intake was associated with a decrease in total stroke among CVD-free women and hemorrhagic stroke among women with CVD history (Rautiainen et al. 2012a).

  14. What quantity of antioxidants should be consumed in the diet?

    Reference dietary intakes (RDI) for vitamins A, C, and E have been defined, but sufficient research data are not available to establish guidelines for measures of total antioxidant capacity in the diet. However, some broad recommendations can be made based upon epidemiology studies and studies of absorption of dietary antioxidants and their effects on in vivo antioxidant status and their ability to prevent postprandial oxidative stress. Prior and coworkers (Prior et al. 2007)` developed a relationship that indicated that 4.6 µmoles Trolox Equivalents (TE) of antioxidant capacity should be consumed for every calorie consumed which is similar to guidelines from the Center for Disease control (http://www.fruitsandveggiesmatter.gov/index.html) based upon fruit and vegetable consumption. This was considered a minimum as it only accounted for oxidative stress associated with energy metabolism. Other sources of free radicals such as dietary pro-oxidants, cigarette smoke, smog, pesticides, drugs, disease situations, etc would increase the need for antioxidants.

    Actual ORAC intakes [1 unit = 1 micromole TE] of 4650 and 5558 units/person/day have been calculated from the food consumption data in the NHANES (2001-02) and USDA CSFII (1994-96) databases, respectively (Wu et al. 2004a). The largest study of ORAC intake comes from an epidemiology study in Sweden (Rautiainen et al. 2012a, Rautiainen et al. 2012b). In this study, the lowest quintile had a median ORAC intake of 8537 and the highest quintile had an ORAC intake of 18,021 expressed as micromoles of Trolox Equivalents per day. Thus, ORAC intakes of 18,000 units are possible from foods if selected properly. In this particular study, the data suggested that “a diet high in total antioxidant capacity is associated with lower risk of incident myocardial infarction.” A Japanese group of researchers (Takebayashi et al. 2010) estimated the average ORAC in “typical vegetables” consumed in Japan was 2080 micromoles Trolox equivalents when 350 g of vegetables were consumed per day.

    Recommendations based upon currently available data are: 1) Increase intake of dietary antioxidant capacity by a minimum of 2000 – 10000 µmol; 2) Consume antioxidants at each meal to prevent postprandial oxidative stress; 3) Consume of a ‘variety’ of dietary sources with high antioxidant capacity.

Don’t see your questions, ask us here….

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Cho, M. J., L. R. Howard, R. L. Prior, and J. R. Clark. 2005. Flavonol glycosides and antioxidant capacity of various blackberry and blueberry genotypes determined by high-performance liquid chromatography/mass spectrometry. J. Sci. Food Agric. 85: 2149-2158.


Cho, M. J., L. R. Howard, R. L. Prior, and T. Morelock. 2008. Flavonoid content and antioxidant capacity of spinach genotypes determined by high-performance chromatography/mass spectrometry. J. Sci. Food Agric. 88: 1099-1106.


De Stefani, E., P. Brennan, P. Boffetta, A. L. Ronco, M. Mendilaharsu, and H. Deneo-Pellegrini. 2000. Vegetables, fruits, related dietary antioxidants, and risk of squamous cell carcinoma of the esophagus: a case-control study in Uruguay. Nutrition & Cancer 38: 23-29.


Del Rio, D., C. Agnoli, N. Pellegrini, V. Krogh, F. Brighenti, T. Mazzeo, G. Masala, B. Bendinelli, F. Berrino, S. Sieri, R. Tumino, P. C. Rollo, V. Gallo, C. Sacerdote, A. Mattiello, P. Chiodini, and S. Panico. 2011. Total antioxidant capacity of the diet is associated with lower risk of ischemic stroke in a large Italian cohort. J Nutr 141: 118-123.


Detopoulou, P., D. B. Panagiotakos, C. Chrysohoou, E. Fragopoulou, T. Nomikos, S. Antonopoulou, C. Pitsavos, and C. Stefanadis. 2009. Dietary antioxidant capacity and concentration of adiponectin in apparently healthy adults: the ATTICA study. Eur J Clin Nutr 64: 161-168.


Ehlenfeldt, M. K., and R. L. Prior. 2001. Oxygen radical absorbance capacity (ORAC) and phenolic and anthocyanin concentrations in fruit and leaf tissues of highbush blueberry. Journal of Agriculture and Food Chemistry 49: 2222-2227.


Fenercioglu, A. K., T. Saler, E. Genc, H. Sabuncu, and Y. Altuntas. 2009. The effects of polyphenol-containing antioxidants on oxidative stress and lipid peroxidation in Type 2 diabetes mellitus without complications. J Endocrinol Invest 33: 118-124.


Hirvonen, T., P. Pietinen, M. Virtanen, M. L. Ovaskainen, S. Hakkinen, D. Albanes, and J. Virtamo. 2001. Intake of flavonols and flavones and risk of coronary heart disease in male smokers. Epidemiology 12: 62-67.


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Rautiainen, S., E. B. Levitan, N. Orsini, A. Åkesson, R. Morgenstern, M. A. Mittleman, and A. Wolk. 2012b. Total Antioxidant Capacity from Diet and Risk of Myocardial Infarction: A Prospective Cohort of Women. The American Journal of Medicine 125: 974-980.


Razquin, C., J. A. Martinez, M. A. Martinez-Gonzalez, M. T. Mitjavila, R. Estruch, and A. Marti. 2009. A 3 years follow-up of a Mediterranean diet rich in virgin olive oil is associated with high plasma antioxidant capacity and reduced body weight gain. Eur J Clin Nutr 63: 1387-1393.


Rissanen, T. H., S. Voutilainen, J. K. Virtanen, B. Venho, M. Vanharanta, J. Mursu, and J. T. Salonen. 2003. Low intake of fruits, berries and vegetables is associated with excess mortality in men: the Kuopio Ischaemic Heart Disease Risk Factor (KIHD) Study. J. Nutr. 133: 199-204.


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