Ginny Bank & Rod Lenoble09.01.02
At first glance, you would think the statement above belongs in an advertisement for one of those "miracle cure" products you might see in the coupon section of the Sunday paper. But it was actually written by Dr. Lester Packer, the world's foremost antioxidant researcher, in his book The Antioxidant Miracle. The word is out-antioxidants are multifaceted and have incredible potential for the prevention of hundreds of diseases. The medical community, the media and even government agencies are bombarding consumers with research about the powerful effects of antioxidants.
However, when it comes to choosing an antioxidant product, most consumers are overwhelmed with the choices, especially when you include not only supplement products among their choices, but functional foods and conventional foods as well. Can they just take two of these pills a day to equal the antioxidant power of a glass of orange juice? How about a serving of broccoli? Blueberries are receiving a lot of media play as having the highest antioxidant capacity of fruits, but how do they compare to taking a supplement product? Fortunately, a method exists that measures antioxidant capacity, whether it is a food product or a pill.
The ORAC (Oxygen Radical Absorbance Capacity) assay is a relatively simple but sensitive method suitable for quantifying the antioxidant capacity of a number of products including whole fruits and vegetables, beverages such as fruit juices and wines, and supplement products. The ORAC assay is used primarily for water-soluble antioxidants. It can also be used to measure the antioxidant capacity of biological samples such as human plasma, blood serum or organ tissue. The chemical assay at the core of the ORAC method was developed by Guohau Cao at the Nutritional Science Department at the University of Connecticut1. In 1994, Dr. Cao brought the method to the USDA Human Nutrition Research Center on Aging to work with Dr. Ronald Prior.
Most measurements of antioxidant activity use either the inhibition time at a fixed degree of inhibition or the extent of inhibition at a fixed time for a basis of quantifying the results. The ORAC method is unique in that it measures both: 1) the degree to which a sample inhibits the action of an oxidizing agent; and 2) how long it takes to do so. These measurements are integrated into a single measurement called the ORAC Value. Trolox, a non-commercial water-soluble derivative of tocopherol, is used as the control standard of antioxidant activity and the units of an ORAC value are expressed as micromoles Trolox equivalents per gram of a substance (mmole TE/g). The chemical assay combines a fluorescent probe with a test sample and an oxidizing agent such as the peroxyl radical, hydroxyl radical or oxidation-catalyzing metal ions. The intensity of the fluorescent probe decreases as it is damaged by free radicals. In the presence of an antioxidant, the fluorescence decreases more slowly as the antioxidant sacrifices itself to the free radicals, protecting the probe. As the antioxidant is spent, the fluorescence eventually drops to zero. By allowing the reaction to go to completion and by measuring the area under the curve (relative fluorescence intensity versus time), the ORAC method combines both inhibition time and inhibition percentage of the free radical damage by the antioxidant into a single quantity.
Although the method is straightforward, it was time consuming to run many samples at once. In 1995, Dr. Cao and Dr. Prior automated this method in order to analyze large numbers of samples2. The automation of the method resulted from adapting the chemical assay to work in a COBAS FARA II analyzer and linking the analyzer to a computer to store the data. Unfortunately, the COBAS FARA II analyzer is no longer produced and therefore, there are very few laboratories that run this method regularly. Today, the ORAC assay has become commonplace in research and in the marketing of antioxidant products, keeping these laboratories considerably busy. A small number of supplement companies have managed to procure this obsolete piece of equipment for measuring ORAC value of their products for quality control and research and development purposes.
In 2001, Dr. Prior and Brunswick Laboratories made improvements to the ORAC assay. The original ORAC assay used b-phycoerythrin (B-PE) as the fluorescent probe. However, B-PE is isolated from a natural source and the purity varied from lot to lot, which often caused poor reproducibility. In addition, B-PE was found to interact with phenolic compounds, usually the very compounds believed to have the antioxidant activity in many samples, and therefore, the values reported were often understated. The improvements made to the assay eliminate the problems associated with using B-PE. By using fluorescein, a synthetic compound, the variability and the phenolic-interference problems were solved3. However the newer method reports ORAC values 2-3 times higher than the original method, which means there is no way to compare the new values to the B-PE method values. This has caused some confusion in the supplement industry as suppliers using the newer method may report significantly higher ORAC values for the same material as a supplier using the original method.
The ORAC assay has already been extensively used to measure the antioxidant capacity of a number of foods and nutritional products. Dr. Prior and Dr. Cao have established an antioxidant database consisting of vegetables, fruits, grains, extracts and other natural products4,5,6. Due to recent media focus on the positive health benefits of antioxidants, the supplement and functional food market has introduced a plethora of antioxidant-based products. The ORAC assay can provide a much-needed system for manufacturers, suppliers and consumers to compare the antioxidant capacity of various supplement products, functional foods and even fruits and vegetables-a new study of fruits and vegetables using the updated ORAC method is in progress. (For a comparison of ORAC values see Figure 1). In fact, a few companies are already using the ORAC concept on their label (see picture below). In addition, the ORAC assay can be used for quality control purposes of antioxidant-based products.
A unique and exciting application for the ORAC assay is in clinical trials. Bioavailability is the latest buzzword in the supplement and functional food industry. Evidence of high antioxidant capacity in vitro does not always guarantee similar results in vivo. Water-soluble antioxidants, for which the ORAC method is primarily used, travel in the blood and therefore have to get into the blood to function in the body. Studies that use ORAC to measure the antioxidant capacity of human plasma and other biological samples following consumption of antioxidant-rich foods or supplements have already been completed 7,8,9,10.
Ten years ago, no one would have predicted that the term "antioxidant" would become a household word. Today there are so many antioxidant products it's difficult for the consumer to sift through them all. So although consumers know they need antioxidants in their diet, actually choosing a product may be a daunting task. By educating consumers about ORAC, the ORAC method can give consumers a means of planning their antioxidant intake in the same way they plan their calcium intake, for example. They can compare what they get from their diet to what they need and make up the difference through supplementation. With the USDA in the process of publishing data on new ORAC values for fruits and vegetables, the consumer will be able to estimate their ORAC value intake per day to determine if their diet measures up to the ORAC units provided in the infamous "five to nine" servings of fruits and vegetables. This is not only very tangible for the consumer (we all know we should be eating our fruits and vegetables), but it's also very relevant as increased consumption of fruits and vegetables has been linked to prevention of numerous diseases, such as cancer and heart disease.
We are probably a long way away from seeing an RDI for antioxidants in ORAC units, but by combining industry use and acceptance, consumer education and continued research using ORAC in the field of antioxidants and oxidative stress, we can certainly help consumers define their individual antioxidant needs.NW
About the authors: Ginny Bank is technical director at RFI Ingredients, Blauvelt, NY, and Rod Lenoble is technical director at Hauser, Inc., Boulder, CO. Ms. Bank can be reached at 720-304-7335; E-mail: ginnyb@rfiingredients.com and Mr. Lenoble can be reached at 720-652-7005; E-mail: rod.lenoble@hauser.com.
References
1. Cao G, Alessio HM, Cutler RG.Oxygen radical absorbance capacity assay for antioxidants.Free Radical Bio Medicine.1993.14, 301-311.
2. Cao G., Verdon C.P., Wu A.H.B., Wang H, Prior R.L.Automated Assay of Oxygen Radical Absorbance Capacity with the COBAS FARA II. Clinical Chemistry.1995.41(12) 1738-1745.
3. Ou B, Hampsch-Woodill M, Prior RL.Development and validation of oxygen radical absorbance activity using fluorescein as the fluorescent probe.Journal of Agricultural and Food Chemistry.2001.49, 4619-4626.
4. Ehlenfeldt MK, Prior RL.Oxygen radical absorbance capacity (ORAC) and phenolic and anthocyanin concentrations in fruit and leaf tissues of highbrush blueberry.Journal of Agricultural and Food Chemistry.2001.49, 2222-2227.
5. Cao, GH, Soffic E, Prior RL.Antioxidant capacity of tea and common vegetables. Journal of Agricultural and Food Chemistry.1996.44, 3426-3431.
6. Cao G, Sanchez-Morena C, Prior RL.Procyanidins, anthocyanins and antioxidant capacity in wines.Faseb J.2000.14, A564.
7. Cao GH; Prior RL, Cutler RG, Yu BP. Effect of dietary restriction on serum antioxidant capacity in rats. Arch. Gerontol. Geriatr.1997. 25, 245-253.
8. Cao GH, Booth SL, Sadowski JA, Prior RL.Increases in human plasma antioxidant capacity after consumption of controlled diets high in fruit and vegetables.American Journal of Clinical Nutrition.1998.68, 1081-1087.
9. Cao GH, Shukitt-Hale B, Bickford PC, Joseph JA, McEwen, J, Prior RL.Hyperoxia induced changes in antioxidant capacity and the effect of dietary antioxidants.Journal of Applied Physiology.1999.86, 1817-1822.
10. Cao GH, Prior RL.Measurement of oxygen radical absorbance capacity in biological samples. Oxidants and Antioxidants, Pt A.1999.299, 50-62.
However, when it comes to choosing an antioxidant product, most consumers are overwhelmed with the choices, especially when you include not only supplement products among their choices, but functional foods and conventional foods as well. Can they just take two of these pills a day to equal the antioxidant power of a glass of orange juice? How about a serving of broccoli? Blueberries are receiving a lot of media play as having the highest antioxidant capacity of fruits, but how do they compare to taking a supplement product? Fortunately, a method exists that measures antioxidant capacity, whether it is a food product or a pill.
The ORAC Assay
The ORAC (Oxygen Radical Absorbance Capacity) assay is a relatively simple but sensitive method suitable for quantifying the antioxidant capacity of a number of products including whole fruits and vegetables, beverages such as fruit juices and wines, and supplement products. The ORAC assay is used primarily for water-soluble antioxidants. It can also be used to measure the antioxidant capacity of biological samples such as human plasma, blood serum or organ tissue. The chemical assay at the core of the ORAC method was developed by Guohau Cao at the Nutritional Science Department at the University of Connecticut1. In 1994, Dr. Cao brought the method to the USDA Human Nutrition Research Center on Aging to work with Dr. Ronald Prior.
Most measurements of antioxidant activity use either the inhibition time at a fixed degree of inhibition or the extent of inhibition at a fixed time for a basis of quantifying the results. The ORAC method is unique in that it measures both: 1) the degree to which a sample inhibits the action of an oxidizing agent; and 2) how long it takes to do so. These measurements are integrated into a single measurement called the ORAC Value. Trolox, a non-commercial water-soluble derivative of tocopherol, is used as the control standard of antioxidant activity and the units of an ORAC value are expressed as micromoles Trolox equivalents per gram of a substance (mmole TE/g). The chemical assay combines a fluorescent probe with a test sample and an oxidizing agent such as the peroxyl radical, hydroxyl radical or oxidation-catalyzing metal ions. The intensity of the fluorescent probe decreases as it is damaged by free radicals. In the presence of an antioxidant, the fluorescence decreases more slowly as the antioxidant sacrifices itself to the free radicals, protecting the probe. As the antioxidant is spent, the fluorescence eventually drops to zero. By allowing the reaction to go to completion and by measuring the area under the curve (relative fluorescence intensity versus time), the ORAC method combines both inhibition time and inhibition percentage of the free radical damage by the antioxidant into a single quantity.
Although the method is straightforward, it was time consuming to run many samples at once. In 1995, Dr. Cao and Dr. Prior automated this method in order to analyze large numbers of samples2. The automation of the method resulted from adapting the chemical assay to work in a COBAS FARA II analyzer and linking the analyzer to a computer to store the data. Unfortunately, the COBAS FARA II analyzer is no longer produced and therefore, there are very few laboratories that run this method regularly. Today, the ORAC assay has become commonplace in research and in the marketing of antioxidant products, keeping these laboratories considerably busy. A small number of supplement companies have managed to procure this obsolete piece of equipment for measuring ORAC value of their products for quality control and research and development purposes.
In 2001, Dr. Prior and Brunswick Laboratories made improvements to the ORAC assay. The original ORAC assay used b-phycoerythrin (B-PE) as the fluorescent probe. However, B-PE is isolated from a natural source and the purity varied from lot to lot, which often caused poor reproducibility. In addition, B-PE was found to interact with phenolic compounds, usually the very compounds believed to have the antioxidant activity in many samples, and therefore, the values reported were often understated. The improvements made to the assay eliminate the problems associated with using B-PE. By using fluorescein, a synthetic compound, the variability and the phenolic-interference problems were solved3. However the newer method reports ORAC values 2-3 times higher than the original method, which means there is no way to compare the new values to the B-PE method values. This has caused some confusion in the supplement industry as suppliers using the newer method may report significantly higher ORAC values for the same material as a supplier using the original method.
Applications for ORAC Assay
The ORAC assay has already been extensively used to measure the antioxidant capacity of a number of foods and nutritional products. Dr. Prior and Dr. Cao have established an antioxidant database consisting of vegetables, fruits, grains, extracts and other natural products4,5,6. Due to recent media focus on the positive health benefits of antioxidants, the supplement and functional food market has introduced a plethora of antioxidant-based products. The ORAC assay can provide a much-needed system for manufacturers, suppliers and consumers to compare the antioxidant capacity of various supplement products, functional foods and even fruits and vegetables-a new study of fruits and vegetables using the updated ORAC method is in progress. (For a comparison of ORAC values see Figure 1). In fact, a few companies are already using the ORAC concept on their label (see picture below). In addition, the ORAC assay can be used for quality control purposes of antioxidant-based products.
A unique and exciting application for the ORAC assay is in clinical trials. Bioavailability is the latest buzzword in the supplement and functional food industry. Evidence of high antioxidant capacity in vitro does not always guarantee similar results in vivo. Water-soluble antioxidants, for which the ORAC method is primarily used, travel in the blood and therefore have to get into the blood to function in the body. Studies that use ORAC to measure the antioxidant capacity of human plasma and other biological samples following consumption of antioxidant-rich foods or supplements have already been completed 7,8,9,10.
Educating the Consumer About ORAC
Ten years ago, no one would have predicted that the term "antioxidant" would become a household word. Today there are so many antioxidant products it's difficult for the consumer to sift through them all. So although consumers know they need antioxidants in their diet, actually choosing a product may be a daunting task. By educating consumers about ORAC, the ORAC method can give consumers a means of planning their antioxidant intake in the same way they plan their calcium intake, for example. They can compare what they get from their diet to what they need and make up the difference through supplementation. With the USDA in the process of publishing data on new ORAC values for fruits and vegetables, the consumer will be able to estimate their ORAC value intake per day to determine if their diet measures up to the ORAC units provided in the infamous "five to nine" servings of fruits and vegetables. This is not only very tangible for the consumer (we all know we should be eating our fruits and vegetables), but it's also very relevant as increased consumption of fruits and vegetables has been linked to prevention of numerous diseases, such as cancer and heart disease.
We are probably a long way away from seeing an RDI for antioxidants in ORAC units, but by combining industry use and acceptance, consumer education and continued research using ORAC in the field of antioxidants and oxidative stress, we can certainly help consumers define their individual antioxidant needs.NW
About the authors: Ginny Bank is technical director at RFI Ingredients, Blauvelt, NY, and Rod Lenoble is technical director at Hauser, Inc., Boulder, CO. Ms. Bank can be reached at 720-304-7335; E-mail: ginnyb@rfiingredients.com and Mr. Lenoble can be reached at 720-652-7005; E-mail: rod.lenoble@hauser.com.
References
1. Cao G, Alessio HM, Cutler RG.Oxygen radical absorbance capacity assay for antioxidants.Free Radical Bio Medicine.1993.14, 301-311.
2. Cao G., Verdon C.P., Wu A.H.B., Wang H, Prior R.L.Automated Assay of Oxygen Radical Absorbance Capacity with the COBAS FARA II. Clinical Chemistry.1995.41(12) 1738-1745.
3. Ou B, Hampsch-Woodill M, Prior RL.Development and validation of oxygen radical absorbance activity using fluorescein as the fluorescent probe.Journal of Agricultural and Food Chemistry.2001.49, 4619-4626.
4. Ehlenfeldt MK, Prior RL.Oxygen radical absorbance capacity (ORAC) and phenolic and anthocyanin concentrations in fruit and leaf tissues of highbrush blueberry.Journal of Agricultural and Food Chemistry.2001.49, 2222-2227.
5. Cao, GH, Soffic E, Prior RL.Antioxidant capacity of tea and common vegetables. Journal of Agricultural and Food Chemistry.1996.44, 3426-3431.
6. Cao G, Sanchez-Morena C, Prior RL.Procyanidins, anthocyanins and antioxidant capacity in wines.Faseb J.2000.14, A564.
7. Cao GH; Prior RL, Cutler RG, Yu BP. Effect of dietary restriction on serum antioxidant capacity in rats. Arch. Gerontol. Geriatr.1997. 25, 245-253.
8. Cao GH, Booth SL, Sadowski JA, Prior RL.Increases in human plasma antioxidant capacity after consumption of controlled diets high in fruit and vegetables.American Journal of Clinical Nutrition.1998.68, 1081-1087.
9. Cao GH, Shukitt-Hale B, Bickford PC, Joseph JA, McEwen, J, Prior RL.Hyperoxia induced changes in antioxidant capacity and the effect of dietary antioxidants.Journal of Applied Physiology.1999.86, 1817-1822.
10. Cao GH, Prior RL.Measurement of oxygen radical absorbance capacity in biological samples. Oxidants and Antioxidants, Pt A.1999.299, 50-62.
