Oxygen radical absorbance capacity

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Oxygen radical absorbance capacity (ORAC) was a method of measuring antioxidant capacities in biological samples in vitro . [1] [2] Because no physiological proof in vivo existed in support of the free-radical theory or that ORAC provided information relevant to biological antioxidant potential, it was withdrawn in 2012. [3] [4]

Contents

Various foods were tested using this method, with certain spices, berries and legumes rated highly in extensive tables once published by the United States Department of Agriculture (USDA). Alternative measurements include the Folin-Ciocalteu reagent, and the Trolox equivalent antioxidant capacity assay.

Method

The assay measures the oxidative degradation of the fluorescent molecule (either beta-phycoerythrin or fluorescein) after being mixed with free radical generators such as azo-initiator compounds. Azo-initiators are considered to produce the peroxyl radical by heating, which damages the fluorescent molecule, resulting in the loss of fluorescence. Antioxidants are considered to protect the fluorescent molecule from the oxidative degeneration. The degree of protection is quantified using a fluorometer. Fluorescein is currently used most as a fluorescent probe. Equipment that can automatically measure and calculate the capacity is commercially available (Biotek, Roche Diagnostics).

The fluorescent intensity decreases as the oxidative degeneration proceeds, and this intensity is typically recorded for 35 minutes after the addition of the azo-initiator (free radical generator). So far, AAPH (2,2’-azobis(2-amidino-propane) dihydrochloride) is the sole free-radical generator used. The degeneration (or decomposition) of fluorescein is measured as the presence of the antioxidant slows the fluorescence decay. Decay curves (fluorescence intensity vs. time) are recorded and the area between the two decay curves (with or without antioxidant) is calculated. Subsequently, the degree of antioxidant-mediated protection is quantified using the antioxidant trolox (a vitamin E analogue) as a standard. Different concentrations of trolox are used to make a standard curve, and test samples are compared to this. Results for test samples (foods) have been published as "trolox equivalents" or TEs. [5] [6]

One benefit of using the ORAC method to evaluate substances' antioxidant capacities is that it takes into account samples with and without lag phases of their antioxidant capacities. This is especially beneficial when measuring foods and supplements that contain complex ingredients with various slow- and fast-acting antioxidants, as well as ingredients with combined effects that cannot be precalculated.

Drawbacks of this method are: 1) only antioxidant activity against particular (probably mainly peroxyl) radicals is measured; however, peroxyl radical formation has never been proven; 2) the nature of the damaging reaction is not characterized; 3) there is no evidence that free radicals are involved in this reaction; and 4) there is no evidence that ORAC values have any biological significance following consumption of any food. Moreover, the relationship between ORAC values and a health benefit has not been established.

Resulting from scientific refutation of the physiological significance of ORAC, the USDA, which had been collating and publishing ORAC data for more than a decade, withdrew its web publication of ORAC values for common American foods in May 2012. [3]

Several modified ORAC methods have been proposed. Most of them employ the same principle (i.e. measurement of AAPH-radical mediated damage of fluorescein); however, ORAC-EPR, electron paramagnetic resonance-based ORAC method directly measures the decrease of AAPH-radical level by the scavenging action of the antioxidant substance. [7]

Regulatory guidance

In the following discussion, the term "antioxidant" refers mainly to non-nutrient compounds in foods, such as polyphenols, which have antioxidant capacity in vitro , so provide an artificial index of antioxidant strength—the ORAC measurement.

Other than for dietary antioxidant vitamins—vitamin A, vitamin C and vitamin E—no food compounds have been proved with antioxidant efficacy in vivo .[ citation needed ] Accordingly, regulatory agencies such as the Food and Drug Administration of the United States and the European Food Safety Authority (EFSA) have published guidance forbidding food product labels to claim or imply an antioxidant benefit when no such physiological evidence exists. [8] [9] This guidance for the United States and European Union establishes it is illegal to imply potential health benefits on package labels of products with high ORAC.

Physiological context

Although research in vitro indicates polyphenols are good antioxidants and probably influence the ORAC value, antioxidant effects in vivo are probably negligible or absent. [3] [10] By non-antioxidant mechanisms still undefined, flavonoids and other polyphenols may reduce the risk of cardiovascular disease and cancer. [11]

As interpreted by the Linus Pauling Institute, EFSA and the USDA, dietary polyphenols have little or no direct antioxidant food value following digestion. [3] [9] [10] [12] Not like controlled test tube conditions, the fate of polyphenols in vivo shows they are poorly conserved (less than 5%), with most of what is absorbed existing as chemically modified metabolites destined for rapid excretion. [13]

The increase in antioxidant capacity of blood seen after the consumption of polyphenol-rich (ORAC-rich) foods is not caused directly by the polyphenols, but most likely results from increased uric acid levels derived from metabolism of flavonoids. [12] [13] According to Frei, "we can now follow the activity of flavonoids in the body, and one thing that is clear is that the body sees them as foreign compounds and is trying to get rid of them." [13]

Food sources

Values are expressed as the sum of the lipid soluble (e.g. carotenoid) and water-soluble (e.g. phenolic) antioxidant fractions (i.e., “total ORAC”) reported as in micromoles trolox equivalents (TE) per 100 gram sample, and are compared to assessments of total polyphenol content in the samples.

These values are considered biologically irrelevant by the EFSA and USDA. [3] [9]

Food ORAC scores - USDA
FoodServing sizeORAC, Trolox equiv., μmol per 100 g
Prune 1 cup14,582
Small Red Bean½ cup dried beans13,727
Wild blueberry 1 cup13,427
Red kidney bean ½ cup dried beans13,259
Pinto bean ½ cup11,864
Cranberry 1 cup raw (whole berries)9,584
Blueberry 1 cup raw (cultivated berries)9,019
Artichoke hearts 1 cup, cooked7,904
Raw unprocessed Cocoa bean 1 oz7,840
Blackberry 1 cup raw (cultivated berries)7,701
Raspberry 1 cup6,058
Strawberry 1 cup5,938
Red Delicious apple 1 apple5,900
Granny Smith apple1 apple5,381
Pecan 1  oz 5,095
Sweet cherry 1 cup4,873
Black plum 1 plum4,844
Russet potato 1, cooked4,649
Chokeberry 1 oz4,497
Black bean ½ cup dried beans4,181
Plum1 plum4,118
Gala apple 1 apple3,903
Pomegranate 100 grams2,860

With nearly all vegetables, conventional boiling can reduce the ORAC value by up to 90%, while steaming retains more of the antioxidants. [14]

Comparisons of ORAC values

The United States Department of Agriculture, previously a publisher of ORAC data, withdrew its web publication of ORAC values for common American foods in 2012 owing to absence of scientific evidence that ORAC has any biological significance. [3]

When comparing ORAC data, care must be taken to ensure the units and food being compared are similar. Some evaluations will compare ORAC units per gram of dry weight of the intact food or its milled powder, others will evaluate ORAC units in fresh or frozen wet weight, and still others will look at ORAC units per serving. Under each evaluation, different foods can appear to have higher ORAC values. For example, although a raisin has no more antioxidant potential than the grape from which it was dried, raisins will appear to have a higher ORAC value per gram of wet weight than grapes due to their reduced water content. Likewise, the large water content in watermelon can make it appear as though this fruit is low in ORAC. Similarly, the typical quantity of food used should be considered; herbs and spices may be high in ORAC, but are applied in much smaller quantities compared to intact whole foods. [15]

Numerous health food and beverage companies and marketers have erroneously capitalized on the ORAC rating by promoting products claimed to be "high in ORAC". As most of these ORAC values have not been independently validated or subjected to peer review for publication in scientific literature, they remain unconfirmed, are not scientifically credible, and may mislead consumers.

See also

Related Research Articles

Antioxidants are compounds that inhibit oxidation, a chemical reaction that can produce free radicals. Autoxidation leads to degradation of organic compounds, including living matter. Antioxidants are frequently added to industrial products, such as polymers, fuels, and lubricants, to extend their usable lifetimes. Foods are also treated with antioxidants to forestall spoilage, in particular the rancidification of oils and fats. In cells, antioxidants such as glutathione, mycothiol or bacillithiol, and enzyme systems like superoxide dismutase, can prevent damage from oxidative stress.

Rancidification is the process of complete or incomplete autoxidation or hydrolysis of fats and oils when exposed to air, light, moisture, or bacterial action, producing short-chain aldehydes, ketones and free fatty acids.

<span class="mw-page-title-main">Flavonoid</span> Class of plant and fungus secondary metabolites

Flavonoids are a class of polyphenolic secondary metabolites found in plants, and thus commonly consumed in the diets of humans.

<span class="mw-page-title-main">Polyphenol</span> Class of chemical compounds

Polyphenols are a large family of naturally occurring phenols. They are abundant in plants and structurally diverse. Polyphenols include flavonoids, tannic acid, and ellagitannin, some of which have been used historically as dyes and for tanning garments.

<span class="mw-page-title-main">Açaí palm</span> Palm tree with many uses, mainly fruit as cash crop

The açaí palm, Euterpe oleracea, is a species of palm tree (Arecaceae) cultivated for its fruit, hearts of palm, leaves, and trunk wood. Global demand for the fruit has expanded rapidly in the 21st century, and the tree is cultivated for that purpose primarily.

<span class="mw-page-title-main">Oxidative stress</span> Free radical toxicity

Oxidative stress reflects an imbalance between the systemic manifestation of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. Disturbances in the normal redox state of cells can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA. Oxidative stress from oxidative metabolism causes base damage, as well as strand breaks in DNA. Base damage is mostly indirect and caused by the reactive oxygen species generated, e.g., O2− (superoxide radical), OH (hydroxyl radical) and H2O2 (hydrogen peroxide). Further, some reactive oxidative species act as cellular messengers in redox signaling. Thus, oxidative stress can cause disruptions in normal mechanisms of cellular signaling.

<span class="mw-page-title-main">ABTS</span> Chemical compound

In biochemistry, ABTS is a chemical compound used to observe the reaction kinetics of specific enzymes. A common use for it is in the enzyme-linked immunosorbent assay (ELISA) to detect the binding of molecules to each other.

Proanthocyanidins are a class of polyphenols found in many plants, such as cranberry, blueberry, and grape seeds. Chemically, they are oligomeric flavonoids. Many are oligomers of catechin and epicatechin and their gallic acid esters. More complex polyphenols, having the same polymeric building block, form the group of tannins.

<span class="mw-page-title-main">Myricetin</span> Chemical compound

Myricetin is a member of the flavonoid class of polyphenolic compounds, with antioxidant properties. Common dietary sources include vegetables, fruits, nuts, berries, tea, and red wine.

<span class="mw-page-title-main">Antioxidant effect of polyphenols and natural phenols</span>

A polyphenol antioxidant is a hypothetized type of antioxidant, in which each instance would contain a polyphenolic substructure; such instances which have been studied in vitro. Numbering over 4,000 distinct chemical structures, such polyphenols may have antioxidant activity {{{1}}} in vitro (although they are unlikely to be antioxidants in vivo). Hypothetically, they may affect cell-to-cell signaling, receptor sensitivity, inflammatory enzyme activity or gene regulation, although high-quality clinical research has not confirmed any of these possible effects in humans as of 2020.

<span class="mw-page-title-main">Folin–Ciocalteu reagent</span>

The Folin–Ciocâlteu reagent (FCR) or Folin's phenol reagent or Folin–Denis reagent, is a mixture of phosphomolybdate and phosphotungstate used for the colorimetric in vitro assay of phenolic and polyphenolic antioxidants, also called the gallic acid equivalence method (GAE). It is named after Otto Folin, Vintilă Ciocâlteu, and Willey Glover Denis. The Folin-Denis reagent is prepared by mixing sodium tungstate and phosphomolybdic acid in phosphoric acid. The Folin–Ciocalteu reagent is just a modification of the Folin-Denis reagent. The modification consisted of the addition of lithium sulfate and bromine to the phosphotungstic-phosphomolybdic reagent.

<span class="mw-page-title-main">Anthocyanin</span> Class of chemical compounds

Anthocyanins, also called anthocyans, are water-soluble vacuolar pigments that, depending on their pH, may appear red, purple, blue, or black. In 1835, the German pharmacist Ludwig Clamor Marquart gave the name Anthokyan to a chemical compound that gives flowers a blue color for the first time in his treatise "Die Farben der Blüthen". Food plants rich in anthocyanins include the blueberry, raspberry, black rice, and black soybean, among many others that are red, blue, purple, or black. Some of the colors of autumn leaves are derived from anthocyanins.

<span class="mw-page-title-main">Trolox</span> Chemical compound

Trolox is a water-soluble analog of vitamin E sold by Hoffman-LaRoche. It is an antioxidant like vitamin E and it is used in biological or biochemical applications to reduce oxidative stress or damage.

The Trolox equivalent antioxidant capacity (TEAC) assay measures the antioxidant capacity of a given substance, as compared to the standard, Trolox. Most commonly, antioxidant capacity is measured using the ABTS Decolorization Assay. Other antioxidant capacity assays which use Trolox as a standard include the diphenylpicrylhydrazyl (DPPH), oxygen radical absorbance capacity (ORAC) and ferric reducing ability of plasma (FRAP) assays. The TEAC assay is often used to measure the antioxidant capacity of foods, beverages and nutritional supplements.

<span class="mw-page-title-main">Dichlorofluorescein</span> Chemical compound

Dichlorofluorescein (DCF) is an organic dye of the fluorescein family, being substituted at the 2 and 7 positions by chloride.

<span class="mw-page-title-main">Phenolic content in tea</span> Natural plant compounds

The phenolic content in tea refers to the phenols and polyphenols, natural plant compounds which are found in tea. These chemical compounds affect the flavor and mouthfeel of tea. Polyphenols in tea include catechins, theaflavins, tannins, and flavonoids.

<span class="mw-page-title-main">Naturally occurring phenols</span> Group of chemical compounds

In biochemistry, naturally occurring phenols are natural products containing at least one phenol functional group. Phenolic compounds are produced by plants and microorganisms. Organisms sometimes synthesize phenolic compounds in response to ecological pressures such as pathogen and insect attack, UV radiation and wounding. As they are present in food consumed in human diets and in plants used in traditional medicine of several cultures, their role in human health and disease is a subject of research. Some phenols are germicidal and are used in formulating disinfectants.

Ferric reducing ability of plasma is an antioxidant capacity assay that uses Trolox as a standard. The FRAP assay was first performed by Iris Benzie and J. J. Strain of the Human Nutrition Research Group at the University of Ulster, Coleraine. The method is based on the formation of O-Phenanthroline-Fe(2+) complex and its disruption in the presence of chelating agents. This assay is often used to measure the antioxidant capacity of foods, beverages and nutritional supplements containing polyphenols.

Antioxidative stress is an overabundance of bioavailable antioxidant compounds that interfere with the immune system's ability to neutralize pathogenic threats. The fundamental opposite is oxidative stress, which can lead to such disease states as coronary heart disease or cancer.

References

  1. ↑ Cao G, Alessio HM, Cutler RG (1993). "Oxygen-radical absorbance capacity assay for antioxidants" (PDF). Free Radic. Biol. Med. 14 (3): 303–11. doi:10.1016/0891-5849(93)90027-R. PMID   8458588. Archived from the original on 2018-07-24. Retrieved 2019-09-11.
  2. ↑ Ou B, Hampsch-Woodill M, Prior RL (2001). "Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe". J. Agric. Food Chem. 49 (10): 4619–26. doi:10.1021/jf010586o. PMID   11599998.
  3. 1 2 3 4 5 6 "Withdrawn: Oxygen Radical Absorbance Capacity (ORAC) of Selected Foods, Release 2 (2010)". United States Department of Agriculture, Agricultural Research Service. 16 May 2012. Retrieved 13 June 2012.
  4. ↑ Gross, P (2009). "New Roles for Polyphenols. A 3-Part report on Current Regulations & the State of Science". Nutraceuticals World. Rodman Media. Retrieved April 11, 2013.
  5. ↑ Huang D, Ou B, Prior RL (2005). "The chemistry behind antioxidant capacity assays". J. Agric. Food Chem. 53 (6): 1841–56. doi:10.1021/jf030723c. PMID   15769103.
  6. ↑ Garrett AR, Murray BK, Robison RA, O'Neill KL (2010). "Measuring antioxidant capacity using the ORAC and TOSC assays". Advanced Protocols in Oxidative Stress II. Methods in Molecular Biology. Vol. 594. pp. 251–62. doi:10.1007/978-1-60761-411-1_17. ISBN   978-1-60761-410-4. PMID   20072922.
  7. ↑ Kohri S, Fujii H, Oowada S, Endoh N, Sueishi Y, Kusakabe M, Shimmei M, Kotake Y (2009). "An oxygen radical absorbance capacity-like assay that directly quantifies the antioxidant's scavenging capacity against AAPH-derived free radicals". Anal. Biochem. 386 (2): 167–71. doi:10.1016/j.ab.2008.12.022. PMID   19150323.
  8. ↑ Guidance for Industry, Food Labeling; Nutrient Content Claims; Definition for "High Potency" and Definition for "Antioxidant" for Use in Nutrient Content Claims for Dietary Supplements and Conventional Foods U.S. Department of Health and Human Services, Food and Drug Administration, Center for Food Safety and Applied Nutrition, June 2008
  9. 1 2 3 EFSA Panel on Dietetic Products, Nutrition and Allergies (2010). "Scientific Opinion on the substantiation of health claims related to various food(s)/food constituent(s) and protection of cells from premature aging, antioxidant activity, antioxidant content and antioxidant properties, and protection of DNA, proteins and lipids from oxidative damage pursuant to Article 13(1) of Regulation (EC) No 1924/20061". EFSA Journal. 8 (2): 1489. doi: 10.2903/j.efsa.2010.1489 .
  10. 1 2 Williams RJ, Spencer JP, Rice-Evans C (April 2004). "Flavonoids: antioxidants or signalling molecules?". Free Radic. Biol. Med. 36 (7): 838–49. doi:10.1016/j.freeradbiomed.2004.01.001. PMID   15019969.
  11. ↑ Arts IC, Hollman PC (2005). "Polyphenols and disease risk in epidemiologic studies". Am. J. Clin. Nutr. 81 (1 Suppl): 317S–325S. doi: 10.1093/ajcn/81.1.317S . PMID   15640497.
  12. 1 2 Lotito SB, Frei B (2006). "Consumption of flavonoid-rich foods and increased plasma antioxidant capacity in humans: cause, consequence, or epiphenomenon?". Free Radic. Biol. Med. 41 (12): 1727–46. doi:10.1016/j.freeradbiomed.2006.04.033. PMID   17157175.
  13. 1 2 3 "Studies force new view on biology of flavonoids", by David Stauth, EurekAlert!. Adapted from a news release issued by Oregon State University
  14. ↑ Ninfali P, Mea G, Giorgini S, Rocchi M, Bacchiocca M (2005). "Antioxidant capacity of vegetables, spices and dressings relevant to nutrition". Br. J. Nutr. 93 (2): 257–66. doi: 10.1079/BJN20041327 . PMID   15788119.
  15. ↑ Tapsell LC, Hemphill I, Cobiac L, Patch CS, Sullivan DR, Fenech M, Roodenrys S, Keogh JB, Clifton PM, Williams PG, Fazio VA, Inge KE (2006). "Health benefits of herbs and spices: the past, the present, the future". Med. J. Aust. 185 (4 Suppl): S4–24. doi:10.5694/j.1326-5377.2006.tb00548.x. hdl: 2440/22802 . PMID   17022438.
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