Measuring Antioxidant Capacity Using the ORAC and TOSC Assays

  • Andrew R. Garrett
  • Byron K. Murray
  • Richard A. Robison
  • Kim L. O’Neill
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 594)

Abstract

Recent epidemiological studies have shown that there may be a link between oxidative stress and the development of several types of chronic diseases. Studies have also shown that diets rich in fruits and vegetables may decrease the incidence of cancer and other chronic diseases. The antioxidant activity of the phytochemicals these foods contain may be partially responsible for the decreased incidence of these diseases in people who regularly consume them. While there are several assays currently used to assess the antioxidant activity of phytochemicals and other antioxidant compounds, two are reviewed here in detail. The first is the oxygen radical absorbance capacity (ORAC) assay, which measures the decrease in fluorescence decay caused by antioxidants, and the second is the total oxyradical scavenging capacity (TOSC) assay, which measures the decrease in ethylene gas production caused by the inhibition of the thermal hydrolysis of ABAP (2,2′-Azobis(2-methyl-(propionamidine) dihydrochloride) by KMBA (α-keto-γ-(methylthio)butyric acid sodium salt) in the presence of antioxidant compounds. These two assays are discussed here, with an in depth review of their methodology and correlation.

Key words

ORAC Oxygen radical absorbance capacity assay TOSC Total oxyradical scavenging capacity assay Antioxidant Phytochemical Oxidative stress Fluorescein Trolox equivalents 

References

  1. 1.
    Oxygen Radical Absorbance Capacity (ORAC) of Selected Foods (2007); Nutrient Data Laboratory, Agricultural Research Service, US Department of Agriculture, United States Department of Agriculture.Google Scholar
  2. 2.
    Ames BN, Gold LS, Willet WC (1995) The Causes and Prevention of cancer. Proc Natl Acad Sci USA 92:5258–5265PubMedCrossRefGoogle Scholar
  3. 3.
    Ames BN, Shigenaga MK, Hagen TM (1993) Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci USA 90:7915–7922PubMedCrossRefGoogle Scholar
  4. 4.
    Christen Y (2000) Oxidative stress and Alzheimer’s disease. Am J Clin Nutr 71:621S–629SPubMedGoogle Scholar
  5. 5.
    Diaz MN, Frei B, Keaney JF Jr (1997) Antioxidants and atherosclerotic heart disease. N Eng J Med 337:408–416CrossRefGoogle Scholar
  6. 6.
    Lang AE, Lozano AM (1998) Parkinson’s disease. First of two parts. N Eng J Med 339:111–114Google Scholar
  7. 7.
    Packer L, Colman C (1999) The anti-oxidant miracle. Wiley, New YorkGoogle Scholar
  8. 8.
    Cao G, Prior RL (1998) Comparison of different analytical methods for assessing total antioxidant capacity of human serum. Clin Chem 44(6):1309–1315PubMedGoogle Scholar
  9. 9.
    Halliwell B, Gutteridge JMC, Cross CE (1992) Free Radicals, antioxidants, and human disease: where are we now? J Clin Lab Med 119:598–620Google Scholar
  10. 10.
    Tomer DP (2003) Measuring parameters that are important in cancer prevention and treatment: assessing antioxidant activity and angiogenesis. Brigham Young University, Master’s ThesisGoogle Scholar
  11. 11.
    McLeman LD (2004) Evidence of synergistic intracellular antioxidant networking and antioxidant regulation of cellular lipid peroxidation in HL-60 acute promyelogenous leukemia. Master’s Thesis. Brigham Young University. 1-37.Google Scholar
  12. 12.
    O’Neill K, Murray B (2002) Power plants. Woodland Publishing, UtahGoogle Scholar
  13. 13.
    Cao G, Booth SL, Sadowski JA, Prior RL (1998) Increases in human plasma antioxidant capacity after consumption of controlled diets high in fruit and vegetables. Am J Clin Nutr 68:1081–1087PubMedGoogle Scholar
  14. 14.
    Huang D, Ou B, Prior RL (2005) The chemistry behind antioxidant capacity assays. J Agric Food Chem 53:1841–1856PubMedCrossRefGoogle Scholar
  15. 15.
    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):4619–4626PubMedCrossRefGoogle Scholar
  16. 16.
    Cao G, Alessio H, Cutler R (1993) Oxygen-radical absorbance capacity assay for antioxidants. Free Radic Biol Med 14(3):303–311PubMedCrossRefGoogle Scholar
  17. 17.
    Cao G, Sofic E, Prior RL (1996) Antioxidant capacity of tea and common vegetables. J Agric Food Chem 44:3426–3431CrossRefGoogle Scholar
  18. 18.
    Huang D, Ou B, Hampsch-Woodill M, Flanagan J, Prior RL (2002) High-throughput assay of oxygen radical absorbance capacity (orac) using a multichannel liquid handling system coupled with a microplate fluorescence reader in 96-well format. J Agric Food Chem 50:4437–4444PubMedCrossRefGoogle Scholar
  19. 19.
    Ou B, Huang D, Hampsch-Woodill M, Flanagan JA, Deemer EK (2002) Analysis of antioxidant of common vegetables employing oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays: a comparative study. J Agric Food Chem 50:3122–3128PubMedCrossRefGoogle Scholar
  20. 20.
    Glazer AN (1990) Phycoerythrin fluorescence based assay for reactive oxygen species. Methods Enzymol 186:161–168PubMedCrossRefGoogle Scholar
  21. 21.
    Cao G, Prior RL (1999) Measurement of oxygen radical absorbance capacity in biological samples. Methods Enzymol 299:50–62PubMedCrossRefGoogle Scholar
  22. 22.
    Pellegrini N, Del Rio D, Colombi B, Bianchi M, Brighenti F (2003) Application of the 2, 2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation assay to a flow injection system for the evaluation of antioxidant activity of some pure compounds and beverages. J Agric Food Chem 51:260–264PubMedCrossRefGoogle Scholar
  23. 23.
    Pulido R, Bravo L, Suara-Calixto F (2000) Antioxidant activity of dietary polyphenols as determined by a modified ferric reducing/antioxidant power assay. J Agric Food Chem 48:3396–3402PubMedCrossRefGoogle Scholar
  24. 24.
    Aruoma OI (2003) Methodological considerations for characterizing potential antioxidant actions of bioactive components in plant foods. Mutat Res 523–524:9–20PubMedGoogle Scholar
  25. 25.
    Gorinstein S, Martin-Belloso O, Katrich E, Lojek A, Ciz M, Gligelmo-Miguel N, Haruenkit R, Park YS, Jung ST, Trakhtenberg S (2003) Comparison of the contents of the main biochemical compounds and the antioxidant activity of some Spanish olive oils as determined by four different radical scavenging tests. J Nutr Biochem 14:154–159PubMedCrossRefGoogle Scholar
  26. 26.
    Yokozawa T, Cho EJ, Hara Y, Kitani K (2000) Antioxidative activity of green tea treated with radical initiator 2, 2’-azobis(2-amidinopropane) dihydrochloride. J Agric Food Chem. 48:5068–5073PubMedCrossRefGoogle Scholar
  27. 27.
    Prior RL, Hoang H, Gu L, Wu X, Bacchiocca M, Howard L, Hampsch-Woodill M, Huang D, Ou B, Jacob R (2003) Assays for hydrophilic and lipophilic antioxidant capacity (oxygen radical absorbance capacity (ORAC(FL))) of plasma and other biological and food samples. J Agric Food Chem 51:3273–3279PubMedCrossRefGoogle Scholar
  28. 28.
    Dugas AJ Jr, Castaneda-Acosta J, Bonin GC, Price KL, Fischer NH, Winston GW (2000) Evaluation of the total peroxyl radical-scavenging capacity of flavonoids: structure-activity relationships. J Nat Prod 63:327–331PubMedCrossRefGoogle Scholar
  29. 29.
    Regoli F, Winston GW (1999) Quantification of total oxidant scavenging capacity of antioxidants for peroxynitrite, peroxyl radicals, and hydroxyl radicals. Toxicol Appl Pharmacol 156:96–105PubMedCrossRefGoogle Scholar
  30. 30.
  31. 31.
    Somogyi A, Rosta K, Pusztai P, Tulassay Z, Nagy G (2007) Antioxidant Measurements. Physiol Meas 28:R41–R55PubMedCrossRefGoogle Scholar
  32. 32.
    Tomer DP, McLeman LD, Ohmine S, Scherer PM, Murray BK, O’Neill KL (2007) Comparison of the total oxyradical scavenging capacity and oxygen radical absorbance capacity assays. J Med Food 10(2):337–344PubMedCrossRefGoogle Scholar
  33. 33.
    Lichtenthaler R, Marx F (2005) Total oxidant scavenging capacities of common european fruit and vegetable juices. J Agric Food Chem 53:103–110PubMedCrossRefGoogle Scholar
  34. 34.
    Lichtenthaler R, Rodrigues RB, Maia JGS, Papagiannopoulos M, Fabricius H, Marx F (2005) Total oxidant scavenging capacities of Euterpe oleracea Mart. (Acai) fruits. Int J Food Sci Nutr 56(1):53–64Google Scholar
  35. 35.
    Company R, Serafim A, Cosson R, Camus L, Shillito B, Fiala-Medioni A, Bebianno MJ (2006) The effect of cadmium on antioxidant responses and the susceptibility to oxidative stress in the hydrothermal vent mussel Bathymodiolus azoricus. Marine Biol 148:817–825CrossRefGoogle Scholar
  36. 36.
    Giessing AMB, Mayer LM (2004) Oxidative coupling during gut passage in marine deposit-feeding invertebrates. Limnol Oceanogr 49(3):716–726CrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Andrew R. Garrett
    • 1
  • Byron K. Murray
    • 1
  • Richard A. Robison
    • 1
  • Kim L. O’Neill
    • 1
  1. 1.Department of Microbiology and Molecular BiologyBrigham Young UniversityProvoUSA

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