Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Total low-molecular-weight antioxidants as a summary parameter, quantified in biological samples by a chemiluminescence inhibition assay

Nature Protocols volume 5, pages 1627–1634 (2010)Cite this article

Subjects

Abstract

Aerobic metabolism requires a complex antioxidative system to balance reactive oxygen species (ROS). When in excess, ROS disrupt cellular activities and molecular structures. Owing to the variety of ROS, there are different antioxidative activities that require various tests for their detection. The so-called 'total antioxidative capacity' inhibition assay presented in this paper can be used to quantify the overall activity of low-molecular-weight antioxidants (AOs) in biological samples. The assay is based on enhanced horseradish peroxidase–catalyzed luminol chemiluminescence. It can be fine-tuned so that the biological samples meet the requirements of the light detector. A detailed protocol describing all relevant parameters is provided. The procedure is quick, inexpensive and reproducible. The assay can be used with diverse biological materials such as plant extracts and blood plasma. Hence, it is applicable to fields as diverse as crop breeding, medical diagnostics or food sciences. The time needed for the assay depends on the number of samples and their AO content. The protocol takes one working day to complete when five samples with five replicates are measured sequentially.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The enhanced HRP cycle.
Figure 2: TAC assay procedure and data evaluation.
Figure 3: On a log-log scale, recovery time and amount of a pure antioxidant are linearly correlated.
Figure 4: TAC of different molecular-weight fractions (whole sample, filtrate and dialysate) from biological materials.
Figure 5: Changes in TAC in Lepidium sativum during abiotic stress treatment (colored columns) and after recovery (gray columns).
Figure 6: TAC of cell culture medium.

Similar content being viewed by others

References

  1. Mittler, R. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7, 405–410 (2002).

    Article  CAS  Google Scholar 

  2. Shao, H.-B., Chu, L.-Y., Lu, Z.-H. & Kang, C.-M. Primary antioxidant free radical scavenging and redox signaling pathways in higher plant cells. Int. J. Biol. Sci. 4, 8–14 (2008).

    Article  CAS  Google Scholar 

  3. Chanvitayapongs, S., Draczynska-Lusiak, B. & Sun, A.Y. Amelioration of oxidative stress by antioxidants and resveratrol in PC12 cells. Neuroreport 8, 1499–1502 (1997).

    Article  CAS  Google Scholar 

  4. Leutner, S., Eckert, A. & Müller, W.E. ROS generation, lipid peroxidation and antioxidant enzyme activities in the aging brain. J. Neural Transm. 108, 955–967 (2001).

    Article  CAS  Google Scholar 

  5. Gogorcena, Y., Iturbe-Ormaetxe, I., Escuredo, P.R. & Becana, M. Antioxidant defenses against activated oxygen in pea nodules subjected to water stress. Plant Physiol. 108, 753–759 (1995).

    Article  CAS  Google Scholar 

  6. Gomez, J.M., Jimenez, A., Olmos, E. & Sevilla, F. Location and effects of long-term NaCl stress on superoxide dismutase and ascorbate peroxidase isoenzymes of pea (Pisum sativum cv. Puget) chloroplasts. J. Exp. Bot. 55, 119–130 (2004).

    Article  CAS  Google Scholar 

  7. Hernandez, J.A. et al. Antioxidant systems and O2•−/H2O2 production in the apoplast of Pea leaves. Its relation with salt-induced necrotic lesions in minor veins. Plant Physiol. 127, 817–831 (2001).

    Article  CAS  Google Scholar 

  8. Sreenivasulu, N., Grimm, B., Wobus, U. & Weschke, W. Differential response of antioxidant compounds to salinity stress in salt-tolerant and salt-sensitive seedlings of foxtail millet (Setaria italica). Physiol. Plant. 109, 435–442 (2000).

    Article  CAS  Google Scholar 

  9. Apak, R., Guclu, K., Ozyurek, M. & Karademir, S.E. Novel total antioxidant capacity index for dietary polyphenols and vitamins C and E, using their cupric ion reducing capability in the presence of neocuproine: CUPRAC method. J. Agric. Food Chem. 52, 7970–7981 (2004).

    Article  CAS  Google Scholar 

  10. Hon-Wing, L., Vang, M.J. & Mavis, R.D. The cooperative interaction between vitamin E and vitamin C in suppression of peroxidation of membrane phospholipids. Biochim. Biophys. Acta 664, 266–272 (1981).

    Article  Google Scholar 

  11. Yeum, K.-J., Russell, R.M., Krinsky, N.I. & Aldini, G. Biomarkers of antioxidant capacity in the hydrophilic and lipophilic compartments of human plasma. Arch. Biochem. Biophys. 430, 97–103 (2004).

    Article  CAS  Google Scholar 

  12. Saleh, L. & Plieth, C. Fingerprinting antioxidative activities in plants. Plant Methods 5, 2 (2009).

    Article  Google Scholar 

  13. Saleh, L. & Plieth, C. A coelenterazine-based luminescence assay to quantify high-molecular-weight superoxide anion scavenger activities. Nat. Protoc., advance online publication, 16 September 2010, doi:10.1038/nprot.2010.121.

  14. Gillespie, K.M. & Ainsworth, E.A. Measurement of reduced, oxidized and total ascorbate content in plants. Nat. Protoc. 2, 871–874 (2007).

    Article  CAS  Google Scholar 

  15. Knörzer, O.C., Burner, J. & Boger, P. Alterations in the antioxidative system of suspension-cultured soybean cells (Glycine max) induced by oxidative stress. Physiol. Plant. 97, 388–396 (1996).

    Article  Google Scholar 

  16. Zhang, J. & Kirkham, M. Antioxidant responses to drought in sunflower and sorghum seedlings. New Phytol. 132, 361–373 (1996).

    Article  CAS  Google Scholar 

  17. Daood, H.G. et al. Antioxidant vitamin content of spice red pepper (paprika) as affected by technological and varietal factors. Food Chem. 55, 365–372 (1996).

    Article  CAS  Google Scholar 

  18. Falk, J., Andersen, G., Kernebeck, B. & Krupinska, K. Constitutive overexpression of barley 4-hydroxyphenylpyruvate dioxygenase in tobacco results in elevation of the vitamin E content in seeds but not in leaves. FEBS Lett. 540, 35–40 (2003).

    Article  CAS  Google Scholar 

  19. Azevedo, A.M. et al. Horseradish peroxidase: a valuable tool in biotechnology. Biotechnol. Annu. Rev. 5, 199–247 (2003).

    Article  Google Scholar 

  20. Díaz, A.N., Sánchez, F.G. & Garcia, J.A.G. Phenol derivatives as enhancers and inhibitors of luminol-H2O2-horseradish peroxidase chemiluminescence. J. Biolumin. Chemilumin. 13, 75–84 (1998).

    Article  Google Scholar 

  21. Nakamura, M. & Nakamura, S. One- and two-electron oxidations of luminol by peroxidase systems. Free Radic. Biol. Med. 24, 537–544 (1998).

    Article  CAS  Google Scholar 

  22. Nicell, J.A. & Wright, H. A model of peroxidase activity with inhibition by hydrogen peroxide. Enzyme Microb. Technol. 21, 302–310 (1997).

    Article  CAS  Google Scholar 

  23. Veitch, N.C. Horseradish peroxidase: a modern view of a classic enzyme. Phytochemistry 65, 249–259 (2004).

    Article  CAS  Google Scholar 

  24. Gillespie, K.M., Chae, J.M. & Ainsworth, E.A. Rapid measurement of total antioxidant capacity in plants. Nat. Protoc. 2, 867–870 (2007).

    Article  CAS  Google Scholar 

  25. Girotti, S. et al. Plasma antioxidant capacity determination: comparative evaluation of chemiluminescent and spectrophotometric assays. Talanta 56, 407–414 (2002).

    Article  CAS  Google Scholar 

  26. Pellegrini, N. et al. Total antioxidant capacity of plant foods, beverages and oils consumed in Italy assessed by three different in vitro assays. J. Nutr. 133, 2812–2819 (2003).

    Article  CAS  Google Scholar 

  27. Speisky, H. et al. Antioxidant screening of medicinal herbal teas. Phytother. Res. 20, 462–467 (2006).

    Article  CAS  Google Scholar 

  28. Harper, L., Nuttall, S.L., Martin, U. & Savage, C.O.S. Adjuvant treatment of patients with antineutrophil cytoplasmic antibody-associated vasculitis with vitamins E and C reduces superoxide production by neutrophils. Rheumatology 41, 274–278 (2002).

    Article  CAS  Google Scholar 

  29. Girotti, S. et al. Chemiluminescent determination of antioxidant capacity of beverages. Ital. J. Food Sci. 2, 113–122 (2002).

    Google Scholar 

  30. Girotti, S. et al. Chemiluminescent determination of total antioxidant capacity during winemaking. Luminescence 21, 233–238 (2006).

    Article  CAS  Google Scholar 

  31. Jaffar, N.-Z. et al. The use of Pholasin as a probe for the determination of plasma total antioxidant capacity. Clin. Biochem. 39, 55–61 (2006).

    Article  CAS  Google Scholar 

  32. Kampa, M. et al. A new automated method for the determination of the Total Antioxidant Capacity (TAC) of human plasma, based on the crocin bleaching assay. BMC Clin. Pathol. 2, 3 (2002).

    Article  Google Scholar 

  33. Kim, D.-O., Lee, K.W., Lee, H.J. & Lee, C.Y. Vitamin C equivalent antioxidant capacity (VCEAC) of phenolic phytochemicals. J. Agric. Food Chem. 50, 3713–3717 (2002).

    Article  CAS  Google Scholar 

  34. Li, L. et al. A fluorometric assay to determine antioxidant activity of both hydrophilic and lipophilic components in plant foods. J. Nutr. Biochem. 20, 219–226 (2009).

    Article  CAS  Google Scholar 

  35. Ou, B., Hampsch-Woodill, M. & Prior, R.L. Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. J. Agric. Food Chem. 49, 4619–4626 (2001).

    Article  CAS  Google Scholar 

  36. Ou, B. et al. Analysis of antioxidant activities of common vegetables employing oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays: a comparative study. J. Agric. Food Chem. 50, 3122–3128 (2002).

    Article  CAS  Google Scholar 

  37. Serpen, A., Gökmen, V., Pellegrini, N. & Fogliano, V. Direct measurement of the total antioxidant capacity of cereal products. J. Cereal Sci. 48, 816–820 (2008).

    Article  CAS  Google Scholar 

  38. Wayner, D.D.M., Burton, G.W., Ingold, K.U. & Locke, S. Quantitative measurement of the total, peroxyl radical-trapping antioxidant capability of human blood plasma by controlled peroxidation: the important contribution made by plasma proteins. FEBS Lett. 187, 33–37 (1985).

    Article  CAS  Google Scholar 

  39. Schlesier, K., Harwat, M., Böhm, V. & Bitsch, R. Assessment of antioxidant activity by using different in vitro methods. Free Radic. Res. 36, 177–187 (2002).

    Article  CAS  Google Scholar 

  40. Cao, G. & Prior, R.L. Comparison of different analytical methods for assessing total antioxidant capacity of human serum. Clin. Chem. 44, 1309–1315 (1998).

    CAS  PubMed  Google Scholar 

  41. Girotti, S., Bolelli, L., Budini, R. & Arfelli, G. Comparison of analytical methods in determining total antioxidant capacity in red wine. Anal. Lett. 35, 747–758 (2002).

    Article  CAS  Google Scholar 

  42. Janaszewska, A. & Bartosz, G. Assay of total antioxidant capacity: comparison of four methods as applied to human blood plasma. Scand. J. Clin. Lab. Invest. 62, 231–236 (2002).

    Article  CAS  Google Scholar 

  43. Said, T.M. et al. Enhanced chemiluminescence assay versus colorimetric assay for measurement of the total antioxidant capacity of human seminal plasma. J. Androl. 24, 676–680 (2003).

    Article  CAS  Google Scholar 

  44. Sanchez-Moreno, C. Review: methods used to evaluate the free radical scavenging activity in foods and biological systems. Food Sci. Technol. Int. 8, 121–137 (2002).

    Article  CAS  Google Scholar 

  45. Wang, C.C. et al. Trolox-equivalent antioxidant capacity assay versus oxygen radical absorbance capacity assay in plasma. Clin. Chem. 50, 952–954 (2004).

    Article  CAS  Google Scholar 

  46. Rice-Evans, C. Measurement of total antioxidant activity as a marker of antioxidant status in vivo: procedures and limitations. Free Radic. Res. 33 Suppl.: S59–S66 (1999).

    Google Scholar 

  47. Vassalle, C. et al. In vivo total antioxidant capacity: comparison of two different analytical methods. Clin. Chem. Lab. Med. 42, 84–89 (2004).

    Article  CAS  Google Scholar 

  48. Whitehead, T.P., Thorpe, G.H.G. & Maxwell, S.R.J. Enhanced chemiluminescent assay for antioxidant capacity in biological fluids. Anal. Chim. Acta 266, 265–277 (1992).

    Article  CAS  Google Scholar 

  49. Sies, H. Total antioxidant capacity: appraisal of a concept. J. Nutr. 137, 1493–1495 (2007).

    Article  CAS  Google Scholar 

  50. Girotti, S. et al. Automated and manual luminescent assay of antioxidant capacity: analytical features by comparison. Talanta 64, 665–670 (2004).

    Article  CAS  Google Scholar 

  51. Ghiselli, A., Serafini, M., Natella, F. & Scaccini, C. Total antioxidant capacity as a tool to assess redox status: critical view and experimental data. Free Radic. Biol. Med. 29, 1106–1114 (2000).

    Article  CAS  Google Scholar 

  52. Nazari, K. et al. Stabilizing and suicide-peroxide protecting effect of Ni2+ on horseradish peroxidase. J. Iranian Chem. Soc. 2, 232–237 (2005).

    Article  CAS  Google Scholar 

  53. Burlingame, J. et al. Total antioxidant capacity and reactive oxygen species in amniotic fluid. Obstet. Gynecol. 101, 756–761 (2003).

    CAS  PubMed  Google Scholar 

  54. Puntarulo, S. & Cederbaum, A.I. Chemiluminescence from acetaldehyde oxidation by xanthine oxidase involves generation of and interactions with hydroxyl radicals. Alcohol Clin. Exp. Res. 13, 84–90 (1989).

    Article  CAS  Google Scholar 

  55. Wippich, N. et al. Comparison between xanthine oxidases from buttermilk and microorganisms regarding their ability to generate reactive oxygen species. Int. J. Mol. Med. 7, 211–216 (2001).

    CAS  PubMed  Google Scholar 

  56. Ziobro, A. & Bartosz, G. A comparison of the total antioxidant capacity of some human body fluids. Cell Mol. Biol. Lett. 8, 415–419 (2003).

    PubMed  Google Scholar 

  57. Evelson, P. et al. Evaluation of total reactive antioxidant potential (TRAP) of tissue homogenates and their cytosols. Arch. Biochem. Biophys. 388, 261–266 (2001).

    Article  CAS  Google Scholar 

  58. Chuang, C.-C. et al. Serum total antioxidant capacity reflects severity of illness in patients with severe sepsis. Crit. Care. 10, 1 (2006).

    Article  Google Scholar 

  59. Lewinska, A., Wnuk, M., Slota, E. & Bartosz, G. Total anti-oxidant capacity of cell culture media. Clin. Exp. Pharmacol. Physiol. 34, 781–786 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank L. Shaw (Kiel) for critically reading the paper. We also thank S. Vollbehr, G. Weppner and S. Anderson for technical assistance; J. Scheller (Institute for Biochemistry, University of Kiel) for providing HepG2 cells; and A. Scheidig (Structural Biology Group, Kiel) and U.-P. Hansen (Biophysics Group, Kiel) for their generous support. We gratefully acknowledge the financial support of the Deutsche Forschungsgemeinschaft (Grant no. PL253/5), as well as access to the core facilities of the Zentrum für Biochemie und Molekularbiologie, Christian-Albrechts-Universität, Kiel.

Author information

Authors and Affiliations

  1. Zentrum für Biochemie und Molekularbiologie, Universität Kiel, Kiel, Germany

    Livia Saleh & Christoph Plieth

Authors
  1. Livia Saleh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christoph Plieth
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

L.S. carried out the experiments reported in the main paper, performed data processing and participated in amending the draft. C.P. conceived of the protocol, carried out the experiments shown in the supplementary information and wrote the paper. Both authors approved the final version.

Corresponding author

Correspondence to Christoph Plieth.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Decay of the antioxidative capacity of pure antioxidants (PDF 15 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Saleh, L., Plieth, C. Total low-molecular-weight antioxidants as a summary parameter, quantified in biological samples by a chemiluminescence inhibition assay. Nat Protoc 5, 1627–1634 (2010). https://doi.org/10.1038/nprot.2010.120

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2010.120

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing