, Volume 26, Issue 10, pp 853–856

Lipid hydroperoxide measurement by oxidation of Fe2+ in the presence of xylenol orange. Comparison with the TBA assay and an iodometric method

  • Zhen-Yue Jiang
  • Alison C. S. Woollard
  • Simon P. Wolff


Study of the role of hydroperoxides and lipid peroxidation in disease requires simple and sensitive methods for direct hydroperoxide measurement. We report on a technique for measuring hydroperoxide which relies upon the rapid hydroperoxide-mediated oxidation of Fe2+ under acidic conditions. Fe3+ forms a chromophore with xylenol orange which absorbs strongly at 560 nm, yielding an apparent E560 (for H2O2, butyl hydroperoxide and cumene hydroperoxide) of 4.3×104 M−1 cm−1. The assay was validated in a study of liposomal lipid peroxidation and shown to give results comparable with those obtained by an iodometric method or by measuring conjugated dienes. The assay involving thiobarbituric acid, by comparison, underestimates lipid peroxidation and does not measure hydroperoxideper se.



butylated hydroxytoluene


conjugated diene


diethylenetriaminepentaacetic acid


ferrous oxidation/xylenol orange


reduced gluthathione




phosphate-buffered saline


thiobarbituric acid


TBA-reactive material


trichloroacetic acid


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Halliwell, B., and Grootveld, M. (1987)FEBS Lett. 213, 9–14.PubMedCrossRefGoogle Scholar
  2. 2.
    Wolff, S.P., Garner, A., and Dean, R.T. (1986)Trends Biochem. Sci. 11, 27–31.CrossRefGoogle Scholar
  3. 3.
    Hicks, M., and Gebicki, J.M. (1979)Anal. Biochem. 99, 249–253.PubMedCrossRefGoogle Scholar
  4. 4.
    Frew, J.E., Jones, P., and Scholes, G. (1983)Anal. Chim. Acta 155, 139–150.CrossRefGoogle Scholar
  5. 5.
    Cathcart, R., Schwiers, E., and Ames, B.N. (1990)Anal. Biochem. 134, 111–116.CrossRefGoogle Scholar
  6. 6.
    Kulmacz, R.J., Miller, J.F., Pendleton, R.B., and Lands, W.E.M. (1990)Methods Enzymol. 186, 431–438.PubMedGoogle Scholar
  7. 7.
    Yamamoto, Y., Frei, B., and Ames, B.N. (1990) inMethods Enzymol. 186, 371–380.Google Scholar
  8. 8.
    Gutteridge, J.M.C. (1986)Free Radical Res. Commun. 1, 173–184.Google Scholar
  9. 9.
    Gutteridge, J.M.C., and Halliwell, B. (1990)Trends Biochem. Sci. 15, 129–135.PubMedCrossRefGoogle Scholar
  10. 10.
    Gutteridge, J.M.C., and Tickner, T.R. (1978)Anal. Biochem. 91, 250–257.PubMedCrossRefGoogle Scholar
  11. 11.
    Yagi, K. (1982) inLipid Peroxides in Biology and Medicine (Yagi, K., ed.) pp. 223–242, Academic Press, New York.Google Scholar
  12. 12.
    Warevdakar, V.S., and Saslaw, L.D. (1959)J. Biol. Chem. 234, 1945–1950.Google Scholar
  13. 13.
    Esterbauer, H., and Cheeseman, K.H. (1990)Methods Enzymol. 186, 407–421.PubMedGoogle Scholar
  14. 14.
    Draper, H.H., and Hadley, M.,Methods Enzymol. 186, 421–431.Google Scholar
  15. 15.
    Gupta, B.L. (1973)Microchem. J. 18, 363–374.CrossRefGoogle Scholar
  16. 16.
    Jiang, Z.-Y., Woollard, A.C.S., and Wolff, S.P. (1990)FEBS Lett. 268, 69–71.PubMedCrossRefGoogle Scholar
  17. 17.
    Pick, U. (1981)Arch. Biochem. Biophys. 212, 186–194.PubMedCrossRefGoogle Scholar
  18. 18.
    Slater, T.F., and Sawyer, B.C. (1971)Biochem. J. 123, 805–814.PubMedGoogle Scholar
  19. 19.
    Pryor, W.A., and Castle, L. (1984)Methods Enzymol. 105, 293–299.PubMedCrossRefGoogle Scholar
  20. 20.
    Grossman,A., and Wendel, A. (1983)Eur. J. Biochem. 135, 549–552.CrossRefGoogle Scholar
  21. 21.
    Sevanian, A., Muakkassah, S.F., and Montestruque, S. (1983)Arch. Biochem. Biophys. 223, 441–452.PubMedCrossRefGoogle Scholar
  22. 22.
    Ohkawa, H., Ohishi, N., and Yagi, K. (1979)Anal. Biochem. 95, 351–358.PubMedCrossRefGoogle Scholar
  23. 23.
    Hunt, J.V., Smith, C.C.T., and Wolff, S.P. (1990)Diabetes 39, 1420–1424.PubMedGoogle Scholar

Copyright information

© American Oil Chemists’ Society 1991

Authors and Affiliations

  • Zhen-Yue Jiang
    • 1
  • Alison C. S. Woollard
    • 1
  • Simon P. Wolff
    • 1
  1. 1.Toxicology Laboratory, Dept. of Clinical PharmacologyUniversity College and Middlesex School of MedicineLondonU.K.

Personalised recommendations