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Determination of 4-hydroxynonenal by high-performance liquid chromatography with electrochemical detection

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Lipids

Abstract

4-Hydroxy-trans-2-nonenal (HNE) is a highly reactive product of lipid peroxidation originating from the breakdown of phospholipid-bound polyunsaturated fatty acids of cellular membranes. Despite its biological relevance, this aldehyde is only occasionally determined due to the complexity of previously described procedures. Here we present a simple and very sensitive method for the detection of HNE in biological samples. The method is based on the measurement of the 2,4-dinitrophenylhydrazone (DNPH) of the aldehyde by electrochemical detection after separation by reverse-phase high-performance liquid chromatography (HPLC). The greater sensitivity of this procedure as compared to the ultraviolet detection method commonly employed to measure DNPH derivatives of aldehydes after HPLC will allow the detection of HNE below the pmol level. The detection of HNE is highly reproducible even in normal tissues and cells. Increased amounts of HNE were detected in the livers of animals intoxicated with prooxidant agents such as carbon tetrachloride, bromotrichloromethane or bromobenzene. An exponential increase in HNE (and in malondialdehyde) was measured in peroxidizing liver microsomes (in the NADPH/Fe-dependent system). The method is also suitable for the study of very small samples, since HNE could be detected in approximately 1 million cultured cells (polyoma virus-transformed baby hamster kidney fibroblasts); the level rose after exposure of the cells to a Fe3+/ADP prooxidant system.

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Abbreviations

DMEM:

Dulbecco's Modified Eagle's Medium

DNPH:

2,4-dinitrophenylhydrazine

ED:

electrochemical detection

EDTA:

ethylenediaminetetraacetic acid

GC/MS:

gas chromatography/mass spectrometry

HNE:

4-hydroxy-trans-2-nonenal

HNE-DNPH:

2,4-dinitrophenylhydrazone of 4-hydroxynonenal

HPLC:

high-performance liquid chromatography

MDA:

malondialdehyde

PBS:

phosphate buffered saline

TBA:

2-thiobarbituric acid

TLC:

thin-layer chromatography

UV:

ultraviolet

References

  1. Benedetti, A., Comporti, M., and Esterbauer, H. (1980)Biochim. Biophys. Acta 620, 281–296.

    PubMed  CAS  Google Scholar 

  2. Esterbauer, H. (1982) inFree Radicals, Lipid Peroxidation and Cancer (McBrien, D.C.H., and Slater, T.F., eds.), pp. 101–128, Academic Press, London.

    Google Scholar 

  3. Esterbauer, H., Benedetti A., Lang, J., Fulceri, R., Fauler, G., and Comporti, M. (1986)Biochim. Biophys. Acta 876, 154–166.

    PubMed  CAS  Google Scholar 

  4. Benedetti, A., and Comporti, M. (1987)Bioelectrochem. Bioenerg. 18, 187–202.

    Article  CAS  Google Scholar 

  5. Esterbauer, H. (1985) inFree Radicals in Liver Injury (Poli, G., Cheeseman, K.H., Dianzani, M.U., and Slater, T.F., eds.) pp. 29–47, IRL Press, Oxford.

    Google Scholar 

  6. Esterbauer, H., Schaur, R.J., and Zollner, H. (1991)Free Radical Biol. Med. 11, 81–128.

    Article  CAS  Google Scholar 

  7. Cajone, F., and Bernelli-Zazzera, A. (1988)Chem. Biol. Interactions 65, 235–246.

    Article  CAS  Google Scholar 

  8. Benedetti, A., Casini, A.F., Ferrali, M., Fulceri, R., and Comporti, M. (1981) inRecent Advances in Lipid Peroxidation and Tissue Injury (Slater, T.F., and Garner, A., eds.), pp. 56–85, Brunel Printing Services, Uxbridge.

    Google Scholar 

  9. Cadenas, E., Müller, A., Brigelius, R., Esterbauer, H., and Sies, H. (1983)Biochem. J. 214, 479–487.

    PubMed  CAS  Google Scholar 

  10. Krokan, H., Grafström, R.C. Sundqvist, K., Esterbauer, H., and Harris, C.C. (1985)Carcinogenesis 6, 755–759.

    Google Scholar 

  11. Hauptlorenz, S., Esterbauer, H., Moll, W., Pümpel, R., Schauenstein, E., and Puschendorf, B. (1985)Biochem. Pharmacol. 34, 3803–3809.

    Article  PubMed  CAS  Google Scholar 

  12. Poot, M., Esterbauer, H., Robinovitch, P.S., and Hoehn, H. (1988)J. Cell Physiol. 137, 421–429.

    Article  PubMed  CAS  Google Scholar 

  13. Brambilla, G., Sciaba, L., Faggin, P., Maura, A., Marinari, U.M., Ferro, M., and Esterbauer, H. (1986)Mutation Res. 171, 169–176.

    Article  PubMed  CAS  Google Scholar 

  14. Esterbauer, H., Cheeseman, K.H., Dianzani, M.U., Poli, G., and Slater, T.F. (1982)Biochem. J. 208, 129–140.

    PubMed  CAS  Google Scholar 

  15. Lang, J., Celoto, C., and Esterbauer, H. (1985)Anal. Biochem. 150, 369–378.

    Article  PubMed  CAS  Google Scholar 

  16. Yoshino, K., Matsura, T., Sano, M., Saito, S.I., and Tomita, I. (1986)Chem. Pharm. Bull. 34, 1694–1700.

    PubMed  CAS  Google Scholar 

  17. Beckman, J.K., Morley, Jr., S.A., and Greene, H.L. (1991)Lipids 26, 155–161.

    PubMed  CAS  Google Scholar 

  18. van Kuijk, F.J.G.M., Thomas, D.W., Stephens, R.J., and Dratz, E.A. (1986)Biochem. Biophys. Res. Commun. 139, 144–149.

    Article  PubMed  Google Scholar 

  19. Tamura, H., and Shibamoto, T. (1991)Lipids 26, 170–173.

    PubMed  CAS  Google Scholar 

  20. Norsten-Höög, C., and Cronholm, T. (1990)Anal. Biochem. 189, 131–137.

    Article  PubMed  Google Scholar 

  21. Benedetti, A., Pompella, A., Fulceri, R., Romani, A., and Comporti, M. (1986)Biochim. Biophys. Acta 876, 658–666.

    PubMed  CAS  Google Scholar 

  22. Casini, A.F., Farrali, M., Pompella, A., Maellaro, E., and Comporti, M. (1986)Am. J. Pathol. 123, 520–531.

    PubMed  CAS  Google Scholar 

  23. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951)J. Biol. Chem. 193, 265–275.

    PubMed  CAS  Google Scholar 

  24. Benedetti, A., Esterbauer, H., Ferrali, M., Fulceri, R., and Comporti, M. (1982)Biochim. Biophys. Acta 711, 345–356.

    PubMed  CAS  Google Scholar 

  25. Burdon, R.H., Gill, V., and Rice-Evans, C. (1990)Free Radical Res. Commun. 11, 65–76.

    CAS  Google Scholar 

  26. Esterbauer, H., and Zollner, H. (1989)Free Radical Biol. Med. 7, 197–203.

    Article  CAS  Google Scholar 

  27. Yoshino, K., Sano, M., Fujita, M., and Tomita, I. (1986)Chem. Pharm. Bull. 34, 5184–5187.

    PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Istituto di Patologia Generale, Università di Siena, Via del Laterino 8, 53100, Siena, Italy

    Christopher Goldring, Alessandro F. Casini, Emilia Maellaro, Barbara Del Bello & Mario Comporti

Authors
  1. Christopher Goldring
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  2. Alessandro F. Casini
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  3. Emilia Maellaro
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  4. Barbara Del Bello
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  5. Mario Comporti
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Additional information

During the course of these studies, Dr. Goldring was on leave from Division of Biochemistry, UMDS Guy's Hospital, London SE1 9RT, UK.

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Goldring, C., Casini, A.F., Maellaro, E. et al. Determination of 4-hydroxynonenal by high-performance liquid chromatography with electrochemical detection. Lipids 28, 141–145 (1993). https://doi.org/10.1007/BF02535778

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  • DOI: https://doi.org/10.1007/BF02535778

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