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Measurement of oxidized nucleobases and nucleosides in human urine by using a GC/MS assay in the selective ion monitoring mode

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Analysis of Free Radicals in Biological Systems

Summary

Urinary excretion of oxidized bases and nucleosides has been suggested as a non-invasive biomarker of oxidative damage and repair. Oxidative adducts, resulting from a variety of reactive oxygen species. are in fact excreted in urine following repair. A method for the analysis of 5-hydroxyuracil (5-OHUra), 5-hydroxy-2’-deoxyuridine (5-OHdUrd). 5-hy- droxymethyluracil (5-HMUra). 5-hydroxymethyl-2’-deoayuridine (5-HMdUrd) in urine has been developed, based on GC/MS quantification. Stable isotopically labelled (M + 4) analogues, synthesized in our laboratory, are used as internal standards, assuring appropriate quantification in complex biological fluids. Urines are first purified by HPLC on a reverse phase column and fractions corresponding to the compounds of interest derivatized for GC/MS analysis. Two different derivatives for GC/MS determination, the trimethylsylyl (TMS) and the tert-butyldimethylsylyl (t-RDMS) derivatives. have been tested. Detection limit in the selective ion monitoring (SIM) mode is generally two-fold lomer with t-BDMS. Preliminary results on four healthy subjects show that the oxidized bases 5-OHUra and 5-HMUra are excreted in urine (approximately 0.7 nmol/kg/day), while the corresponding nucleosides are not detectable. The developed method could have important applications in the analysis of oxidative lesions in DNA exposed to oxidative stress or in DNA from biological samples.

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Author information

Authors and Affiliations

  1. CEA/Département de Recherche Fodamentale sur la Matiére Condensée-SESAM/LAN, F-38054, Cédex 9, Grenoble, France

    M.-F. Incardona, F. Bianchini & J. Cadet

  2. Laboratoire de Biochimie C, CHRU de Grenohle, Hopital Michallon, B.P. 217 X, F-38043, La Tronche, France

    A. E. Favier

Authors
  1. M.-F. Incardona
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  2. F. Bianchini
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  3. A. E. Favier
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  4. J. Cadet
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Editor information

Editors and Affiliations

  1. Laboratoire de Biochimie C, CHU Albert Michallon, BP 217, F-38043, Grenoble Cédex 9, France

    A. E. Favier

  2. CEA/Département de Recherche Fondamentale sur la Matière, Condensée, SESAM/LAN, 17, rue des Martyrs, F-38054, Grenoble Cédex 9, France

    J. Cadet

  3. Biophysics Research Institute, Medical College of Wisconsin, 8701 Watertown Plank Road, PO Box 26509, Milwaukee, WI, 53226-0509, USA

    B. Kalyanaraman

  4. Laboratoire de Biochimie Recherche, Domaine Universitaire, F-38400, Saint Martin d’Hères, France

    M. Fontecave

  5. Laboratoire de Chimie Biomimétique LEDSS II, Domaine Universitaire, F-38400, Saint Martin d’Hères, France

    J.-L. Pierre

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© 1995 Birkhäuser Verlag Basel/Switzerland

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Incardona, MF., Bianchini, F., Favier, A.E., Cadet, J. (1995). Measurement of oxidized nucleobases and nucleosides in human urine by using a GC/MS assay in the selective ion monitoring mode. In: Favier, A.E., Cadet, J., Kalyanaraman, B., Fontecave, M., Pierre, JL. (eds) Analysis of Free Radicals in Biological Systems. Birkhäuser Basel. https://doi.org/10.1007/978-3-0348-9074-8_18

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  • DOI: https://doi.org/10.1007/978-3-0348-9074-8_18

  • Publisher Name: Birkhäuser Basel

  • Print ISBN: 978-3-0348-9895-9

  • Online ISBN: 978-3-0348-9074-8

  • eBook Packages: Springer Book Archive

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