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Lipid hydroperoxide analysis by reverse-phase high-performance liquid chromatography with mercury cathode electrochemical detection

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Book cover Analysis of Free Radicals in Biological Systems

Summary

Lipid hydroperoxides (LOOHs) are important intermediates generated during peroxidative degeneration of unsaturated lipids in cell membranes and other organized assemblies. Such degeneration typically occurs under conditions of oxidative stress. Since LOOHs are subject to rapid turnover in the presence of peroxidases, reductants, or metal ions, their levels may be very low and difficult to measure in complex systems such as peroxidizing cells or lipoproteins. This has prompted the development of new approaches for the high sensitivity/high specificity detection of biological LOOHs. One such approach is reverse-phase high-performance liquid chromatography with electrochemical detection using a renewable mercury drop cathode [HPLC-EC(Hg)]. Using HPLC-EC(Hg) under optimal mobile phase conditions, we have been able to achieve baseline separation of several cholesterol hydroperoxides (ChOOHs). not only from one another, but also from phospholipid hydroperoxide species (PLOOHs). ChOOHs and PLOOHs in model systems, e.g. photodynamically treated liposomes or erythrocyte membranes, could be readily resolved and quantified by this means, with detection limits of <0.5 pmol and <3Opmol, respectively. Moreover, with HPLCEC( Hg) it was possible to determine the relative susceptibility of membrane ChOOHs and PLOOHs to selenoperoxidase-catalyzed reduction (detoxification). HPLC-EC( Hg) analysis of LOOHs has been extended to more complex systems. viz. (i) murine leukemia L1210 cells subjected to photooxidative stress; and (ii) oxidatively modified low density lipoprotein (LDL). Discrete peroxide families could be identified and quantified in each case. On the strength of these observations, and because of its low cost. ease of operation. and extraordinary accuracy/precision, HPLC-EC(Hg) is a highly recommended technique for ultrasensitive LOOH analysis.

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Author information

Authors and Affiliations

  1. Institute of Molecular Biology, Jagiellonian University, Krakow, Poland

    W. Korytowski

  2. Department of Biochemistry, Medical College of Wisconsin, 53226, WI, Milwaukee, USA

    W. Korytowski & A. W. Girotti

Authors
  1. W. Korytowski
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  2. A. W. Girotti
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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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Korytowski, W., Girotti, A.W. (1995). Lipid hydroperoxide analysis by reverse-phase high-performance liquid chromatography with mercury cathode electrochemical detection. 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_12

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  • DOI: https://doi.org/10.1007/978-3-0348-9074-8_12

  • 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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