Quantifying Nanomolar Levels of Nitrite in Biological Samples by HPLC-Griess Method : Special Reference to Arterio-Venous Difference in vivo

Access this Article

Search this Article

Author(s)

    • MIWA TOMOKO
    • Department of Pharmacology, School of Medicine, Kanazawa Medical University
    • SHINKAWA IKUMI
    • Department of Pharmacology, School of Medicine, Kanazawa Medical University
    • NISHIZAWA NAOKI
    • Department of Pharmacology, School of Medicine, Kanazawa Medical University
    • NOMURA MIHOKO
    • Department of Pharmacology, School of Medicine, Kanazawa Medical University
    • YOSHIDA JUNKO
    • Department of Pharmacology, School of Medicine, Kanazawa Medical University
    • KAWADA TOMIE
    • Department of Clinical Pharmacy, Faculty of Pharmacy, Musashino University
    • NISHIO MATOMO
    • Department of Pharmacology, School of Medicine, Kanazawa Medical University

Abstract

Nitrite (NO<sub>2</sub><sup>-</sup>) is assumed to play an important role in regulation of vascular tone as a reservoir of nitric oxide (NO). To examine its physiological contribution, however, a sensitive method is required for determination of the true level of NO<sub>2</sub><sup>-</sup> in biological samples. To this end, practical consideration to avoid NO<sub>2</sub><sup>-</sup> contamination through the quantification procedure is important. We present here a highly sensitive and accurate method for determining NO<sub>2</sub><sup>-</sup> in plasma by improving the HPLC-Griess system with minimal NO<sub>2</sub><sup>-</sup> contamination in the samples. The system achieved high sensitivity (detection limit of 2 nM and sensitivity to 1 nM) and complete separation of the NO<sub>2</sub><sup>-</sup> signal peak by modifying the system setup and mobile phase. Using this method, we achieved acceptable quantification of low NO<sub>2</sub><sup>-</sup> levels in plasma. Deproteinization by ultrafiltration and exposure to atmosphere before measurement were identified as the major sources of NO<sub>2</sub><sup>-</sup> contamination during sample processing. We addressed these issues by the use of methanol for deproteinization and gas-tight caps. These countermeasures allowed us to detect small arterio-venous NO<sub>2</sub><sup>-</sup> differences in rabbit plasma that may indicate kinetic difference of NO<sub>2</sub><sup>-</sup> in a small number of samples (<i>n</i> = 6). This difference became prominent when NO<sub>2</sub><sup>-</sup> or a NO releasing agent, NOR1, was intravenously applied. Our results indicate that application of a sensitive method with careful handling is important for accurate determination of NO<sub>2</sub><sup>-</sup> and that our method is applicable for further examination of the kinetic features of NO<sub>2</sub><sup>-</sup> <i>in vivo</i>.

Journal

  • The Tohoku Journal of Experimental Medicine

    The Tohoku Journal of Experimental Medicine 215(1), 1-11, 2008-05-01

    Tohoku University Medical Press

References:  29

Cited by:  2

Codes

  • NII Article ID (NAID)
    10024167728
  • NII NACSIS-CAT ID (NCID)
    AA00863920
  • Text Lang
    ENG
  • Article Type
    Journal Article
  • ISSN
    00408727
  • Data Source
    CJP  CJPref  J-STAGE 
Page Top