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

Abstract

Nitrite (NO₂^-) 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 NO₂^- in biological samples. To this end, practical consideration to avoid NO₂^- contamination through the quantification procedure is important. We present here a highly sensitive and accurate method for determining NO₂^- in plasma by improving the HPLC-Griess system with minimal NO₂^- contamination in the samples. The system achieved high sensitivity (detection limit of 2 nM and sensitivity to 1 nM) and complete separation of the NO₂^- signal peak by modifying the system setup and mobile phase. Using this method, we achieved acceptable quantification of low NO₂^- levels in plasma. Deproteinization by ultrafiltration and exposure to atmosphere before measurement were identified as the major sources of NO₂^- 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 NO₂^- differences in rabbit plasma that may indicate kinetic difference of NO₂^- in a small number of samples (n = 6). This difference became prominent when NO₂^- 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 NO₂^- and that our method is applicable for further examination of the kinetic features of NO₂^- in vivo.