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

Abstract

Nitrite (NO(2)(-)) 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(2)(-) in biological samples. To this end, practical consideration to avoid NO(2)(-) contamination through the quantification procedure is important. We present here a highly sensitive and accurate method for determining NO(2)(-) in plasma by improving the HPLC-Griess system with minimal NO(2)(-) 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(2)(-) signal peak by modifying the system setup and mobile phase. Using this method, we achieved acceptable quantification of low NO(2)(-) levels in plasma. Deproteinization by ultrafiltration and exposure to atmosphere before measurement were identified as the major sources of NO(2)(-) 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(2)(-) differences in rabbit plasma that may indicate kinetic difference of NO(2)(-) in a small number of samples (n = 6). This difference became prominent when NO(2)(-) 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(2)(-) and that our method is applicable for further examination of the kinetic features of NO(2)(-) in vivo.