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. NO 2

The critical role of nitric oxide (NO) in regulating vascular tone is now widely recognized, and the search for reliable and practical indices of endothelial NO formation have been received much attention. 1) Early studies that attempted to use plasma nitrate (NO 3 Ϫ ) or NO x (nitrite [NO 2 Ϫ ] plus NO 3 Ϫ ) as stable metabolites of NO 2,3) encountered too high a degree of interference, resulting from numerous confounding factors (including contamination), for their results to be applied practically. [4][5][6] In addition, as-yet unexplained and paradoxical decreases in NO x following increases in NO formation have been reported. 7,8) In contrast, plasma NO 2 Ϫ has been the source of much interest lately as a promising indicator of NO production following reports that approximately 70-90% of circulating plasma NO 2 Ϫ is derived from endothelial nitric oxide synthase (eNOS) activity in humans and animals. 9,10) Indeed, several studies have shown a close relationship between changes in endothelium-dependent blood flow and plasma NO 2 Ϫ levels. 9,11,12) Furthermore, plasma NO 2 Ϫ is now believed to be a storage site for NO activity. Plasma NO 2 Ϫ is catalyzed by the nitrite reductase activity of deoxygenated hemoglobin (deoxyHb) 13) ; evidence in support of this includes the enhancement of vasodilatory activity of NO 2 Ϫ or production of NO in the presence of deoxyHb. [14][15][16][17][18] To further examine and evaluate these roles for plasma NO 2 Ϫ , accurate and highly sensitive methods for quantifying NO 2 Ϫ and precise information regarding kinetics in vivo are essential. Although there have been some preliminary and incomplete reports regarding NO 2 Ϫ kinetics in vivo, 19,20) to date, there are no systematic data based on standardized methods.Therefore, the goal of the present study was to clarify the kinetic features of plasma NO 2 Ϫ in vivo using a canonical method (NO 2 Ϫ loading study) with an established highly sensitive quantifying technique, 21) taking possible arteriovenous (A-V) differences into consideration. That steady-state NO 2 Ϫ levels might differ between veins and arteries has been subject to debate for many years. 7,12,[21][22][23] MATERIALS AND METHODS Measurement of NO 2؊ and NO 3 ؊We determined NO x levels using a high-performance liquid chromatography (HPLC)-Griess system (ENO10 and ENO20; Eicom, Kyoto, Japan) consisting of a separation column, a flow reactor (with Griess reagent), a reduction column, and a detector at 540 nm, as described elsewhere. 24) Operating under default conditions, the detection limit and sensitivity was 0.1 mM for both NO 2 Ϫ and NO 3 Ϫ with a loading volume of 10 m1. To determine nanomolar concentrations of NO 2 Ϫ , we removed the reduction column and increased the loading volume to 100 m1, which improved the sensitivity and detection limits for NO 2 Ϫ to 1 nM and 2 nM, respectively. 21) In addition, a modified aqueous mobile phase was applied to improve the recovery time of the system. Special attention was paid to excluding possible sources of NO 2 Ϫ co...

Nitric oxide (NO) from endothelial cells is now widely accepted as a key substance in the regulation of vascular tone. 1) However, due to its short half-life and susceptibility to many substances in vivo, the important roles of stable carriers or a reservoir of NO bioactivity in circulating blood are receiving a great deal of attention. [2][3][4] One of these is nitrite (NO 2 Ϫ ), which is catalyzed by the nitrite reductase activity of deoxygenated hemoglobin to form NO. 2,5) Then, it will cause vasodilation to increase blood flow, leading to improve oxygen supply at the region of oxygen deficiency. Accumulating evidence 2,5) under steady-state conditions and pathophysiological conditions support this idea. S-Nitrosothiols (R-SNOs) in plasma are also considered carriers of NO bioactivity and are expected to transport the activity downstream. 3,[6][7][8] However, plasma levels of R-SNOs, even under unstimulated conditions, vary widely by species, institute, and methods, ranging from non-detectable to 9200 nM. 3,9) As the assessment for RSNOs is problematic and not free from artifacts, 9-11) these methodological difficulties are likely responsible for divergent data and for the fragile basis of a regulatory role for plasma R-SNOs. Therefore, the presence and quantity of RSNOs under steady-state conditions have been subject to reevaluation with reliable methods. 9,[12][13][14][15] Another concern is the involvement of R-SNO in pharmacological modifications of vascular tone (blood pressure in vivo). Endothelial stimulation by acetylcholine will cause NO formation, 1) and therefore, R-SNOs may be involved in the vasodilatory effect. However, as far as we surveyed, no such report was found. Nitrovasodilators such as glyceryl trinitrate (GTN), isosorbide dinitrate (ISDN) and sodium nitroprusside (SNP) require respective unique metabolic activation before developing NO bioactivity, 16) and the possible involvement of RSNOs as intermediates is indicated. 17,18) However, no report has systematically compared all of these NO-related vasodilators in relation to plasma R-SNOs in one species. Therefore, we systematically examined the possible involvement of R-SNOs in the regulation of vascular tone under steady-state conditions and pharmacological stimulations by acetylcholine and nitrovasodilators. For this aim, we employed quantitative devices for R-SNOs with high sensitivity avoiding artifacts as is described elsewhere. 10) MATERIALS AND METHODSAnimal Experiments Japanese white rabbits weighing 2.3-3.5 kg (17-23 weeks old) were anesthetized with intravenous sodium pentobarbital (30 mg/kg). Cannulae were inserted into the jugular vein (for drug administration), the carotid artery (to monitor blood pressure), and the femoral vein and artery (for blood sampling). A tachometer to measure pulse rate was triggered by pulse waves of arterial pressure. Experimental procedures were performed after a stabilization period of 20-60 min. When required, several minutes were allowed to elapse for the recovery of hemodynamic parameters af...