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...

This study compared the concentrations of components such as nitrate, organic acids, free amino acids, cations and sugars in komatsuna (Brassica campestris var. perviridis) grown with carbonate, sulfate and chloride application (CO 3 -TK, SO 4 -TK and Cl-TK, respectively). The komatsuna was cultivated in 1/2000-a Wagner pots (6 hills per pot) filled with light-colored Andosol in a glasshouse for 37 days at soil water potential (SWP) right before harvesting of −6.2 and −62 kPa. And 26.5 mmol c of carbonate, sulfate or chloride in the form of potassium salts was applied to each pot. Chloride application to the komatsuna induced relatively high concentrations of potassium, calcium and magnesium ions, and the low concentrations of malate, glucose and fructose on a dry weight basis (DWB). Carbonate application induced relatively high concentrations of malate and low concentrations of nitrate. Sulfate was almost between chloride and carbonate. Concentration variation was not substantial in free amino acids. Those tendencies were almost the same in both SWPs. Most of the variations in component concentrations were attributed to the regulation of ionic and osmotic balance to respond to chloride, nitrate and sulfate absorption, as judged from quantitative relationships among the components and water in the komatsuna. It seems that the absorption of nitrate is influenced by the pH of the soil. There were clear differences in glucose, fructose and malate concentrations among CO 3 -TK, SO 4 -TK and Cl-TK on a fresh weight basis just as on a DWB.