Detailed methods for the quantification of nitric oxide in aqueous solutions using either an oxygen monitor or EPR

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• 作者：Venkataraman ; Sujatha ; Martin ; Sean M. ; Schafer ; Freya Q. ; Buettner ; Garry R.
• 关键词：Nitric oxide ; Nitrite ; N-methyl-D-glucamine dithiocarbamate (MGD) ; EPR ; Oxygen monitor ; Free radicals ; 3-CP ; 3-carboxy proxyl ; DDI ; De-ionized distilled water ; EPR ; Electron Paramagnetic Resonance ; a^N ; Hyperfine splitting constant of nitrogen
• 刊名：Free Radical Biology and Medicine
• 出版年：2000
• 期刊代码：105_08915849
• 类别：med
• 出版时间：September 15, 2000
• 卷：29
• 期：6
• 页码：580-585
• 文件大小：75.5 K

摘要

The interest in nitric oxide has grown with the discovery that it has many biological functions. This has heightened the need for methods to quantify nitric oxide. Here we report two separate methods for the quantification of aqueous stock solutions of nitric oxide. The first is a new method based on the reaction of nitric oxide with oxygen in liquid phase (4mg align=center border=0 SRC=/images/glyphs/BO0.GIF>NO + O₂ + 2H₂O → 4HNO₂); an oxygen monitor is used to measure the consumption of oxygen by nitric oxide. This method offers the advantages of being both simple and direct. The presence of nitrite or nitrate, frequent contaminants in nitric oxide stock solutions, does not interfere with the quantification of nitric oxide. Measuring the disappearance of dissolved oxygen, a reactant, in the presence of known amounts of nitric oxide has provided verification of the 4:1 stoichiometry of the reaction. The second method uses electron paramagnetic resonance spectroscopy (EPR) and the nitric oxide trap {Fe²⁺-(MGD)₂}, (MGD = N-methyl-D-glucamine dithiocarbamate). The nitrosyl complex is stable and easily quantitated as a room temperature aqueous solution. These two methods are validated with Sievers 280 Nitric Oxide Analyzer and cross-checked with standards using UV-Vis spectroscopy. The practical lower limits for measuring the concentration of nitric oxide using the oxygen monitor approach and EPR are approximately 3 μM and 500 nM, respectively. Both methods provide straightforward approaches for the standardization of nitric oxide in solution.