An improved method to measure nitrate/nitrite with an NO-selective electrochemical sensor

被引:24
作者
Boo, Yong Chool
Tressel, Sarah L.
Jo, Hanjoong
机构
[1] Georgia Tech, Wallace H Coulter Dept Biomed Engn, Atlanta, GA 30322 USA
[2] Emory Univ, Div Cardiol, Atlanta, GA 30322 USA
[3] Kyungpook Natl Univ, Sch Med, Dept Mol Med, Taegu 702701, South Korea
来源
NITRIC OXIDE-BIOLOGY AND CHEMISTRY | 2007年 / 16卷 / 02期
关键词
endothelial cells; eNOS; nitric oxide; NO assay; NO sensor;
D O I
10.1016/j.niox.2006.09.004
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Nitric oxide (NO) produced from NO synthase(s) (NOS) is an important cell signaling molecule in physiology and pathophysiology. It remains challenging, however, to measure NO accurately and reproducibly in many cell types producing relatively low levels of NO from the enzymes such as endothelial NO synthase (eNOS). In the present study, we describe a very sensitive and convenient analytical method that affords measurement of 1 to 2 nM concentration of NO, (nitrite plus nitrate) in culture media. In the present study, we used an ultra-sensitive NO-selective electrochemical sensor (AmiNO700) in combination with a highly efficient nitrate conversion method, which coupled the nitrate reductase step with the glucose-6-phosphate dehydrogenase system. An aliquot of conditioned culture media was first treated with nitrate reductase, NADPH, glucose-6-phosphate dehydrogenase and glucose-6-phosphate to convert nitrate to nitrite quantitatively. The nitrite (that is present originally plus the reduced nitrate) was then reduced to equimolar NO in an acidic iodide bath while NO was being detected by the sensor. With this analytical method, we can quantitatively and reliably measure basal and stimulated NO release from cultured endothelial cells. We believe this improved assay should be useful in measuring a wide range of NO levels, especially the low but physiologically relevant levels, in many cell types. (c) 2006 Elsevier Inc. All rights reserved.
引用
收藏
页码:306 / 312
页数:7
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