Fluorometric determination of nitric oxide

Document Type

Article

Publication Date

1-1-1995

Abstract

A variety of different spectrophotometric methods have been developed to quantify nitric oxide (NO)-derived metabolites such as nitrate and nitrite. Some of these methods measure the formation of colored complexes such as azo dyes (e.g., Griess reaction), whereas other methods measure the formation of fluorometric compounds. Virtually all of these methods require the rapid and spontaneous decomposition of NO in the presence of molecular oxygen to yield potent N-nitrosating agents which ultimately N-nitrosate the various detector compounds. The N-nitrosation of 2,3-diaminonaphthalene (DAN) to yield the highly fluorescent 2,3-naphthotriazole offers the advantages of specificity, sensitivity, and versatility. This assay is capable of detecting as little as 10-30 nM (10-30 picomol/ml) of NO and may be used to quantify NO generated in a physiologically relevant environment (e.g., neutral pH) with minimal interference due to nitrite decomposition. The trapping efficiency of DAN for NO is approximately 80% and the enhanced sensitivity of this assay provides a valuable method for the determination of small amounts of NO. This chapter details the spectrofluorometric methods used in our laboratory to quantify NO generated by several different NO-releasing compounds and by a variety of different cell types in culture. In addition, we describe how this method may be modified to quantify nitrite and nitrate, the stable decomposition products of NO oxidation in aqueous solution. © 1995 Academic Press Limited.

Publication Source (Journal or Book title)

Methods: A Companion to Methods in Enzymology

First Page

40

Last Page

47

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