A Spectrophotometric Method for the Direct Detection and Quantitation of Nitric Oxide, Nitrite, and Nitrate in Cell Culture Media
✍ Scribed by Lisa A. Ridnour; Julia E. Sim; Michael A. Hayward; David A. Wink; Sean M. Martin; Garry R. Buettner; Douglas R. Spitz
- Publisher
- Elsevier Science
- Year
- 2000
- Tongue
- English
- Weight
- 117 KB
- Volume
- 281
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A method for the spectrophotometric determination of nitric oxide, nitrite, and nitrate in tissue culture media is presented. The method is based on the nitric oxide-mediated nitrosative modification of sulfanilic acid that reacts with N-(1-naphthyl)ethylenediamine dihydrochloride forming an orange-colored product absorbing at 496 nm. Nitric oxide levels were determined in culture media from this absorbance measurement using chemiluminescence standardization. Extinction coefficients of 5400 and 6600 M ؊1 cm ؊1 were determined for the nitric oxide product in assay solutions containing 0.1 or 100 mM KPO 4 buffer (pH 7.4), respectively, with a limit of detection of 1 M. Acidification of these reactions (pH 2.4) generated a pinkcolored product absorbing at 540 nm allowing for quantitation of total nitric oxide/nitrite levels using extinction coefficients of 38,000 and 36,900 M ؊1 cm ؊1 , for the assay solutions described. The limit of detection of this assay was approximately 300 nM. Using the 100 mM KPO 4 buffer system, nitrate levels were determined following reduction to nitrite using a coppercoated cadmium reagent with an extinction coefficient of 29,500 M ؊1 cm ؊1 and a detection limit of 0.5 M. The utility of these assays was demonstrated in the standardization of nitric oxide-saturated cell culture media, and the release of nitric oxide by the NONOate compound DEA/NO.