Journal article
A Spectrophotometric Method for the Direct Detection and Quantitation of Nitric Oxide, Nitrite, and Nitrate in Cell Culture Media
Analytical biochemistry, Vol.281(2), pp.223-229
06/01/2000
DOI: 10.1006/abio.2000.4583
PMID: 10870839
Abstract
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 KPO4 buffer (pH 7.4), respectively, with a limit of detection of 1 μM. Acidification of these reactions (pH 2.4) generated a pink-colored 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 KPO4 buffer system, nitrate levels were determined following reduction to nitrite using a copper-coated cadmium reagent with an extinction coefficient of 29,500 M−1 cm−1 and a detection limit of 0.5 μM. 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.
Details
- Title: Subtitle
- A Spectrophotometric Method for the Direct Detection and Quantitation of Nitric Oxide, Nitrite, and Nitrate in Cell Culture Media
- Creators
- Lisa A Ridnour - Section of Cancer Biology, Radiation Oncology Center, Washington University, St. Louis, Missouri, 63108Julia E Sim - Section of Cancer Biology, Radiation Oncology Center, Washington University, St. Louis, Missouri, 63108Michael A Hayward - Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226David A Wink - Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, 20892Sean M Martin - Free Radical Research Institute, University of Iowa, Iowa City, Iowa, 52242-1101Garry R Buettner - Free Radical Research Institute, University of Iowa, Iowa City, Iowa, 52242-1101Douglas R Spitz - Section of Cancer Biology, Radiation Oncology Center, Washington University, St. Louis, Missouri, 63108
- Resource Type
- Journal article
- Publication Details
- Analytical biochemistry, Vol.281(2), pp.223-229
- Publisher
- Elsevier Inc
- DOI
- 10.1006/abio.2000.4583
- PMID
- 10870839
- ISSN
- 0003-2697
- eISSN
- 1096-0309
- Language
- English
- Date published
- 06/01/2000
- Academic Unit
- Pathology; Radiation Oncology
- Record Identifier
- 9984047871402771
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