An Assay Method for Nitric Oxide-Related Compounds in Whole Blood

An Assay Method for Nitric Oxide-Related Compounds in Whole Blood

Recent evidence suggests that nitric oxide (NO) generatedin vivowill be converted into the forms of nitrite/nitrate, nitrosyl hemoproteins, nitrosyl metal complexes, and S-nitroso-compounds in the circulation. Nitrosothiols have also been reported to be relatively stable metabolites with micromolar...

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Bibliographic Details
Published in:Analytical biochemistry Vol. 247; no. 2; pp. 417 - 427
Main Authors: Sonoda, Masaru, Kobayashi, Jun, Takezawa, Masue, Miyazaki, Takashi, Nakajima, Takanori, Shimomura, Hiroji, Koike, Kazuyuki, Satomi, Akira, Ogino, Hiroshi, Omoto, Ryozo, Komoda, Tsugikazu
Format: Journal Article
Language:English
Published: United States Elsevier Inc 01-05-1997
Subjects:
Animals
Blood Chemical Analysis - methods
Drug Stability
Evaluation Studies as Topic
Hot Temperature
Humans
In Vitro Techniques
Lipopolysaccharides - toxicity
Luminescent Measurements
Male
Nitrates - blood
Nitric Oxide - blood
Nitrites - blood
Nitroso Compounds - blood
Oxidation-Reduction
Rats
Rats, Sprague-Dawley
Spectrometry, Fluorescence
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Summary:Recent evidence suggests that nitric oxide (NO) generatedin vivowill be converted into the forms of nitrite/nitrate, nitrosyl hemoproteins, nitrosyl metal complexes, and S-nitroso-compounds in the circulation. Nitrosothiols have also been reported to be relatively stable metabolites with micromolar levels in plasma. We hypothesized, therefore, that the determinations of all the NO-related compounds in blood would be of diagnostic significance. The assay method described here consists of the thermolysis of all the NO-related compounds in whole blood and the detection of resulting nitrate by fluorometry or chemiluminescence after an enzymatic reduction.S-Nitroso-albumin and nitrosyl hemoglobin can be easily thermolysed to nitrate, and relatively stableS-nitroso-glutathione is also degraded to nitrate in the presence of blood constituents with high molecular mass (above 30 kDa). Concentrations of NO-related compounds in blood from healthy human as well as control or lipopolysaccharide-stimulated rats were determined. We found that membrane-bound NO which showed the augmented levels under the pathophysiological states could also be detected. Together with electron spin resonance spectra, our data indicate that the fraction of NO diffused and metabolized within red cells and the other NO-metabolites in plasma such as nitrite/nitrate and S-nitroso-compounds, both of which can reflect NO-productionin vivo,would be recovered and detected quantitatively by this method.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.1997.2056