High-performance liquid chromatography method with radiochemical detection for measurement of nitric oxide synthase, arginase, and arginine decarboxylase activities

Nitric oxide has been shown to be involved in numerous biological processes, and many studies have aimed to measure nitric oxide synthase (NOS) activity. Recently, it has been demonstrated that arginase and arginine decarboxylase (ADC), two enzymes that also employ arginine as a substrate, may regul...

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Published inMethods and findings in experimental and clinical pharmacology Vol. 28; no. 1; p. 3
Main Authors Volke, A, Wegener, G, Vasar, E, Volke, V
Format Journal Article
LanguageEnglish
Published Spain 01.01.2006
Subjects
Agmatine - analysis
Animals
Arginase - metabolism
Arginine - analysis
Arginine - metabolism
Brain - enzymology
Carboxy-Lyases - metabolism
Chromatography, High Pressure Liquid - methods
Citrulline - analysis
Escherichia coli - enzymology
In Vitro Techniques
Male
Nitric Oxide Synthase - metabolism
Ornithine - analysis
Radiochemistry
Rats
Rats, Wistar
Reproducibility of Results
Tritium
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Summary:Nitric oxide has been shown to be involved in numerous biological processes, and many studies have aimed to measure nitric oxide synthase (NOS) activity. Recently, it has been demonstrated that arginase and arginine decarboxylase (ADC), two enzymes that also employ arginine as a substrate, may regulate NOS activity. We aimed to develop a HPLC-based method to measure simultaneously the products of these three enzymes. Traditionally, the separation of amino acids and related compounds with HPLC has been carried out with precolumn derivatization and reverse phase chromatography. We describe here a simple and fast HPLC method with radiochemical detection to separate radiolabeled L-arginine, L-citrulline, L-ornithine, and agmatine. 3H-labeled L-arginine, L-citrulline, agmatine, and 14C-labeled L-citrulline were used as standards. These compounds were separated in the normal phase column (Allure Acidix 250 x 4.6 mm i.d.) under isocratic conditions in less than 20 min with good sensitivity. Using the current method, we have shown the formation of L-citrulline and L-ornithine in vitro using brain tissue homogenate of rats and that of agmatine by Escherichia coli ADC.
ISSN:0379-0355
DOI:10.1358/mf.2006.28.1.962769