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 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.
Volke
A
A
Department of Dermatology and Venereology, University of Tartu, Tartu, Estonia.
Wegener
G
G
Vasar
E
E
Volke
V
V
D000376:Agmatine / Q000032:analysis; D000818:Animals; D001119:Arginase / Q000378:metabolism*; D001120:Arginine / Q000032:analysis / Q000378:metabolism; D001921:Brain / Q000201:enzymology*; D002262:Carboxy-Lyases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002956:Citrulline / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology; D066298:In Vitro Techniques; D008297:Male; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D009952:Ornithine / Q000032:analysis; D011854:Radiochemistry; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D014316:Tritium
D014316:Tritium; D002956:Citrulline; D000376:Agmatine; D001120:Arginine; D009952:Ornithine; D019001:Nitric Oxide Synthase; D001119:Arginase; D002262:Carboxy-Lyases; C019722:arginine decarboxylase