High-performance liquid chromatographic determination of nitric oxide synthase-related arginine derivatives in vitro and in vivo.
In this paper we present a sensitive and reproducible method for the extraction and quantification of the nitric oxide (NO) synthase (NOS)-related basic amino acids L-hydroxyarginine (L-NHA), L-arginine (L-Arg), L-monomethylarginine (L-NMA), and L-dimethylarginine (L-NDA) in human serum samples by high-performance liquid chromatography (HPLC) analysis. We demonstrate that the serum level of L-NHA can be used as a sensitive and highly specific index of a systemic increase in NOS activity in vivo whose serum concentration, unlike that of the NO degradation products nitrite and/or nitrate, is not influenced by dietary intake. First, we measured L-NHA formation by a recombinant NOS preparation and by lipopolysaccharide-stimulated alveolar macrophages to demonstrate that this amino acid is produced by NOS in vitro. HPLC determination of L-NHA in human serum, however, proved to be difficult due to the presence of amino acids interfering with its detection. Therefore, we developed a clean-up procedure for the extraction of basic amino acids from these serum samples by using a cation-exchange cartridge. The isolated amino acids were subjected to precolumn derivatization with o-pthaldialdehyde and analyzed using a short reversed-phase column which allowed the baseline separation of L-NHA, L-Arg, L-NMA, and L-NDA within 16 min. By using this technique, the average concentrations of L-NHA, L-Arg, L-NMA, and L-NDA in the serum of healthy human subjects were determined to be 9.1, 96.1, 0.1, and 0.4 microM, respectively.
Meyer
J
J
Center of Physiology, J. W. Goethe University Clinic Frankfurt/M, Germany.
Richter
N
N
Hecker
M
M
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000706:statistics & numerical data; D002852:Chromatography, Ion Exchange; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D016676:Macrophages, Alveolar / Q000378:metabolism; D008297:Male; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D051381:Rats; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019323:omega-N-Methylarginine / Q000032:analysis / Q000097:blood
D019323:omega-N-Methylarginine; D009569:Nitric Oxide; C068309:N(omega)-hydroxyarginine; C018524:N,N-dimethylarginine; D001120:Arginine; D019001:Nitric Oxide Synthase