HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC DETERMINATION OF NITRIC-OXIDESYNTHASE-RELATED ARGININE DERIVATIVES IN-VITRO AND IN-VIVO

In this paper we present a sensitive and reproducible method for the e xtraction and quantification of the nitric oxide (NO) synthase (NOS)-r elated basic amino acids L-hydroxyarginine (L-NHA), L-arginine (L-Arg) , L-monomethylarginine (L-NMA), and L-dimethylarginine (L-NDA) in huma n serum samples by highperformance Liquid chromatography (RPLC) analys is. We demonstrate that the serum level of L-NHA can be used as a sens itive 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 niti ate, is not influenced by dietary intake. First, we measured L-NHA formation by a recombinant NOS preparation a nd by lipopolysaccharide-stimulated alveolar macrophages to demonstrat e 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 p resence of amino acids interfering with its detection. Therefore, we d eveloped a clean-up procedure for the extraction of basic amino acids from these serum samples by using a cation-exchange cartridge. The iso lated amino acids were subjected to precolumn derivatization with o-pt haldialdehyde and analyzed using a short reversed-phase column which a llowed the baseline separation of L-NHA, L-Arg, L-NMA, and L-NDA withi n 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 mu M, respectively. (C) 1997 Academic Press.