Determination of Arginine, Asymmetric Dimethylarginine, and Symmetric Dimethylarginine in Human Plasma and Other Biological Samples by High-Performance Liquid Chromatography

Abstract

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), may be related to reduced biosynthesis of nitric oxide in diseases associated with accelerated atherosclerosis. The closely related compound symmetric dimethylarginine (SDMA) does not inhibit NOS, but may compete with arginine for cellular uptake, thereby limiting substrate availability for NOS. We report on a method for the simultaneous measurement of arginine, ADMA, and SDMA as a tool to gain insight in the role of these compounds in the regulation of NOS activity. Sample cleanup was performed by solid-phase extraction on polymeric cation-exchange columns using monomethylarginine as internal standard. After derivatization with ortho-phthaldialdehyde reagent containing 3-mercaptopropionic acid, analytes were separated by isocratic reversed-phase HPLC with fluorescence detection. The stable derivatives were separated with near baseline resolution. Using a sample volume of 0.2 ml, linear calibration curves were obtained with limits of quantification of 0.08 μM for arginine and 0.01 μM for ADMA and SDMA. Analytical recovery was 98–102%, and interassay CV was better than 3%. Plasma from healthy volunteers (n = 53) contained 94 ± 26 μM arginine, 0.42 ± 0.06 μM ADMA, and 0.47 ± 0.08 μM SDMA. Due to its high precision and sensitivity this method is a valuable tool in research on the metabolism of dimethylated arginines and their role in the regulation of NOS activity.