High-performance liquid chromatographic analysis of muscular interstitial arginine and norepinephrine kinetics. A microdialysis study in rats

Abstract

Complex interactions between the L-arginine/nitric oxide synthase (NOS) pathway and the sympathetic nervous system have been reported. Methods capable of measuring L-arginine and norepinephrine (NE) have mainly been reported for plasma. We report the use of the microdialysis technique combined with high-performance liquid chromatography (HPLC) for measurement of both L-arginine and NE within the same tissue microdialysis sample. The microdialysis probe consisted of linear flexible probes (membrane length: 10 mm, outside diameter: 290 microm, molecular weight cut-off 50 kDa). The method used for L-arginine measurement was HPLC with fluorescence detection, giving a within-run and a between-day coefficient of variation of 2.9 and 12.8%, respectively. The detection limit was 0.5 pM/20 microl injected for L-/D-arginine. The method used for NE measurement was HPLC with electrochemical detection. The coefficients of variation were 4% for within-assay precision and 7.5% for between-assay precision. The detection limit for NE was 1 fmol/20 microl injected. The microdialysis technique coupled with HPLC system was validated in vivo to measure muscular interstitial concentrations of both arginine and NE under baseline conditions and after intravenous infusion of 500 mg/kg of L-arginine or D-arginine. In conclusion, the microdialysis technique coupled to HPLC allows the simultaneous measurements of both L-arginine and NE within the same tissue microenvironment and will enable the study of the complex interactions between the L-arginine/NO pathway and sympathetic nervous system within the interstitial space of different organs.