Nanomolar quantification and identification of various nitrosothiols by high performance liquid chromatography...

Nitrosothiols (RS-NOs) appear to be critically involved in various signal transduction mechanisms. We describe here a specific and highly sensitive quantification method for RS-NOs by using high performance liquid chromatography (HPLC) combined with a flow reactor system. RS-NOs were applied to an HPLC system of C18-reverse phase or a gel filtration column and eluted with 10 mM sodium acetate buffer (pH 5.5) plus 0.5 mM diethylenetriamine pentaacetic acid with or without either 0-7% methanol or 0.15 M NaCl. The eluate from the HPLC column was mixed with a solution containing 1.75 mM HgCl2 or 1.75 mM CuSO4 for RS-NO decomposition in a reaction coil via a three-way connector. NO2- generated via the metal-induced RS-NO decomposition was then reacted with Griess reagent, which was infused through a second three-way connector, yielding a diazo-compound detected at 540 nm. In a separate experiment, a copper particle-loaded column was used for RS-NO degradation instead of the metal-ion flow reactor. In all RS-NOs tested, i.e., nitrosoglutathione (GS-NO), nitroso-L-cysteine, and nitrosoalbumin, the nitroso- group was converted to NO2- by the Hg2+-reaction system as well as copper-loaded column, and the recovery was almost 100%. The Cu2+-solution flow reaction system, however, yielded only 30% recovery of RS-NOs as NO2-. Also, the RS-NOs could be identified at nanomolar concentrations: detection limit, 3.0 nM in a 150-microl aliquot. These RS-NOs showed well-resolved elution profiles even in the presence of NO2- and NO3-. More importantly, biological generation of GS-NO was quantitatively demonstrated with RAW264 cells in culture incorporating free GSH in the medium. In conclusion, our novel RS-NO assay will be useful to examine the formation and functions of RS-NOs in biological systems.