Electrochemical detection of nitroso-arginine as an intermediate between N-hydroxy-arginine and citrulline. An in vitro versus in vivo study using microcarbon electrodes in neuronal nitric oxide synthase and mice brain.

The aim of the study was to describe in vivo and in vitro the transformation of N-hydroxy-arginine (NHA) into nitrite and citrulline. The products of NHA oxidation were studied by electrochemical methods. Cyclic voltammetry of NHA on microcarbon electrode showed an oxidation in two steps with one electron and one proton exchanged at each step. The first step gave a radical species NHA(.) with a half-life shorter than 1 micros and the second step gave nitroso-arginine (NA) with a half-life of about 1 s (1.5 s). Coulometric oxidation of NHA gave citrulline and nitrite. Differential pulse voltammetry (DPV) in vivo and in vitro gave a peak in reduction at -1.66 V vs Ag/AgCl for NA. After reductive adsorption of NA on the microelectrode surface in mice brain it gave the two peaks of NHA in oxidation plus another peak identified as nitrite. DPV in native and recombinant rat brain nitric oxide (NO)-synthase gave NA signal permitting K(m) and V(max) determination. All these results showed that NA was synthetized by NO-synthases before the final products, citrulline and nitrite.