Assessment of nitric oxide synthase activity in vitro and in vivo by gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric method for the determination of nitric oxide synthase activity is described. The method is based on the gas chromatographic–mass spectrometric measurement of l-[15N2]arginine-derived [15N]nitrite as its pentafluorobenzyl derivative in the negative-ion chemical ionization mode. Application of the method to the analysis of [15N]nitrite formation by purified neuronal nitric oxide synthase revealed KM values of 3.1 μM by Hanes and 4.6 μM by Lineweaver–Burk for l-[15N2]arginine. The corresponding Vmax values were 0.204 and 0.228 μmol [15N]nitrite min−1 mg−1 NOS, respectively. NG-Nitro-l-arginine and NG,NG-dimethylarginine (asymmetric dimethylarginine) were identified by this method as the most potent enzyme inhibitors. Nitric oxide synthase activity was also assessed in vivo by i.v. injection of l-[15N2]arginine in a rat and determination of plasma [15N]nitrite and [15N]nitrate. The assay described in this work allows for accurate, specific and highly sensitive determination of nitric oxide synthase activity in vitro and in vivo.