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-[N-15(2)]arginine-derived [N-15]nitrite as its pentafluorobenzyl derivative in the negative-ion chemical ionization mode. Application of the method to the analysis of [N-15]nitrite formation by purified neuronal nitric oxide synthase revealed K-M values of 3.1 mu M by Hanes and 4.6 mu M by Lineweaver-Burk for L-[N-15(2)]arginine. The corresponding V-max values were 0.204 and 0.228 mu mol [N-15]nitrite min(-1) mg(-1) NOS, respectively. N-G-Nitro-L-arginine and N-G,N-G-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-[N-15(2)]arginine in a rat and determination of plasma [N-15]nitrite and [N-15]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. (C) 2000 Elsevier Science BN. All rights reserved.