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 ni tric oxide synthase activity is described. The method is based on the gas c hromatographic-mass spectrometric measurement of L-[N-15(2)]arginine-derive d [N-15]nitrite as its pentafluorobenzyl derivative in the negative-ion che mical ionization mode. Application of the method to the analysis of [N-15]n itrite formation by purified neuronal nitric oxide synthase revealed K-M va lues of 3.1 mu M by Hanes and 4.6 mu M by Lineweaver-Burk for L-[N-15(2)]ar ginine. The corresponding V-max values were 0.204 and 0.228 mu mol [N-15]ni trite min(-1) mg(-1) NOS, respectively. N-G-Nitro-L-arginine and N-G,N-G-di methylarginine (asymmetric dimethylarginine) were identified by this method as the most potent enzyme inhibitors. Nitric oxide synthase activity was a lso 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 describe d in this work allows for accurate, specific and highly sensitive determina tion of nitric oxide synthase activity in vitro and in vivo. (C) 2000 Elsev ier Science BN. All rights reserved.