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 K(M) values of 3.1 microM by Hanes and 4.6 microM by Lineweaver-Burk for L-[15N2]arginine. The corresponding Vmax values were 0.204 and 0.228 micromol [15N]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-[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.
Tsikas
D
D
Institute of Clinical Pharmacology, Hannover Medical School, Germany. tsikas.dimitros@mh-hannover.de
Sandmann
J
J
Savva
A
A
Luessen
P
P
Böger
R H
RH
Gutzki
F M
FM
Mayer
B
B
Frölich
J C
JC
D000818:Animals; D001120:Arginine / Q000097:blood; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D066298:In Vitro Techniques; D008297:Male; D009566:Nitrates / Q000097:blood; D019001:Nitric Oxide Synthase / Q000097:blood*; D052248:Nitric Oxide Synthase Type I; D009573:Nitrites / Q000097:blood; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity
D009566:Nitrates; D009573:Nitrites; D001120:Arginine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat