Journal of Chromatography B, 742 (2000) 143–153 www.elsevier.com / locate / chromb Assessment of nitric oxide synthase activity in vitro and in vivo by gas chromatography–mass spectrometry a, a a a a * ¨ ¨ Dimitrios Tsikas , Jorg Sandmann , Athanasia Savva , Piet Lueßen , Rainer H. Boger , a b a ¨ ¨ Frank-Mathias Gutzki , Bernd Mayer , Jurgen C. Frolich a Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30623 Hannover, Germany b Institute of Pharmacology and Toxicology, Karl-Franzens-University Graz, 8010 Graz, Austria Received 15 December 1999; received in revised form 16 February 2000; accepted 17 February 2000 Abstract A gas chromatographic–mass spectrometric method for the determination of nitric oxide synthase activity is described. 15 15 The method is based on the gas chromatographic–mass spectrometric measurement of L-[ N ]arginine-derived [ N]nitrite 2 as its pentafluorobenzyl derivative in the negative-ion chemical ionization mode. Application of the method to the analysis of 15 [ N]nitrite formation by purified neuronal nitric oxide synthase revealed K values of 3.1 mM by Hanes and 4.6 mM by M 15 15 21 21 Lineweaver–Burk for L-[ N ]arginine. The corresponding V values were 0.204 and 0.228 mmol [ N]nitrite min mg 2 max G G G NOS, respectively. N -Nitro-L-arginine and N ,N -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 15 15 15 L-[ N ]arginine in a rat and determination of plasma [ N]nitrite and [ N]nitrate. The assay described in this work allows 2 for accurate, specific and highly sensitive determination of nitric oxide synthase activity in vitro and in vivo.  2000 Elsevier Science B.V. All rights reserved. Keywords: Enzymes; Nitric oxide synthase; Asymmetric dimethylarginine 1. Introduction shown to be derived from molecular oxygen and not G from water [2,3]. NOS is inhibited by various N - G Nitric oxide synthases (NOSs, EC 1.14.13.39) are substituted L-arginine analogs such as N -nitro-L- G a family of enzymes which catalyze the oxidation of arginine ( L-NNA), N -nitro-L-arginine methylester G L-arginine to nitric oxide (NO) and citrulline via ( L-NAME), N -methyl-L-arginine ( L-NMA), and G G G intermediate formation of N -hydroxy-L-arginine N ,N -dimethyl-L-arginine (ADMA, asymmetric di- 21 [1]. In this reaction, NADPH, Ca , calmodulin methylarginine) [4–8]. L-NMA and ADMA are (CaM), FAD, FMN and tetrahydro-biopterin (H B) endogenous NOS inhibitors [6,7]. NO plays a critical 4 are required as cofactors [1]. The oxygen atom role in signal transduction pathways in the car- incorporated into both citrulline and NO has been diovascular and nervous systems and is a key component of the cytostatic / cytotoxic function of the immune system [8,9]. *Corresponding author. Tel.: 149-511-5323-959; fax: 149- Assessment of NOS activity in vitro and in vivo 511-5322-750. E-mail address: tsikas.dimitros@mh-hannover.de (D. Tsikas) allows for the determination of factors influencing 0378-4347 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0378-4347(00)00142-0