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Measurement of nitric oxide synthase activity in vivo and in vitro by gas chromatography-mass spectrometry.
Methods Mol Biol. 2004; 279:81-103.MM

Abstract

Measurement of the nitric oxide (NO) metabolites nitrite and nitrate in biological matrices is a reliable method to assess NO synthase (NOS) activity. Unlike the "L-citrulline assay," the "nitrite/nitrate assay" is generally applicable and permits acquisition of maximum information about what NOS did actually produce. From the various analytical approaches and methods available so far for the quantitation of nitrite and nitrate, mass-spectrometry-based methods, notably gas chromatography-mass spectrometry (GC-MS), offer the highest reliability in terms of accuracy, precision, specificity, minimum of interferences, sensitivity, and no limitation regarding biological matrix. Among the GC-MS methods, that one utilizing the derivatization reagent pentafluorobenzyl bromide is the single assay permitting measurement of nitrite and nitrate. This method possesses the unique opportunity to measure simultaneously stable-isotope-labeled nitrite and nitrate (i.e. [15N]nitrite and [15N]nitrate), which are exclusively produced by NOS-catalyzed oxidation of L-[guanidino-15N2]-arginine, the stable-isotope-labeled L-arginine analog. *NOS may concomitantly produce *NO and O2*-, thus forming peroxynitrite (ONOO-), which also degrades to nitrite and nitrate. Thus, the GC-MS nitrite/nitrate assay provides information about biologically relevant NOS activity This chapter describes GC-MS protocols for the measurement of NOS activity in vitro (e.g., of isolated NOS preparations and in cells) and in vivo (in animals and humans).

Authors+Show Affiliations

Institute of Clinical Pharmacology, Hannover Medical School, Germany.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15199238

Citation

Tsikas, Dimitrios. "Measurement of Nitric Oxide Synthase Activity in Vivo and in Vitro By Gas Chromatography-mass Spectrometry." Methods in Molecular Biology (Clifton, N.J.), vol. 279, 2004, pp. 81-103.
Tsikas D. Measurement of nitric oxide synthase activity in vivo and in vitro by gas chromatography-mass spectrometry. Methods Mol Biol. 2004;279:81-103.
Tsikas, D. (2004). Measurement of nitric oxide synthase activity in vivo and in vitro by gas chromatography-mass spectrometry. Methods in Molecular Biology (Clifton, N.J.), 279, 81-103.
Tsikas D. Measurement of Nitric Oxide Synthase Activity in Vivo and in Vitro By Gas Chromatography-mass Spectrometry. Methods Mol Biol. 2004;279:81-103. PubMed PMID: 15199238.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Measurement of nitric oxide synthase activity in vivo and in vitro by gas chromatography-mass spectrometry. A1 - Tsikas,Dimitrios, PY - 2004/6/17/pubmed PY - 2004/9/17/medline PY - 2004/6/17/entrez SP - 81 EP - 103 JF - Methods in molecular biology (Clifton, N.J.) JO - Methods Mol. Biol. VL - 279 N2 - Measurement of the nitric oxide (NO) metabolites nitrite and nitrate in biological matrices is a reliable method to assess NO synthase (NOS) activity. Unlike the "L-citrulline assay," the "nitrite/nitrate assay" is generally applicable and permits acquisition of maximum information about what NOS did actually produce. From the various analytical approaches and methods available so far for the quantitation of nitrite and nitrate, mass-spectrometry-based methods, notably gas chromatography-mass spectrometry (GC-MS), offer the highest reliability in terms of accuracy, precision, specificity, minimum of interferences, sensitivity, and no limitation regarding biological matrix. Among the GC-MS methods, that one utilizing the derivatization reagent pentafluorobenzyl bromide is the single assay permitting measurement of nitrite and nitrate. This method possesses the unique opportunity to measure simultaneously stable-isotope-labeled nitrite and nitrate (i.e. [15N]nitrite and [15N]nitrate), which are exclusively produced by NOS-catalyzed oxidation of L-[guanidino-15N2]-arginine, the stable-isotope-labeled L-arginine analog. *NOS may concomitantly produce *NO and O2*-, thus forming peroxynitrite (ONOO-), which also degrades to nitrite and nitrate. Thus, the GC-MS nitrite/nitrate assay provides information about biologically relevant NOS activity This chapter describes GC-MS protocols for the measurement of NOS activity in vitro (e.g., of isolated NOS preparations and in cells) and in vivo (in animals and humans). SN - 1064-3745 UR - https://www.unboundmedicine.com/medline/citation/15199238/Measurement_of_nitric_oxide_synthase_activity_in_vivo_and_in_vitro_by_gas_chromatography_mass_spectrometry_ L2 - https://dx.doi.org/10.1385/1-59259-807-2:081 DB - PRIME DP - Unbound Medicine ER -