Abstract
A simple and sensitive method is presented to measure the unstable molecule nitric oxide (NO) by reconversion of nitrate/nitrite to NO. Nitrate and nitrite are the stable degradation products of NO that accumulate in supernatants of biological samples that release nitric oxide. First, nitrate is enzymatically converted to nitrite using nitrate reductase. In a second step, nitrite is reduced to equimolar NO concentrations by an acidic iodide solution and quantified with an amperometric Clark-type electrode. This method provides the ability to assess basal-and agonist-stimulated cumulative NO formation in different biological models and is a sensitive alternative to the widely used Griess assay.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ricciardolo, F. L. (2003) Multiple roles of nitric oxide in the airways. Thorax 58, 175–182.
Anderson, T. J. (2003) Nitric oxide, atherosclerosis and the clinical relevance of endothelial dysfunction. Heart Fail. Rev. 8, 71–86.
Shimokawa, H. (1999) Primary endothelial dysfunction: atherosclerosis. J. Mol. Cell Cardiol. 31, 23–37.
Forstermann, U., Closs, E. I., Pollock, J. S., et al. (1994) Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions. Hypertension 23, 1121–1131.
Moncada, S., Palmer, R. M., and Higgs, E. A. (1991) Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43, 109–142.
Archer, S. (1993) Measurement of nitric oxide in biological models. FASEB J. 7, 349–360.
Gryglewski, R. J., Moncada, S., and Palmer, R. M. (1986) Bioassay of prostacyclin and endothelium-derived relaxing factor (EDRF) from porcine aortic endothelial cells. Br. J. Pharmacol. 87, 685–694.
Kelm, M., Feelisch, M., Spahr, R., et al. (1988) Quantitative and kinetic characterization of nitric oxide and EDRF released from cultured endothelial cells. Biochem. Biophys. Res. Commun. 154, 236–244.
Henry, Y., Ducrocq, C., Drapier, J. C., et al. (1991) Nitric oxide, a biological effector. Electron paramagnetic resonance detection of nitrosyl-iron-protein complexes in whole cells. Eur. Biophys. J. 20, 1–15.
Palmer, R. M., Ferrige, A. G., and Moncada, S. (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327, 524–526.
Kelm, M., Preik-Steinhoff, H., Preik, M., et al. (1999) Serum nitrite sensitively reflects endothelial NO formation in human forearm vasculature: evidence for biochemical assessment of the endothelial L-arginine-NO pathway [in process citation]. Cardiovasc. Res. 41, 765–772.
Thomsen, L. L., Ching, L. M., and Baguley, B. C. (1990) Evidence for the production of nitric oxide by activated macrophages treated with the antitumor agents flavone-8-acetic acid and xanthenone-4-acetic acid. Cancer Res. 50, 6966–6970.
Kikura-Hanajiri, R., Martin, R. S., and Lunte, S. M. (2002) Indirect measurement of nitric oxide production by monitoring nitrate and nitrite using microchip electrophoresis with electrochemical detection. Anal. Chem. 74, 6370–6377.
Tsukahara, H., Gordienko, D. V., and Goligorsky, M. S. (1993) Continuous monitoring of nitric oxide release from human umbilical vein endothelial cells. Biochem. Biophys. Res. Commun. 193, 722–729.
Berkels, R., Mueller, A., Roesen, R., et al. (1999) Nifedipine and Bay K 8644 induce an increase of [Ca2+]i and NO in endothelial cells. J. Cardiovasc. Pharmacol. 4, 175–181.
Malinski, T., Mesaros, S., Patton, S. R., et al. (1996) Direct measurement of nitric oxide in the cardiovascular system. Physiol. Res. 45, 279–284.
Berkels, R., Purol-Schnabel, S., and Roesen, R. (2001) A new method to measure nitrate/nitrite with a NO-sensitive electrode. J. Appl. Physiol. 90, 317–320.
Bannenberg, G., Xue, J., Engman, L., et al. (1995) Characterization of bronchodilator effects and fate of S-nitrosothiols in the isolated perfused and ventilated guinea pig lung. J. Pharmacol. Exp. Ther. 272, 1238–1245.
Berkels, R., Bertsch, A., Breitenbach, T., et al. (1996) The calciumantagonist nifedipine stimulates endothelial NO release in therapeutical concentrations. Pharm. Pharmacol. Lett. 6, 75–78.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc.
About this protocol
Cite this protocol
Berkels, R., Purol-Schnabel, S., Roesen, R. (2004). Measurement of Nitric Oxide by Reconversion of Nitrate/Nitrite to NO. In: Hassid, A. (eds) Nitric Oxide Protocols. Methods in Molecular Biology™, vol 279. Humana Press. https://doi.org/10.1385/1-59259-807-2:001
Download citation
DOI: https://doi.org/10.1385/1-59259-807-2:001
Publisher Name: Humana Press
Print ISBN: 978-1-58829-237-7
Online ISBN: 978-1-59259-807-6
eBook Packages: Springer Protocols