Simple and reliable measurement of nitric oxide metabolites in plasma.
The history of nitric oxide (NO*) is a remarkable one. In the early 1980s it was established that nitric oxide was produced in the human body. Shortly afterward it became clear that nitric oxide had important functions in the regulation of vascular tone, and it was demonstrated that nitric oxide was identical to endothelium-derived relaxing factor, a factor derived from endothelial cells that induced relaxation of smooth muscle cells. Nitric oxide was selected as the 1992 Molecule of the Year by the journal Science, and the 1998 Nobel Prize for Medicine or Physiology was awarded to Louis Ignarro, Ferid Murad, and Robert Furchgott, the founders of nitric oxide research. Since its discovery, the gaseous radical nitric oxide has elicited much attention from the scientific community, and nitric oxide has been implicated in many diverse processes ranging from the regulation of vascular tone and male erectile function to neurotransmission and microbiocidal activity.
Soon after the discovery that nitric oxide was produced in the body, nitric oxide was reported to be the product of the enzyme nitric oxide synthase (NOS), of which 3 isoforms exist: neuronal NOS, inducible NOS, and endothelial NOS. Neuronal NOS and endothelial NOS, constitutively expressed calcium/calmodulindependent isoforms, demonstrate that nitric oxide plays an important role in normal homeostasis. On the other hand, inducible NOS is the inducible isoform expressed during inflammatory conditions in response to cytokines such as tumor necrosis factor [alpha], interleukin 1, and interferon [gamma]. Regulation of this isoform is under the control of the transcription factor nuclear factor [kappa]B.
The importance of nitric oxide spurred the development of assays to monitor its production (1). The site of nitric oxide production was initially determined by the detection of NOS isoforms. The activity of NOS was first determined by use of enzyme histochemical assays and after the development of antibodies by immunohistochemical methods, although the development and use of antibodies proved to be challenging (2).
Nitric oxide is a short-lived radical and readily reacts with small scavenger molecules (e.g., uric acid, glutathione) and macromolecules (proteins, DNA) or is oxidized into nitrite and nitrate. Therefore, the detection of the radical itself proved very difficult, and only labor-intensive, sophisticated spin-trapping methods measured actual production of the nitric oxide radical. Assays for the stable reaction products of nitric oxide were also developed. Nitric oxide radical readily reacts with reactive oxygen species such as superoxide anions to yield peroxynitrite, an extremely reactive and toxic compound that is able to nitrosylate tyrosine residues in proteins, leading to the formation of modified proteins with changed or inhibited function. This nitrotyrosine modification can be detected using specific antibodies.
We were interested in nitric oxide's role in the liver during systemic inflammatory conditions (endotoxemia, a model of sepsis) and in acute rejection after liver transplantation (3, 4). More specifically, we were interested in the effects of NOS inhibitors on liver function in these conditions (3). To assess the effect of NOS inhibitors, we needed a reliable method to measure nitric oxide metabolites. We opted for an assay based on the Griess reagent, which is specific for nitrite, and we validated this assay for use with plasma samples. Our main findings were: (a) nitric oxide was predominantly and rapidly oxidized to nitrate rather than nitrite, and therefore it was useless to apply the Griess assay without prior reduction of nitrate to nitrite using nitrate reductase; (b) plasma and serum samples needed to be deproteinized to eliminate artifacts; and (c) the assay proved to be reliable and reproducible, and the recovery of nitrate and nitrite from plasma by use of our Griess-based assay was approximately 90%. We subsequently modified this assay for other body fluids such as urine and sputum (5). In addition to the Griess assay, HPLC methods were developed to measure nitrite and nitrate. The disadvantage of methods measuring stable end products is that actual nitric oxide production from NOS cannot be determined. Methods have also been developed that use stable isotopes to measure nitrite and nitrate derived from nitric oxide. These methods are, however, rather labor-intensive and complicated.
Although the interest in nitric oxide may have somewhat diminished over the last decade, methods to measure nitric oxide (metabolites) are still used extensively.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: H. Moshage, University Medical Center Groningen, Groningen, the Netherlands.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: H. Moshage, Dutch Digestive Diseases Foundation.
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
(1.) Moshage H. Nitric oxide determinations: much ado about NO-thing? [Editorial] Clin Chem 1997;43:553-6.
(2.) Coers W, Kempinga C, Klok PA, Timens W, Moshage H. Specificity of antibodies to nitric oxide synthase isoforms in human, guinea pig, rat and mouse tissues. J Histochem Cytochem 1998;46:1385-91.
(3.) Vos TA, Gouw ASH, Klok PA, Havinga R, van Goor H, Huitema S, Roelofsen H, et al. Differential effects of nitric oxide (NO) synthase inhibitors on endotoxin-induced liver damage in rats. Gastroenterology 1997;113:1323-33.
(4.) Van der Hoeven JA, Lindell S, van Suylichem PT, Vos T, Groothuis GG, Moshage H, Ploeg RJ. Extended preservation and effect of nitric oxide production in liver transplantation. Transplant Int 1998;11(Suppl 1):S171-3.
(5.) Moshage H, Stegeman CA, Jansen PL. Determination of nitrite and nitrate in stored urine. Clin Chem 1998;44:1780-1.
Han Moshage 
 Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.
 This article has been cited 370 times since publication.
Address correspondence to the author at: Department of Gastroenterology and Hepatology, University Medical Center Groningen, PO Box 30.001, 9700 RB Groningen, the Netherlands. E-mail firstname.lastname@example.org.
Received June 2, 2009; accepted June 9, 2009.
Previously published online at DOI: 10.1373/clinchem.2009.128710
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