Nitric Oxide to Nitrite/Nitrate

The endogenous production of nitric oxide (NO) by nitric oxide synthase (NOS) has been established as playing an important role in vascular homeostasis, neurotransmission, and immunological  host defense mechanisms (Moncada, Palmer et al. 1991). A methodology for NO detection is complex and the focus of many debates and discussion concerning the biochemistry of NO. NO is involved in many physiological processes and also becomes perturbed during disease. Therefore its accurate detection and quantification is critical to understanding health and disease. Due to the extremely short physiological half life of this gaseous free radical, alternative strategies for the detection of the reaction products of NO biochemistry have been developed. The quantification of NO metabolites in biological samples provides valuable information with regards to in vivo NO production, bioavailability, and metabolism. The major pathway for NO metabolism is the stepwise oxidation to nitrite (NO2and nitrate NO3 ) (Yoshida, Kasama et al. 1983). In plasma or other physiological fluids or buffers NO is oxidized almost completely to nitrite, where it remains stable for several hours (Kelm, Feelisch et al. 1992; Grube, Kelm et al. 1994). The oxidation of NO by molecular oxygen is second order with respect to NO:

2NO +  O2 -> 2NO2 (1)

2NO + 2NO2 -> 2N2O3 (2)

2N2O3 + 2H2O -> 4NO2 + 4H+ (3)

NO and nitrite are rapidly oxidized to nitrate in whole blood. The half life of NO2- in human blood is about 110 seconds (Kelm 1999). Nitrate on the other hand has a circulating half life of 5-8 hours (Tannenbaum 1994; Kelm and Yoshida 1996). Although the mechanisms by which NO and NO2- are converted to NO3- in vivo are not entirely clear, there are several possibilities. One mechanism suggests that the NO2- derived from NO autoxidation is rapidly converted to NO3- via its oxidation by certain oxyhemoproteins (P-Fe2+O2) such as oxyhemoglobin or oxymyoglobin (Ignarro, Fukuto et al. 1993):

2P-Fe2+O2 + 3NO2 + 2H+-> 2P-Fe3+ + 3NO3 + H2O (4)

or

4P-Fe2+O2 + 4NO2 + 4H+ -> 4P-Fe3+ + 4NO3 + O2 + 2H2O (5)

NO2- would in turn react with the hemoproteins, this reaction is quite slow, requiring 2-3 hr. The presence of predominately NO3- in vivo (Bryan, Rassaf et al. 2004) may have to do with the fact that the levels of NO produced by nitric oxide synthase (NOS) in vivo are relatively small and thus the half-life of NO would be much longer. In this case, NO would react directly and very rapidly with oxyhemoproteins (P-Fe2+O2) to yield NO3before it has an opportunity to autoxidize to NO2 as described above in reactions 1-3.

P-Fe2+O2 + NO > P-Fe3+ + NO3  (6)

These mechanisms of autoxidation of NO would also be important in tissues and cell culture samples where NO may interact with a multitude of hemoproteins. Therefore detection and quantification of nitrite and nitrate provides an index of NO bioavailability or production.

From Nitrite/Nitrate to NO

During fasting conditions with low intake of nitrite/nitrate, enzymatic NO formation from NOS accounts for the majority of nitrite (Rhodes, Leone et al. 1995). On the basis of these studies, it was believed that NO is acutely terminated by oxidation to nitrite and nitrate. However, it is now appreciated that nitrite or nitrate can be recycled to produce NO in various ways. NO production has been described in infarcted heart tissue from nitrite (Zweier, Wang et al. 1995). The nitrite reductase activity in mammalian tissues has been linked to the mitochondrial electron transport system (Walters, Casselden et al. 1967), protonation (Zweier, Wang et al. 1995; Hunter, Dejam et al. 2004), deoxyhemoglobin (Cosby, Partovi et al. 2003; Hunter, Dejam et al. 2004), and xanthine oxidase (Alikulov, L’vov et al. 1980; Li, Samouilov et al. 2004; Webb, Bond et al. 2004). Both nitrite and nitrate have been shown to be reduced ultimately back to NO by commensal bacteria (Lundberg, Weitzberg et al. 2004) and bacteria in the urogenital tract (Lundberg, Carlsson et al. 1997). These pathways have been extensively reviewed (Lundberg and Weitzberg 2005) in the vascular compartment but extend to all organ systems. So not only can nitrite and nitrate determination reflect NO production but may also serve as an alternative source of NO. Thus accurate detection of both anions becomes crucial in NO biology.

The Biological Activity of Nitrite and Nitrate

Nitrite and nitrate in blood is widely used as an index of endothelial NO synthase activity (Lauer, Preik et al. 2001; Kleinbongard, Dejam et al. 2003) as routine indirect measures of NO levels. Up until recently nitrite was thought to be an inert oxidative breakdown product of endogenous NO synthesis. Nitrite is now considered a central homeostatic molecule in NO biology and may serve as an important signaling molecule (Bryan, Fernandez et al. 2005; Bryan 2006). Nitrite has been investigated as a vasodilator in mammals for over 125 years and is a known by-product of organic nitrate metabolism. Most recently nitrite has emerged as an endogenous signaling molecule and regulator of gene expression that can serve as a diagnostic marker and also as a potential therapy for cardiovascular disease. The recent discoveries that nitrite can be reduced back to NO under appropriate physiological conditions and nitrite itself can directly nitrosate thiols to form RSNOs (Bryan, Fernandez et al. 2005), has caused intense interest in this molecule (Gladwin, Schechter et al. 2005).  A recent report by Kleinbongard et al. (Kleinbongard, Dejam et al. 2006), demonstrated that plasma nitrite levels progressively decrease with increasing cardiovascular risk load. Risk factors considered include age, hypertension, smoking, and hypercholesterolemia revealing the biological importance of nitrite status in human health. This data together with the recent discovery of nitrite as a signaling molecule have opened a new avenue for the diagnostic and therapeutic application of nitrite, especially in cardiovascular diseases, using nitrite as marker as well as an active agent. Furthermore, dietary nitrate has been recently shown to reduce diastolic blood pressure in healthy volunteers (Larsen, Ekblom et al. 2006). Therefore it is prudent at this juncture to carefully and accurately account for all nitrite and nitrate in biological samples.

-written by Dr. Nathan S. Bryan
The University of Texas Houston Health Science Center

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