This alternate route involves the provision of nitrate and nitrite reductively recycled to NO. Nitrite reduction to NO can occur in a much simpler mechanism than nitrate. The 1-electron reduction of nitrite can occur by ferrous heme proteins (or any redox active metal) through the following reaction:
NO2- + Fe(II) + H+ ↔ NO + Fe(III) + OH-
This is the same biologically active NO as that produced by NOS, with nitrite rather than L-arginine as the precursor and is a relatively inefficient process.24 Therefore, for this reaction to occur, the tissues or biological compartment must have a sufficient pool of nitrite stored. Since plasma nitrite is a direct measure of NOS activity,25 a compromised NOS system can also affect downstream nitrite production and metabolism, which can perhaps exacerbate any condition associated with decreased NO bioavailability. Replenishing nitrate and nitrite through dietary means may then act as a protective measure to compensate for insufficient NOS activity under conditions of hypoxia or in a number of conditions characterized by NO insufficiency. A number of pre-clinical and clinical studies have demonstrated that this dietary strategy is very effective at treating and preventing a number of chronic age-related diseases. Furthermore enriching dietary intake of nitrite and nitrate translates into significantly less injury from heart attack.26 Previous studies also demonstrated that nitrite therapy given intravenously prior to reperfusion protects against hepatic and myocardial ischemia/reperfusion injury.27 Moreover, inhalation of nitrite selectively dilates the pulmonary circulation under hypoxic conditions in vivo in sheep.28 Topical application of nitrite improves skin infections and ulcerations.29 Furthermore, in the stomach, nitrite-derived NO seems to play an important role in host defense30 and in regulation of gastric mucosal integrity.31
All of these studies together along with the observation that nitrite can act as a marker of NOS activity25 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. Oral nitrite has also been shown to reverse N(G)-Nitro-L-arginine-methyl ester (L-NAME)-induced hypertension and serve as an alternate source of NO in vivo.32 In fact, a report by Kleinbongard et al33 demonstrates that plasma nitrite levels progressively decrease with increasing cardiovascular risk. Since a substantial portion of steady state nitrite concentrations in blood and tissue are derived from dietary sources,23 modulation of nitrite and/or nitrate intake may provide a first line of defense for conditions associated with NO insufficiency.34 Indeed, it has been reported that dietary nitrate reduces blood pressure in healthy volunteers.35,36 What is clearly emerging is that there are two pathways for NO production, one through endothelial production via the L-arginine pathway and one through dietary sources of nitrite, nitrate, and antioxidants. This is illustrated in Figure 2. The L-arginine pathway becomes dysfunctional with age, and we, therefore, need a back up system to compensate. Eating a diet rich in NO potential, i.e., sufficient nitrite and nitrate along with antioxidants to facilitate reduction to NO, can appear to overcome an insufficiency in endothelium-derived NO. This dietary pathway does not appear to be affected by age.
Figure 2. The two NO pathways
NITRIC OXIDE DIAGNOSTICS
Now that we have recognized and proven ways to restore NO in humans, how does one recognize NO insufficiency before the manifestation of disease? This is a critically important issue if we want to prevent disease. Understanding the normal metabolism of NO allows us to develop novel diagnostics. The major pathway for NO metabolism is the stepwise oxidation to nitrite and nitrate. For years, both nitrite (NO2-) and nitrate (NO3-) (collectively, NOx) have been used as surrogate markers of NO production in biological tissues, but there have not been any new developments in the use of NO biomarkers in the clinical setting for diagnostic or prognostic utility. In fact, NO status is still not part of the standard blood chemistry routinely used for diagnostic purposes. This is simply unacceptable given the critical nature of NO in many disease processes, thus new technologies should be developed.43 The only true measure of endothelial NO production (endothelial function) is through flow mediated dilatation (FMD). FMD is a non-invasive ultrasound-based method where arterial diameter is measured in response to an increase in shear stress, which causes release of NO from the endothelium and consequent endothelium dependent dilatation. FMD has been shown to correlate with invasive measures of endothelial function, as well as with the presence and severity of the major traditional vascular risk factors.44 NOx have recently been shown to be biomarkers for cardiovascular and other diseases from both diagnostic and therapeutic aspects.45 However, it is not known if levels of nitrite and nitrate correlate with FMD. In addition to blood, urinary levels of NOx provide a means to assess systemic NO production in vivo, or renal handling of these anions, which may be compromised in the geriatric patient.46 A report by Kleinbongard et al47 demonstrated that plasma nitrite levels in humans progressively decrease with increasing cardiovascular risk load. Risk factors considered included age, hypertension, smoking, and hypercholesterolemia, conditions all known to reduce the bioavailability of NO. Although a correlation exists in the plasma, it is not known whether the situation is mirrored in the heart or other tissue of interest in specific disease. The recent recognition of a human nitrogen cycle whereby nitrate and nitrite are reduced to NO by an enterosalivary circulation of nitrate13 opens up the potential for using saliva as a potential biomarker for NO status in certain diseases. These novel NO diagnostic test strips are now commercially available through Neogenis Labs (www.neogenis.com).
NATURAL PRODUCT CHEMISTRY FROM PLANTS GENERATE NITRIC OXIDE
Traditional herbal medicines used for thousands of years in Asia and other regions have been proven effective in certain cardiovascular disorders. Some of the herbal medicines have profound NO bioactivity primarily due to the nitrate-nitrite-NO reduction pathway, as they contain very large amounts of NOx in the extracts given to patients.32 The described benefits of these ancient medications may be attributed to their inherent NOx content combined with their robust NOx reductase activity to generate NO independent of the L-arginine-NO pathway.14,26,32,50 The therapeutic benefits of these herbal medicines are providing an alternative source of NO to patients that may be unable to make NO from L-arginine owing to endothelial dysfunction.
There is an endogenous nitrite reductase activity in animal tissues, such as the liver and aorta, but this inherent biological capacity is low (around 1 pmol/mg protein). The reductase activity in some of these herbal medicines may exceed that detected in the animal tissues.32 It is estimated that the increased reductase activity may occur by orders of magnitude, almost 1000-times higher than endogenous production of NO. This would equate to 300 nmoles per day of NO from a single herbal preparation. The average NO production in the human body (70 kg) is 1.68 mmol NO per day (based on an NO production rate of 1 μmol/kg/h). By supplying the exogenous NOx and reductase activities, herbal medicines offer an alternative therapeutic strategy to combat or treat any condition related to NO insufficiency including heart disease and hypertension.
Maintaining NO homeostasis requires the repletion of NOx through which the ability to generate NO can be restored to compensate for the inability of the endothelium to convert L-arginine to NO in coronary heart disease. This concept has recently been tested through the development of a rationally designed dietary supplement with natural products selected for their NO activity based on their NOx content as well as an oxygen independent nitrite reductase. In a double-blinded, placebo-controlled study in patients over the age of 40 with at least 3 cardiovascular risk factors, this type of technology was found to