The first pathway to be discovered for the endogenous production of NO was involving L
from a group of enzymes call nitric oxide synthase (NOS). NOS enzymes produce NO by catalyzing a five electron oxidation of the guanidino nitrogen of L
-arginine. Oxidation of L
-arginine to L
-citrulline occurs via two successive mono-oxygenation reactions producing NG
-arginine as an intermediate. Two moles of O2
and 1.5 moles of nicotinamide adenine dinucleotide phosphate (NADPH) are consumed per mole of NO formed.
NOS enzymes are the only enzymes known to simultaneously require multiple bound cofactors/prosthetic groups: flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), heme, glutathione, NADPH, tetrahydrobiopterin (BH4
) and Ca2+
-calmodulin. There are three isoforms of NOS, the genetic sequence of each residing on three distinct chromosomes. One type is constitutive, Ca2+
/calmodulin dependent and releases NO for short time periods in response to receptor or physical stimulation. NO released by this enzyme acts as a transduction mechanism underlying several physiological responses. The other enzyme type is induced after activation of macrophages, endothelial cells and a number of other cells by cytokines and once expressed, synthesizes NO for long periods of time. Furthermore, this enzyme is Ca2+
independent since calmodulin is already bound to the enzyme, and its induction is inhibited by glucocorticoids.
Endothelial NOS (eNOS), neuronal NOS (nNOS) which are both constitutively expressed in mammalian cells have now been well characterized in the cardiovascular system and nervous system respectively, and an inducible NOS (iNOS) which was first believed to be expressed only when activated by an immune response. Now it is appreciated that eNOS is found in other cells and tissues besides the endothelium, iNOS is found constitutively in some tissues, and there are inducible forms of both eNOS and nNOS, adding confusion to the nomenclature as it was first described. In an attempt to clarify the nomenclature, the three different isoforms are now commonly referred to as NOSI, NOSII, and NOSIII for neuronal, inducible and endothelial isoforms, respectively, based on the order in which they were first purified and cloned.
For years, scientists and physicians have investigated L
-arginine supplementation as a means to enhance NO production. This strategy has been shown to work effectively in young healthy individuals with functional endothelium or in older patients with high levels of asymmetric dimethyl L
-arginine (ADMA) where the supplemental L
-arginine could outcompete this natural inhibitor of NO production. Patients with endothelial dysfunction, however, by definition, are unable to convert L
-arginine to NO and, therefore, this strategy has failed in clinical trials. Schulman et al.
found that L
-arginine, when added to standard postinfarction therapies, did not improve vascular stiffness measurements or ejection fraction and was associated with higher postinfarction mortality. L
-arginine should not be recommended following acute myocardial infarction (MI). However, there are also a number of studies showing benefit to patients taking L
-arginine just as many showing no benefit, no harm.
Understanding the complex and complicated reaction pathway for NOS mediated production of NO from L
-arginine helps us define the context for rational interventions. Using L
-arginine supplementation therapy alone may not be effective due to oxidative stress in geriatric patients resulting in constitutive NOS uncoupling by redox-based post translational modifications. Supplementing L
-arginine with anti-oxidants to prevent oxidation of reduced co-factors such as BH4
, might prevent NOS uncoupling and lead to better results. In a study by Taddei et al.
, the role of oxidative stress on NO availability and endothelial dysfunction was examined in both younger and older aged populations. They found that NO availability was profoundly restored in older patients when oxidative stress is removed by antioxidants such as vitamin C. In older individuals (age > 60 years) characterized by a profound alteration in NO availability, vitamin C not only enhanced the response to the endothelial agonist but also restored the inhibitory effect of L
-NMMA on vasodilation to acetylcholine. Although, it is demonstrated that anti-oxidant supplemen tation can be very beneficial for those experiencing oxidative stress and endothelial dysfunction, it showed no benefit in younger (age < 60) or healthy individuals with no endothelial dys func tion. Collectively, the literature suggests that strategies to enhance NO production through L
-arginine supple mentation are equivocal at best.
Although the L
-arginine-NO pathway was the first to be discovered, it does not necessarily mean it is the primary pathway for the endogenous production of NO. In fact nitrogen cycling in bacteria and production of NO as an intermediate in denitrification may be one of the most primitive pathways known, dating back to the Archaean era.
The now recognized human nitrate-nitrite-nitric oxide pathway that still relies on bacteria may be a redundant system for overcoming the body's inability to make NO from L
It appears that we have at least two systems for affecting NO production/homeostasis. The first is through the classical L
-arginine-NO pathway. This is a complex and complicated five-electron oxidation of L
-arginine and if any of the co-factors become limiting, then NO production from NOS shuts down, and in many cases, NOS produces superoxide instead.
The enzymatic production of NO normally proceeds very efficiently. However, in disease characterized by oxidative stress where essential NOS cofactors become oxidized, NOS uncoupling, or conditions of hypoxia where oxygen is limiting, this process can no longer maintain NO production.
This process is illustrated in . Therefore, one can argue saliently that there has to be an alternate route for NO production. It is highly unlikely that nature devised such a sophisticated mechanism of NO production as a sole source of a critical molecule. This alternate route involves the provision of nitrate and nitrite reductively recycled to NO (). The two-electron reduction of nitrate to nitrite occurs through symbiosis with facultative anaerobic bacteria that reside in the crypts of our tongue.
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−
↔ NO + Fe(III)
Figure 2. NO production and biochemistry. There are a number of critical steps for the NOS production of NO from L-arginine. Under healthy conditions (top), enzymatic function proceeds normally. Under disease conditions (bottom), there can be a number of problems (more ...)
Figure 3. Two pathways for endogenous nitric oxide (NO) production. The L-arginine NO pathway can be enhanced through regular exercise, which becomes dysfunctional with age. The dietary pathway through reduction of nitrate and nitrite is not affected by age but (more ...)
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.
Much of the recent focus on nitrite physiology is due to its ability to be reduced to NO during ischemic or hypoxic events.–
Nitrite reductase activity in mammalian tissues has been linked to the mitochondrial electron transport system,,
and xanthine oxidase.,
Therefore, for this reaction to occur, the tissues or biological compart ment must have a sufficient pool of nitrite stored. Since plasma nitrite is a direct measure of NOS activity,
a compromised NOS system can also affect downstream nitrite production and metabolism, which can perhaps exacerbate any condition associated with decreased NO bioavailability. Considerable published data support the notion that exogenous nitrite contributes to whole body NO production: NO produced from nitrite in the upper intestine is up to 10,000 times the concentrations that occur in tissues from enzymatic synthesis,
nitrite can act as a circulating NO donor,
and nitrite can itself perform many actions previously attributable to NO
without the intermediacy of NO.
Experiments in primates revealed a beneficial effect of long-term application of nitrite on cerebral vasospasm.
Moreover, inhalation of nitrite selectively dilates the pulmonary circulation under hypoxic conditions in vivo
Topical application of nitrite improves skin infections and ulcerations.
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. Since a substantial portion of steady state nitrite concen trations in blood and tissue are derived from dietary sources,
modulation of nitrite and/or nitrate intake may provide a first line of defense for conditions associated with NO insufficiency.
The recognition of this mammalian nitrogen cycle has led researchers to explore the role of dietary nitrate and nitrite in physiological processes that are known to be regulated by NO.
Nitrite can transiently form nitrosothiols (RSNOs) under both normoxic and hypoxic conditions,
and a recent study by Bryan et al.
demonstrates that steady state concentrations of tissue nitrite and nitroso are affected by changes in dietary nitrite and nitrate (collectively, NOx) intake. Furthermore, enriching dietary intake of nitrite and nitrate translates into significantly less injury from heart attack.
Previous studies demonstrated that nitrite therapy given intravenously prior to reperfusion protects against hepatic and myocardial ischemia/reperfusion (I/R) injury.
Additionally, oral nitrite has also been shown to reverse L
-NAME induced hypertension and serve as an alternate source of NO in vivo
These results have since been corroborated in humans. In fact, it has been reported that dietary nitrate reduces blood pressure in healthy volunteers.,
Commercial development of nitrite and nitrate enriched dietary supplements has been shown to impact important cardiovascular risk factors in the aging population leading to a reduction in triglycerides and restoration of NO homeostasis.
Furthermore, in the stomach, nitrite-derived NO seems to play an important role in host defense
and in regulation of gastric mucosal integrity.
However, this is pH dependent. Since stomach acid production declines with age and many patients are prescribed proton pump inhibitors, this pathway may be disrupted in the geriatric patient causing additional problems with maintaining NO homeostasis.
Nitrite and nitrate therapy may then offer an all natural, over the counter and cost effective regimen for conditions associated with NO insufficiency. This has the potential to provide the basis for new preventive or therapeutic strategies and new dietary guidelines for optimal health. From a public health perspective, we may be able to make better recommendations on diet and dramatically affect the incidence and severity of cardiovascular disease and the subsequent clinical events.