Nitric Oxide: The Overlooked Molecule in Patient Care and Chronic Disease Prevention
Although often overlooked or not considered in clinical medicine, nitric oxide (NO) is one of the most important signaling molecules in the body. It is involved in virtually every organ system, and is responsible for modulating an astonishing variety of effects. Thus, it is no surprise to learn that a host of diseases or conditions may be caused or affected by the body’s dysregulation of NO production/signaling. Maintaining NO homeostasis is critical for optimal health and disease prevention, and developing diagnostics and therapeutics to accomplish this will have a profound effect on public health. The aim of this paper is to introduce the reader to the importance of NO, and the age-dependent decline in NO production and its consequences. NO diagnostics and therapeutic strategies for maintaining NO homeostasis will also be considered.
Chronic diseases including heart disease, diabetes, Alzheimer’s, and cancer account for 61% of deaths worldwide. Almost 45% of these deaths occur prematurely before the age of 70. Fortunately though, most of these deaths are preventable by diet and lifestyle modification. The common factor causal for these chronic diseases may be insufficient nitric oxide (NO). Science has now shown that certain diets and moderate exercise can restore NO and positively affect these chronic diseases. The discovery in the 1980’s of the mammalian biosynthesis of NO and its roles in the immune1,2 cardiovascular3-5 and nervous6 systems established a startling new paradigm in the history of cellular signaling mechanisms. In fact, the discovery of NO was so profound that a Nobel Prize was awarded in 1998 to the 3 US scientists responsible for its discovery.
NO is one of the most important signaling molecules in the body, and is involved in virtually every organ system where it is responsible for modulating an astonishing variety of effects. NO has been shown to be involved in and affect every biological system in humans. One can then imagine the host of diseases or conditions that may be caused or affected by the body’s dysregulation of NO production/signaling. Maintaining NO homeostasis is critical for optimal health and disease prevention, and developing diagnostics and therapeutics to accomplish this will have a profound effect on public health. NO has a number of clinical applications including: inhaled NO for premature babies with pulmonary hypertension, treatment for erectile dysfunction and systemic hypertension, and many others. In fact, the release of NO from nitroglycerin is the mechanism of action for this drug, which has been used for over 160 years for the treatment of acute angina in cardiac patients.
The first pathway to be discovered for the endogenous production of NO was that involving L-arginine. 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 can outcompete this natural inhibitor of NO production. However, patients with endothelial dysfunction, by definition, are unable to convert L-arginine to NO and, therefore, this strategy has failed in clinical trials. In fact, the Vascular Interaction With Age in Myocardial Infarction (VINTAGE MI) randomized clinical trial, concluded that L-arginine therapy (when added to standard postinfarction therapies) did not improve vascular stiffness measurements or ejection fraction, and was associated with higher postinfarction mortality.7 Thus, 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 and just as many showing no benefit, no harm. Collectively, the literature suggests that strategies to enhance NO production through L-arginine supplementation are equivocal at best. Therefore there must be an alternative pathway for repleting NO in patients.
AGE-DEPENDENT DECLINE IN NITRIC OXIDE PRODUCTION
When we are young and healthy, the endothelial production of NO through L-arginine is efficient and sufficient to produce NO; however, as we age we lose our ability to synthesize endothelial derived NO. Most of the works on the activity of NO in cells and tissues agree that the bioavailability or the generation of nitric oxide synthase (NOS) derived NO decreases with aging. It has been proposed that superoxide can scavenge NO to form peroxynitrite and thereby reduce its effective concentrations in cells.8 It has also been reported that there is decreased NOS expression with aging both in constitutive and inducible isoforms.9,10 Berkowitz et al11 observed the upregulation of arginase (an enzyme that degrades the natural substrate for NOS, L-arginine) in aged blood vessels and the corresponding modulation of NOS activity. Taddei et al12 have shown that there is a gradual decline in endothelial function due to aging, with greater than 50% loss in endothelial function in the oldest age group tested as measured by forearm blood flow assays. Egashira et al13 reported more dramatic findings in the coronary circulation of aging adults whereby there was a loss of 75% of endothelium-derived nitric oxide in 70-80 year-old patients compared to young, healthy 20-year-olds. Vita et al14 demonstrated that increasing age was one predictor of abnormal endothelium-dependent vasodilation in atherosclerotic human epicardial coronary arteries. Whilst Gerhard et al15 concluded from their 1996 study that age was the most significant predictor of endothelium-dependent vasodilator responses by multiple stepwise regression analysis. Collectively, these important findings illustrate that endothelium-dependent vasodilation in resistance vessels declines progressively with increasing age. This abnormality is present in healthy adults who have no other cardiovascular risk factors, such as diabetes, hypertension, or hypercholesterolemia. Most of these studies found that impairment of endothelium-dependent vasodilation was clearly evident by the fourth decade. In contrast, endothelium-independent vasodilation does not change significantly with aging, demonstrating that the responsiveness to NO does not change; only the ability to generate it does.
These observations enable us to conclude that reduced availability of endothelium-derived NO occurs as we age and to speculate that this abnormality may create an environment that is conducive to atherogenesis and other vascular disorders. It appears that aging interrupts NO signaling at every conceivable level, from production to inactivation. Given that NO is a necessary molecule for maintenance of health and prevention of disease, restoration of NO homeostasis may provide a new treatment modality for age and age related disease.
The core of what we are dealing with is referred to as endothelial dysfunction, which is by definition the physiological dysfunction of normal biochemical processes carried out by the endothelium, the cells that line the inner surface of all blood vessels, including arteries and veins (as well as the innermost lining of the heart and lymphatics). Loss of endothelial NO function is associated with several cardiovascular disorders, including atherosclerosis, which is due either to decreased production or to increased degradation of NO.16 Experimental and clinical studies provide evidence that defects of endothelial NO function is not only associated with all major cardiovascular risk factors, such as hyperlipidemia, diabetes, hypertension, smoking, and severity of atherosclerosis, but also has a profound predictive value for the future atherosclerotic disease progression.17-20 The dysfunctional endothelial NOS/NO pathway is considered as an early marker or a common mechanism for various cardiovascular disorders.
It appears that the inability to produce sufficient NO under the right preclinical conditions enhances the risk for a number of diseases that plague the older population. If this is true, then there exists an opportunity to intervene early during this process, and to implement strategies to restore NO homeostasis, and, perhaps, delay or prevent the onset and progression of certain diseases. This gradual loss of NO activity with age can be sped up or slowed down based on individual lifestyle and diet. This idea is illustrated in the hypothetical graphical representation in Figure 1. Adopting healthy habits such as a good diet and exercise can prolong the precipitous drop in NO production that occurs with age. To the contrary, a poor diet along with physical inactivity can accelerate the process and lead to a faster decline in NO production at a younger age. Therapeutic strategies directed at improving endothelial function or providing an alternative source of NO should be the primary focus because they may reduce the incidence of atherosclerosis or other diseases that occur with aging, even perhaps Alzheimer’s disease.
Figure 1. Hypothetical representation of NO production based on diet and lifestyle.
HUMAN NITROGEN CYCLE
Until recently it was thought that NO acted only as an autocrine or paracrine mediator due to its extremely short half-life (
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 pathway, and if any of the co-factors become limiting, then NO production from NOS shuts down, and in many cases, NOS then produces superoxide instead. The enzymatic production of NO is, indeed, a very complex and coordinated effort that 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. 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. 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 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. 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. NOx have recently been shown to be biomarkers for cardiovascular and other diseases from both diagnostic and therapeutic aspects. 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. A report by Kleinbongard et al33 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 nitrate37 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 Nitric Oxide Diagnostics (www.nitricoxidediagnostics.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.38 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. 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.38 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 significantly restore plasma levels of NOx, reduce triglycerides by 27%, and modestly reduce blood pressure and C-reactive protein thereby modifying the cardiovascular risk profile of patients after only 30 days.39 Harnessing the NO activity of natural plant-based products may provide a first line of defense for conditions associated with NO insufficiency.
The role of diet in the prevention and control of morbidity and premature mortality due to non-communicable diseases has been well established by vast population-based epidemiological studies carried out during the last decade.40 Nothing affects our health more than what we choose to eat. NO is essential for maintaining normal blood pressure, preventing adhesion of blood cells to the endothelium, and preventing platelet aggregation; it may, therefore, be argued that this single abnormality – the inability to generate NO – puts us at risk for diseases that plague us later in life, such as atherosclerosis, myocardial infarction, stroke, and peripheral vascular disease. Therefore developing strategies and new technologies designed to restore NO availability is essential for inhibiting the progression of certain common chronic diseases. NO should be a primary consideration in any personalized lifestyle medicine for prevention of chronic disease. We now have the tools and resources for diagnosing NO insufficiency and repleting NO in patients.
Bryan NS. Application of nitric oxide in drug discovery and development. Expert Opin Drug Discov. 2011 Nov;6(11):1139-54.
Bryan NS. Pharmacological therapies, lifestyle choices and nitric oxide deficiency: A perfect storm. Pharmacol Res. 2012 Dec;66(6):448-56.
Bryan NS (Editor): Food, Nutrition and the Nitric Oxide Pathway. DesTech Publishing – Pennsylvania ISBN: 978-1-932078-84-8, September 2009.
Bryan NS and Loscalzo J (Editors) Nitrite and Nitrate in Human Health and Disease – Springer Humana Press New York ISBN: 978-1-60761-615-3, May 2011.
Nathan S. Bryan & Janet Zand with Bill Gottlieb: The Nitric Oxide (NO) Solution. Good for you Books Publishing – ISBN: 978-0-615-41713-4; November 2010
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