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April 04, 2013

What's Wrong With Nitric Oxide - Part 2


Given the sometimes overwhelming complexity of biological systems, there’s an almost unavoidable tendency, even among top researchers, and medical professionals, to simplify matters by labeling certain biochemicals simplistically as either “good” or “bad.”

But rarely in biology do substances wear only black or white hats. More realistically, we find that certain substances produced within the body exert protective roles in some situations, but in other situations, when regulatory and stabilizing systems fail, these same substances may be harmful if produced in excess, or metabolized inefficiently. In other words, in an ironic twist of fate, many biological molecules have the tendency to exacerbate the very bodily damage they were initially produced to protect against.

As an example, many researchers believe that the cholesterol which leads to the production of atherosclerotic plaque in the arteries is initially a protective substance. The thinking is, that as a vital component of cellular structure, cholesterol is drawn to the artery to “patch up” microscopic injuries in the arterial wall. It’s only when various cell–signaling and inflammatory systems go awry, however, that cholesterol is altered from a protective to a pathological molecule, eventually resulting in the build–up of the cholesterol–laden plaque associated with heart disease.

Another example of such a two–faced biological chemical is the gaseous signaling molecule, nitric oxide.

Produced as a protective molecule under periods of stress and trauma, nitric oxide is most well–known as a substance integral for the proper dilation of blood vessels. The blood vessels of people with cardiovascular disease, high blood pressure, insulin resistance, and diabetes are almost always dangerously resistant to the vasodilating effects of nitric oxide; and as such, strategies to increase nitric oxide levels and to restore proper nitric oxide signaling in these patients represent a major focus of current nutritional and pharmaceutical intervention.

But so far, the results of such strategies have failed to deliver much in the way of meaningful benefit – and some have even proven deadly. Such puzzling outcomes speak to the dual nature of nitric oxide, as well as the intricate complexity of its metabolism.

As the big picture of nitric oxide becomes clearer, we now know that while a deficiency of nitric oxide function is strongly implicated in cardiovascular disorders, an excess of nitric oxide, and inefficient nitric oxide metabolism, are known to exacerbate the vascular damage associated with heart disease. Nitric oxide and its metabolites also play particularly crucial roles in the spread of cancer, and the development of degenerative brain disorders like Alzheimer’s and Parkinson’s disease.

In recent years, many companies within the nutritional supplement industry have offered up “nitric oxide–boosting” formulas containing amino acid precursors of nitric oxide such as arginine and citrulline. The marketing behind such products obviously focuses on the supposedly “good” roles of nitric oxide, but the research that exists indicates that such products may possess the potential to do serious harm in certain populations, or if taken over extended periods of time.

So, rather than simply finding ways of “boosting” or suppressing nitric oxide levels to treat or prevent different disorders, our goal will be to find nutritional and lifestyle strategies to modulate nitric oxide production, ensuring proper, healthy, nitric oxide signaling and metabolism in all tissues of the body for a lifetime.

To do this requires that we first take a look at the biological “assembly line” responsible for the production of nitric oxide. Though complicated at first, we’ll find that the answers we seek can be found only by a glimpse into the inner workings of our molecular biochemistry.

The Nitric Oxide Synthases

It’s well–known that our body produces nitric oxide (NO) from the amino acid arginine, and that the production of nitric oxide from arginine is catalyzed by a group of enzymes known as nitric oxide synthases (NOS). At least three (major) types of NOS exist, each classified by the types of tissues and cells in which they are found (the fact that nitric oxide can be produced by several different enzymes, and can be used for many different biological reactions, may be our first clue as to why NO may be beneficial in some instances, and harmful in others).

Endothelial nitric oxide synthase, or eNOS, can be found in the lining of the blood vessels, called the endothelium. The nitric oxide produced by eNOS triggers vasodilation, and is an important factor in regulating blood pressure. Endothelial nitric oxide further supports cardiovascular health by inhibiting the “stickiness” of blood cells called platelets, and preventing platelets from adhering to the endothelium – an early phenomenon in the development of atherosclerosis. It has been noted that all known or suspected risk factors for cardiovascular disease – including high cholesterol, high blood pressure, high homocysteine, high triglycerides, and smoking – involve the reduced bioavailability of nitric oxide within the endothelium.

Neuronal nitric oxide, or nNOS, is found in neurons in the brain and nervous system. The nitric oxide produced by nNOS acts as a neurotransmitter and signaling molecule, and, in precise amounts, may play a role in memory and learning. Disorders of nitric oxide production by nNOS, on the other hand, may play a role in many neurological diseases like Parkinson’s and Alzheimer’s.

Inducible nitric oxide, or iNOS, is produced by cells of the immune system called macrophages. Our immune system makes use of the nitric oxide’s toxic free radical–generating capacity to kill invading pathogens like viruses and bacteria. But unlike eNOS and nNOS, which produce nitric oxide on demand for seconds, or minutes at the most, iNOS is able to chronically stimulate the production of nitric oxide for hours or even days.

Although nitric oxide is a potential free radical regardless of where and how it’s produced, nitric oxide produced by the immune system (in macrophages via iNOS) may be particularly apt to inflict dangerous collateral damage to the tissues with which it comes in contact. As the name inducible nitric oxide synthase indicates, the activity of iNOS is greatly increased under conditions of stress or trauma. It’s now thought that many of the potentially harmful effects of NO may be due to the excess nitric oxide produced from iNOS in the macrophages.

In this edition of the Integrated Supplements Newsletter, we’ll begin by focusing on nitric oxide’s role in the cardiovascular system. We’ll see precisely why trying to “boost” nitric oxide levels via the indiscriminate intake of nitric oxide precursors isn’t advisable. Instead, we’ll find that nitric oxide modulation is akin to performing a tune–up on a high–performance engine, and that we’ll need to make subtle, calculated adjustments to keep our nitric oxide metabolism running smoothly. Given what is now known about nitric oxide (but which many supplement companies continue to ignore), we’ll develop a unique strategy to ensure the proper, healthy, production and metabolism of nitric oxide within the cardiovascular system.

NO and the Cardiovascular System

In recent decades, research has made it clear that all known cardiovascular risk factors (including elevated cholesterol, elevated triglycerides, elevated homocysteine, and smoking) impair nitric oxide metabolism. As such, it’s now well–accepted that disorders of nitric oxide activity underlie the development of cardiovascular disease. Decreased bioavailability of NO is not only a direct cause of the “silent killer” known as high blood pressure (as blood vessels dilate under the influence of NO), but faulty NO signaling can also lead to cell adhesion, proliferation, and ultimately, to the acceleration of arteriosclerotic lesions within the endothelium. Impaired nitric oxide activity has also been associated with insulin resistance and diabetes, and some researchers believe that nitric oxide may be the molecular key to the well–established link between diabetes and cardiovascular disease.

Study Link – Is type 2 diabetes mellitus a vascular disease (atheroscleropathy) with hyperglycemia a late manifestation? The role of NOS, NO, and redox stress.

Quote from the above study:

Cardiovascular disease accounts for at least 85 percent of deaths for those patients with type 2 diabetes mellitus (T2DM). Additionally, 75 percent of these deaths are due to ischemic heart disease. . . The vulnerable three arms of the eNOS reaction responsible for the generation of eNO is discussed in relation to the hypothesis: (1) The L–arginine substrate. (2) The eNOS enzyme. (3) The BH4 cofactor.

In the early days of nitric oxide research (which wasn’t all that long ago), scientists logically assumed that the nitric oxide precursor, the amino acid arginine, when added to the diet, would increase levels of nitric oxide. They therefore reasoned that supplying the body with large amounts of arginine would likely restore proper vascular function (including vasodilation). This hypothesis was often validated in animal studies, and even in some short–term human studies:

Study Link – Oral L–arginine improves endothelium–dependent dilation in hypercholesterolemic young adults.

Quote from the above study:

After oral L–arginine, plasma L–arginine levels rose from 115+/–103 to 231+/–125 micromol/liter (P<0.001), and [ endothelium–dependent dilation] improved from 1.7+/–1.3 to 5.6+/–3.0% (P<0.001).

But as longer–term studies on supplemental arginine began to be conducted in patients with pre–existing heart disease, a seemingly strange trend began to emerge. In many of these studies, the patients taking arginine often fared notably worse than those not taking the amino acid. One important trial even had to be stopped prematurely because of an increase in death in the group taking arginine supplements:

Study Link – L–Arginine Therapy in Acute Myocardial Infarction The Vascular Interaction With Age in Myocardial Infarction (VINTAGE MI) Randomized Clinical Trial.

Quote from the above study:

6 participants (8.6%) in the L–arginine group died during the 6–month study period vs none in the placebo group (P = .01). Because of the safety concerns, the data and safety monitoring committee closed enrollment. . . L–Arginine, when added to standard postinfarction therapies, does not improve vascular stiffness measurements or ejection fraction and may be associated with higher postinfarction mortality. L–Arginine should not be recommended following acute myocardial infarction.

Study Link – L–Arginine Supplementation in Peripheral Arterial Disease – No Benefit and Possible Harm

Quote form the above study:

Although absolute claudication distance improved in both L–arginine– and placebo–treated patients, the improvement in the L–arginine–treated group was significantly less than that in the placebo group (28.3% versus 11.5%; P=0.024). . . As opposed to its short–term administration, long–term administration of L–arginine is not useful in patients with intermittent claudication and PAD.

Study Link – Dietary Supplementation With L–Arginine Fails to Restore Endothelial Function in Forearm Resistance Arteries of Patients With Severe Heart Failure.

Study Link – Oral L–Arginine in Patients With Coronary Artery Disease on Medical Management.

Quote from the above study:

Oral L–arginine therapy does not improve NO bioavailability in CAD patients on appropriate medical management and thus may not benefit this group of patients.

In retrospect, it’s not surprising that adding additional arginine to the diet of people with cardiovascular disease often produced negative results. The reason is that, whatever the underlying causes of nitric oxide dysfunction in heart disease, one thing’s for certain – it is NOT due to an arginine deficiency (as arginine is abundantly supplied in the vast majority of diets).

It’s likely that individuals with pre–existing heart disease (or a tendency towards heart disease) suffer from disorders involving several of the enzymes and cofactors which are needed to convert arginine into nitric oxide efficiently. In other words, if nitric oxide production is faulty, then adding arginine to the diet is destined to make matters worse in the long–run, as was found in the above studies. It’s highly likely that overwhelming the nitric oxide–producing system with supplemental doses of arginine may actually impair nitric oxide production, and lead to the production of other, more harmful substances – even in healthy people (we’ll see exactly how this phenomenon takes place later).

ADMA – The First Clue

As supplementing the diet with additional arginine produced many unpredictable and harmful effects, researchers began to wonder what factors could be responsible for impairing nitric oxide production in those suffering from cardiovascular disease and diabetes. One current suspect is an arginine analog called asymmetrical dimethyl arginine, or ADMA, for short. Acting as arginine’s “evil twin”, so to speak, ADMA can “tie up” nitric oxide synthase enzymes and can significantly inhibit NO production. People with heart disease and diabetes almost always exhibit very high levels of ADMA, and ADMA has proven to be a very strong independent risk factor for cardiovascular disease and insulin resistance:

Study Link – Risk of acute coronary events and serum concentration of asymmetrical dimethylarginine.

Quote from the above study:

In an analysis of men who did not smoke, those who were in the highest quartile for ADMA (>0.62 μ mol/L) had a 3.9–fold (95% CI 1.25–12.3, p=0.02) increase in risk of acute coronary events compared with the other quartiles. Our findings suggest that ADMA is a predictor of acute coronary events.

Elevated ADMA levels have even been implicated in erectile dysfunction, a finding which isn’t terribly surprising considering the role of nitric oxide and vasodilation in facilitating the erectile response. Given how important proper NO metabolism is to cardiovascular health, researchers now believe that erectile dysfunction may be among the earliest physical manifestations of heart disease:

Study Link – Elevation of asymmetrical dimethylarginine (ADMA) and coronary artery disease in men with erectile dysfunction.

Quote from the above study:

As elevation of ADMA has been found to be associated with many risk factors for both CAD [coronary artery disease] and ED [erectile dysfunction], our data provide further strong evidence for the close interrelation of CAD and ED. Determination of ADMA may help to identify underlying cardiovascular disease in men with ED.

Where Does ADMA Come From?

In simple terms, ADMA is a byproduct of protein metabolism. Healthy people are usually able to metabolize and eliminate it properly, but in aging and disease, ADMA levels tend to rise. Because kidney disease patients excrete protein metabolites like ADMA less efficiently than healthy individuals, ADMA levels are known to be particularly high in those with kidney disease. And because ADMA is able to increase heart disease risk by interfering with NO production, an elevated ADMA level has been proposed to be the key “non–traditional” risk factor for heart disease in those with kidney disease. In other words, even kidney patients with normal cholesterol, blood pressure, and triglycerides are still very much prone to heart disease simply because of their elevated levels of ADMA.

In patients with heart disease and/or diabetes, but without overt kidney disease, it’s a little more difficult to say exactly why ADMA levels are almost invariably elevated. We do know, however, that the enzyme which breaks down ADMA, called dimethylarginine dimethylaminohy­drolase, or DDAH, is known to be particu­larly susceptible to oxidative damage in­flicted by known cardiovascular toxins like oxidized cholesterol, oxidized polyun­saturated fatty acids, and homocysteine.

For many months now, we at Integrated Supplements have been warning you of the dangers of oxidized cholesterol – found in many processed cholesterol–containing foods and powders, or produced in the body under conditions of oxidative stress. Researchers have recently shown that oxidized cholesterol caused a much greater elevation in ADMA levels than native, unoxidized cholesterol, due to oxidized cholesterol’s (oxLDL) unique ability to impair DDAH function.

Study Link – Novel Mechanism for Endothelial Dysfunction. Dysregulation of Dimethylarginine Dimethylaminohydrolase.

Quote from the above study:

The addition of oxLDL or TNF–a to ECV304 significantly increased the level of ADMA in the conditioned medium. The effect of oxLDL or TNF–a was not due to a change in DDAH expression but rather to the reduction of DDAH activity.

And the following study showed that a lipid peroxidation product produced from the omega–6 fat linoleic acid, called 4–HNE, significantly impaired nitric oxide production by interfering with DDAH activity. The effect was only partially reversed by arginine, but completely reversed by supplying increased amounts of DDAH along with antioxidants:

Study Link – Role of DDAH–1 in lipid peroxidation product–mediated inhibition of endothelial NO generation.

Quote from the above study:

We show that the lipid hydroperoxide degradation product 4–hydroxy–2–nonenal (4–HNE) causes a dose–dependent decrease in NO generation from bovine aortic endothelial cells, accompanied by a decrease in DDAH enzyme activity. The inhibitory effects of 4–HNE (50 µM) on endothelial NO production were partially reversed with L–Arg supplementation (1 mM). Overexpression of human DDAH–1 along with antioxidant supplementation completely restored endothelial NO production following exposure to 4–HNE (50 µM). These results demonstrate a critical role for the endogenous methylarginines in the pathogenesis of endothelial dysfunction. Because lipid hydroperoxides and their degradation products are known to be involved in atherosclerosis, modulation of DDAH and methylarginines may serve as a novel therapeutic target in the treatment of cardiovascular disorders associated with oxidative stress.

Study Link – Lipid peroxidation and nitric oxide inactivation in postmenopausal women.

Quote from the above study:

NO inactivation and the increase in lipid peroxidation may contribute to endothelial dysfunction and to the greater risk for atherosclerosis in postmenopausal women.

And, illustrating just how far–reaching the effects of lipid peroxide–induced disruption of nitric oxide metabolism can be, patients suffering from major depression have been shown to exhibit elevated levels of 4–HNE, decreased activity of DDAH and subsequently, increased levels of ADMA, and decreased plasma nitric oxide.

Study Link – Increased (E)–4–hydroxy–2–nonenal and asymmetric dimethylarginine concentrations and decreased nitric oxide concentrations in the plasma of patients with major depression. 

Quote from the above study:

There is an increase in circulating HNE in major depression. HNE inactivates the cysteine residue in the active site of endothelial DDAH leading to the accumulation of ADMA in the circulation. The ADMA then decreases the production of eNOS. This could reduce the amount of NO diffusing from cerebral blood vessels to nearby neurons and influence the release of neurotransmitters. ADMA also constricts cerebral blood vessels and may contribute to the decreased regional perfusion in major depression. The accumulation of ADMA could explain the increased risk of CHD in major depression. The preservation of DDAH activity and the reduction of ADMA accumulation may represent a novel therapeutic approach to the treatment of major depression.

In the above study, we also find that the potent cellular antioxidant glutathione was able to significantly reduce the level of lipid peroxides and protect the damage inflicted on the DDAH enzyme:

The effects of HNE on DDAH activity were significantly attenuated by the addition of glutathione (P<0.0001).

Taken together, these studies give us good reason to believe that the fundamental disorder of nitric oxide bioavailability seen in aging and disease is due to oxidative stress – particularly oxidative stress driven by products of lipid peroxidation (i.e. oxidized fat and cholesterol).

And researchers in the field are beginning to come to this same conclusion:

Article Link – When the endothelium cannot say ‘NO’ anymore.

Quote from the above article:

The mechanism by which ADMA is elevated in some patients may relate to oxidative stress. ADMA is inactivated by an enzyme named dimethylarginine dimethylaminohydrolase (DDAH); most investigators agree that DDAH plays an important role in the regulation of ADMA levels. DDAH activity is downregulated by oxidative stress, as it is associated with high cholesterol, high glucose, and high homocysteine levels. In these settings, accumulation of ADMA can be prevented by addition of antioxidants in experimental models. Inhibition of DDAH, in turn, leads to elevated ADMA levels, which in turn promote further generation of oxidants, possibly by uncoupling NO synthase. This vicious circle provides an integrative explanation for the interrelation between lack of NO, excess of oxygen–derived free radicals, and progression of vascular lesion formation.

A Nutritional Plan of Attack

To lower our level of oxidative stress, reducing our intake of oxidized cholesterol (from cholesterol–containing powders like powdered eggs, powdered cheese, and whey protein concentrate), as well as dramatically reducing our intake of dietary polyunsaturated fatty acids (omega–6– and omega–3–containing fats) is the only reasonable place to start. In addition, it is likely prudent to supplement with known inhibitors of lipid peroxidation, like Vitamin E, coenzyme Q10 and lipoic acid.

As noted, the above study on nitric oxide and depression showed that the cellular antioxidant, glutathione, significantly reduced the harmful effects of HNE on DDAH, so a supplement of undenatured whey protein isolate (which contains the “building blocks” of glutathione) along with the mineral selenium (also important for glutathione production) are likely to be very helpful as well.

Note: For more information on the role of lipids in oxidative stress, see the November and December 2007 issues of the Integrated Supplements Newsletter.

Homocysteine and Nitric Oxide

In addition to byproducts produced from unsaturated fat and cholesterol, the protein–derived substance, homocysteine, has also been shown to be an important contributor to the burden of oxidative stress. Homocysteine is an amino acid produced in high amounts due to the inefficient metabolism of the amino acid methionine, and elevated homocysteine levels have increasingly been implicated as a major heart disease risk factor in recent decades. If homocysteine does, in fact, cause an elevation in ADMA (and a subsequent decrease in NO production), as is shown in the following study, this would clearly lend molecular–level support to the homocysteine hypothesis of heart disease.

Study Link – Homocysteine Impairs the Nitric Oxide Synthase Pathway Role of Asymmetric Dimethylarginine.

Quote from the above study:

Homocysteine post–translationally inhibits DDAH enzyme activity, causing ADMA to accumulate and inhibit nitric oxide synthesis. This may explain the known effect of homocysteine to impair endothelium–mediated nitric oxide–dependent vasodilatation.

Vitamins B6, B12, and folic acid can reliably reduce homocysteine, which seems to be an important piece of the puzzle given what we now know about homocysteine’s role in impairing nitric oxide production. Folic acid in particular may play several roles in ensuring proper nitric oxide metabolism (more on this later).

And as we look beyond ADMA and DDAH, deeper into the various pathways involved in nitric oxide metabolism, we begin to see yet again, that oxidative stress is the common thread responsible for disrupting all of them.

Nitric Oxide, Superoxide, and Peroxynitrite

As we outlined in the previous Integrated Supplements Newsletter, nitric oxide, being a gaseous chemical, often doesn’t stick around long once it’s produced. Nitric oxide is known to rapidly react with the free radical superoxide (O2–), producing the powerful oxidant, peroxynitrite (OONO–) (remember that oxidizing chemicals like superoxide and peroxynitrite are potent molecular–level drivers of oxidative stress).

Researchers now believe that much of the cellular damage associated with cardiovascular disease (and other diseases in which nitric oxide plays a major role) involves the over–production of superoxide and peroxynitrite from faulty nitric oxide metabolism. In fact, much of the reason that nitric oxide levels are low in cardiovascular disease is because, under conditions of oxidative stress, arginine is converted to these harmful oxidants instead of nitric oxide – yet another reason why supplying additional arginine to the body is wrought with potential danger.

Article Link – Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly.

Quote from the above article:

The direct toxicity of nitric oxide is modest but is greatly enhanced by reacting with superoxide to form peroxynitrite (ONOO–). Nitric oxide is the only biological molecule produced in high enough concentrations to out–compete superoxide dismutase for superoxide.

As we’ve seen, the oxidative stress of aging and disease can damage the fragile enzyme DDAH, causing nitric oxide’s “evil twin,” ADMA to become elevated. ADMA competes with arginine for NOS and causes a reduction in nitric oxide production.

But this isn’t even the only way in which oxidative stress can impair nitric oxide production. The synthesis of nitric oxide from NOS requires a cofactor called tetrahydrobiopterin, or BH4, for short. When BH4 is damaged under conditions of oxidative stress (i.e. when it is oxidized), NOS then converts arginine directly to superoxide, and ultimately to the harmful reducing agent, peroxynitrite. Scientists call this effect an uncoupling effect, as damage to BH4 is able to uncouple, or divert arginine synthesis away from nitric oxide and towards superoxide and peroxynitrite. In a vicious downward spiral, peroxynitrite causes the oxidation of even more BH4, and the synthesis of nitric oxide is then even further impaired.

Study Link – Oxidation of Tetrahydrobiopterin by Peroxynitrite: Implications for Vascular Endothelial Function.

Quote from the above study:

Nitric oxide and superoxide react rapidly to form peroxynitrite, which may be the reactive species responsible for many of the toxic effects of nitric oxide. Here we show that BH4 is a primary target for peroxynitrite–catalyzed oxidation because at pH 7.4, physiologically relevant concentrations of BH4 are oxidized rapidly by low concentrations of peroxynitrite. . . Thus, abnormally low levels of BH4 can promote a cycle of its own destruction mediated by nitric oxide synthase–dependent formation of peroxynitrite. This mechanism might contribute to vascular endothelial dysfunction induced by oxidative stress.

Not surprisingly then, damage to tetrahydrobiopterin has been shown to cause all of the cardiovascular risk factors associated with impaired nitric oxide bioavailability; and repairing or preventing damage to tetrahydrobiopterin represents a key strategy in restoring healthy nitric oxide production.

As an example, while it’s common knowledge that high cholesterol levels represent a threat to cardiovascular health (especially when coupled with an environment of oxidative stress, in which significant amounts of cholesterol are prone to oxidation), few people realize that (oxidized) cholesterol may do cardiovascular damage largely through its ability to damage tetrahydrobiopterin, and thus, healthy nitric oxide production. Infused tetrahydrobiopterin has been shown to counter this effect and restore endothelial function in patients with high cholesterol:

Study Link– Tetrahydrobiopterin restores endothelial function in hypercholesterolemia.

Quote from the above study:

In hypercholesterolemia, impaired nitric oxide activity has been associated with increased nitric oxide degradation by oxygen radicals. Deficiency of tetrahydrobiopterin, an essential cofactor of nitric oxide synthase, causes both impaired nitric oxide activity and increased oxygen radical formation. . . this study demonstrates restoration of endothelial dysfunction by tetrahydrobiopterin suppletion in hypercholesterolemic patients.

It’s also been shown that other cardiovascular risk factors, like smoking, may do their damage by impairing tetrahydrobiopterin function as well:

Study Link – Tetrahydrobiopterin Improves Endothelium–Dependent Vasodilation in Chronic Smokers Evidence for a Dysfunctional Nitric Oxide Synthase.

Quote from the above study:

These data support the concept that in addition to the free radical burden of cigarette smoke, a dysfunctional [eNOS] due to BH4 depletion may contribute at least in part to endothelial dysfunction in chronic smokers.

And while we can’t realistically inject ourselves with tetrahydrobiopterin as was done in the above studies, there may be several nutritional strategies which will allow us to optimize the function of tetrahydrobiopterin within our bodies.

Supplementation with the well–known antioxidant, vitamin C, has been shown to protect tetrahydrobiopterin from oxidation, and to restore proper NOS activity:

Study Link – Long–Term Vitamin C Treatment Increases Vascular Tetrahydrobiopterin Levels and Nitric Oxide Synthase Activity.

Quote from the above study:

In vivo, beneficial effect of vitamin C on vascular endothelial function appears to be mediated in part by protection of tetrahydrobiopterin and restoration of eNOS enzymatic activity.

Nitrate Tolerance – More Clues on The Importance of Tetrahydrobiopterin

The nitric oxide–boosting drug, nitroglycerin, has been used for over a century as a vasodilator in coronary artery disease, but a troubling phenomenon, called nitrate tolerance, almost invariably arises with the drug’s long–term use. Researchers have wondered for years why it is that nitroglycerin quickly loses its efficacy, and many theories have been proposed to explain this occurrence. The most likely scenario appears to be that nitroglycerin gradually increases the formation of reactive oxygen species (or, ROS – the molecular–level drivers of oxidative stress) by impairing the bioavailability of tetrahydrobiopterin. Two of the ROS produced in the development of nitrate tolerance are the aforementioned superoxide and peroxynitrite radicals, which, as we’ve seen, further impair nitric oxide production in a vicious downward spiral. Ironically, considering the fact that nitrate drugs are so commonly used as short–term treatments for heart disease symptoms, nitric oxide drugs are known to increase mortality in those with existing heart disease; and the production of superoxide and peroxynitrite from nitric oxide helps to explain why:

Study Link – Long–term nitrate use may be deleterious in ischemic heart disease: A study using the databases from two large–scale postinfarction studies. Multicenter Myocardial Ischemia Research Group.

Quote from the above study:

The Cox analyses with all the variables retained revealed that nitrates were associated with a significantly increased mortality risk (MSMI: hazard ratio 3.78, P =.011; MDPIT: hazard ratio 1.61, P =.019) in patients who had recovered from an acute coronary event. . . These analyses raise concern about the potential adverse effects of long–acting nitrate therapy in chronic coronary disease.

It’s worth noting, too, that the phenomenon of nitrate tolerance – where a nitric oxide boosting substance “works” in the short–term, but is harmful in the long–term – parallels many of the same surprisingly harmful effects noticed in long–term studies where arginine was administered as a nitric oxide precursor. Many nitric oxide–boosting products are often “cycled,” or taken for relatively short periods of time, with a subsequent “layoff” of arbitrary duration. But, if these products “stop working” with continued use (as empirical evidence indicates is indeed the case), it’s fair to assume that the arginine they contain is no longer being converted to nitric oxide efficiently, and is instead being converted into harmful superoxide and peroxynitrite. No supplement company can say with any certainty precisely when this toxic phenomenon begins, or whether “cycling” the product makes it less harmful in the long–term. Thus it’s probably safe to say that the more a person consumes supplemental arginine or nitric oxide–boosting supplements (cycled or not), the greater the potential harm he or she is doing to his or her body.

But, of course, even those of us not consuming nitrate drugs or nitric oxide–boosting supplements can still learn a valuable lesson from what is now known about nitrate tolerance.

If progressive damage to tetrahydrobiopterin (both by nitric oxide itself and its ROS metabolites) is responsible for nitrate tolerance, then many of the same strategies which help to prevent nitrate tolerance may do so by protecting tetrahydrobiopterin (which is our goal as well). Studies show that this is, in fact, the case.

Vitamin C has been used with success to attenuate nitrate tolerance:

Study Link – Randomized, double–blind, placebo–controlled study of the preventive effect of supplemental oral vitamin C on attenuation of development of nitrate tolerance.

Quote from the above study:

These results indicate that combination therapy with vitamin C is potentially useful for preventing the development of nitrate tolerance.

And folic acid, a nutrient which may be able to “pinch hit” for tetrahydrobiopterin, has been shown to prevent nitroglycerin–induced nitrate tolerance as well:

Study Link – Folic Acid Prevents Nitroglycerin–Induced Nitric Oxide Synthase Dysfunction and Nitrate Tolerance.

Quote from the above study:

Our data demonstrate that supplemental folic acid prevents both nitric oxide synthase dysfunction induced by continuous [nitroglycerin] and nitrate tolerance in the arterial circulation of healthy volunteers. We hypothesize that the reduced bioavailability of tetrahydrobiopterin is involved in the pathogenesis of both phenomena. Our results confirm the view that oxidative stress contributes to nitrate tolerance.

And, beyond its role in nitrate tolerance, we find several unique roles of folic acid in ensuring proper NO production and cardiovascular health. The active form of folic acid, known as 5–methyltetrahydrofolate, has been shown to prevent the vascular disruption caused by high cholesterol:

Study Link – 5–Methyltetrahydrofolate, the Active Form of Folic Acid, Restores Endothelial Function in Familial Hypercholesterolemia.

Quote from the above study:

These results show that the active form of folic acid restores in vivo endothelial function in FH. It is suggested from our in vitro experiments that this effect is due to reduced catabolism of NO.

Folic acid is also known to play a role in reducing levels of the previously–mentioned cardiovascular toxin, homocysteine. And homocysteine has been shown to inhibit tetrahydrobiopterin functioning:

Study Link – Homocysteine induces oxidative stress by uncoupling of no synthase activity through reduction of tetrahydrobiopterin.

Quote from the above study:

The results show that the oxidative stress and inhibition of NO release induced by homocysteine depend on eNOS uncoupling due to reduction of intracellular tetrahydrobiopterin availability.

Tetrahydrobiopterin and Depression

It’s also interesting to note that, in addition to its role in producing nitric oxide, tetrahydrobiopterin is also a known co–factor in the production of the neurotransmitters noradrenalin, serotonin, and dopamine.

We saw previously how lipid peroxides can impair proper nitric oxide production; and we referenced studies in which depressed patients were shown to exhibit elevated lipid peroxide levels and decreased nitric oxide levels in their plasma.

Other studies show that reduced availability of tetrahydrobiopterin may not only impair nitric oxide production, but also the production of important brain chemicals (called monoamines in the quote below) in depression.

Study Link – The role of pterins in depression and the effects of antidepressive therapy.

Quote from the above study:

As a raised N:B ratio implies failure to convert neopterin to biopterin, it is possible that reduced availability of tetrahydrobiopterin, the essential cofactor for the formation of noradrenaline, serotonin and dopamine, may exert rate–limiting control over the synthesis of monoamines implicated in the pathogenesis of depressive illness.

Add to this the fact that depression is very strongly correlated with the development of heart disease:

Study Link – Depression as a predictor for coronary heart disease. A review and meta–analysis.

Quote from the above study:

It is concluded that depression predicts the development of [coronary heart disease] in initially healthy people. The stronger effect size for clinical depression compared to depressive mood points out that there might be a dose–response relationship between depression and [coronary heart disease].

And it’s tempting to theorize that disorders of tetrahydrobiopterin function, leading to both impaired nitric oxide function, and impaired neurotransmitter synthesis, may act as a common biological thread tying together both heart disease and depression.

Unlike the symptoms of heart disease, the burden of psychological depression often manifests during the first three or four decades of life – so even young people who are not often concerned with their heart disease risks should still take note of the research presented here. And considering that psychological depression is not only a major health challenge in and of itself, but is also a clear warning sign of impending cardiovascular disease, we can clearly see how valuable integrated and biologically sound nutritional strategies are if we can indeed combat both disorders simultaneously.

As is so common in biology, addressing fundamental defects and nutrient imbalances on a cellular and molecular level, imparts a beneficial “ripple effect” throughout the entire body, whereas treating merely symptoms (with pharmaceuticals, and the “wrong” nutrients) often leads to a whole host of negative side effects.

Further Neutralizing ROS

Many of the nutrients we’ve already mentioned have potent antioxidant and cell–protective effects, and synergistically, these nutrients work together to ensure proper nitric oxide production at each step of the biological “assembly line.”

To Review:

Nutrients to prevent lipid peroxidation and to lower ADMA by preventing oxidative damage to DDAH:

• Vitamin E

• Lipoic Acid

• Whey Protein Isolate

• Selenium

Nutrients for lowering homocysteine levels:

• Folic Acid

• Vitamin B–6

• Vitamin B–12

• Trimethylglycine

Nutrients for protecting tetrahydrobiopterin

• Vitamin C

• Folic Acid

And in addition to these nutrients, several others may play important roles in ensuring healthy nitric oxide metabolism within the cardiovascular system and beyond. Plant chemicals like polyphenols and flavanols (found in such foods as tea, wine, and chocolate), may be able to scavenge superoxide and peroxynitrite radicals, potentially neutralizing these reactive oxygen species before they can trigger NOS uncoupling and the downward spiral of faulty nitric oxide metabolism.

Of course, even though polyphenols are able to quickly deactivate ROS in vitro (in a test tube), there’s still some debate as to how well these plant chemicals do so within our bodies. Our livers usually do an excellent job of “deactivating” these “foreign” chemicals before they reach the bloodstream, and surprisingly few polyphenols actually reach general circulation. It’s been proposed that either these polyphenols stimulate our own antioxidant systems (like those involving glutathione), or that the polyphenol conjugates (conjugation, or the “attachment” of one substance to another in order to improve elimination, is what the liver does to detoxify such compounds) may have antioxidant effects in and of themselves.

For more info, see:

Study Link – How should we assess the effects of exposure to dietary polyphenols in vitro?

But whatever their mechanisms of action, some plant chemicals appear to have remarkable effects on nitric oxide production in vivo (in the body) when consumed orally. The flavanols (a type of polyphenol) in unprocessed cocoa, for example, have been shown to significantly lower blood pressure via nitric oxide–mediated action.

Study Link – Flavanol–rich cocoa induces nitric–oxide–dependent vasodilation in healthy humans.

Quote from the above study:

In healthy humans, flavanol–rich cocoa induced vasodilation via activation of the nitric oxide system, providing a plausible mechanism for the protection that flavanol–rich foods induce against coronary events.

And subsequent studies by the same researchers showed that older individuals had a greater response to cocoa flavanols than younger individuals – indicating that the flavanols may be able to partly correct the decline in nitric oxide function which occurs in aging.

Study Link – Aging and vascular responses to flavanol–rich cocoa.

Quote from the above study:

Flavanol–rich cocoa enhanced several measures of endothelial function to a greater degree among older than younger healthy subjects. Our data suggest that the NO–dependent vascular effects of flavanol–rich cocoa may be greater among older people, in whom endothelial function is more disturbed.

And besides various plant polyphenols, and the vitamins we’ve already mentioned, another, more “non–traditional” antioxidant may have the unique ability to protect against the free radical damage associated with nitric oxide.

Creatine and Cardiovascular Health

Though it’s often though of only as a sports supplement useful for enhancing muscle size and strength, the high energy molecule, creatine (which, unlike some polyphenols, is very well–absorbed) has been shown to scavenge both superoxide and peroxynitrite – the molecules largely responsible for nitric oxide–induced damage.

Study Link – Direct Antioxidant Properties of Creatine.

Quote from the above study:

. . .creatine displayed a significant ability to remove [superoxide] and [peroxynitrite] when compared with controls. . . To our knowledge, this is the first evidence that creatine has the potential to act as a direct antioxidant against aqueous radical and reactive species ions.

We looked briefly at the role homocysteine plays in inhibiting DDAH, and tetrahydrobiopterin – two cofactors required for proper nitric oxide production. So, logically, lowering homocysteine will be an important part of ensuring proper nitric oxide metabolism and cardiovascular health. It’s known that the neutralization of homocysteine requires a chemical process called methylation, and that substances which donate methyl groups (CH3), such as betaine (TMG), SAMe, and choline – all available as dietary supplements – have been shown to lower homocysteine levels.

Because creatine can produce energy substrates quickly, without the metabolic demands of breaking down glucose or fatty acids for fuel, creatine can be thought of as an “emergency” energy molecule. As such, creatine is in very high demand by the most metabolically active tissues like the muscles, brain, and heart (even in non–athletes).

But the production of creatine within the body just so happens to “use up” methyl groups at an astonishingly high rate. It turns out that taking “pre–formed” creatine as a supplement (meaning, that our body doesn’t have to go through the metabolic steps of making it) spares the valuable methyl groups which would otherwise be used for creatine production – methyl groups which can then be used to neutralize homocysteine. And this effect is more than mere biological speculation – oral creatine supplements have, in fact, been shown to lower homocysteine levels significantly:

Study Link – Oral creatine supplements lower plasma homocysteine concentrations in humans.

Quote from the above study:

After four weeks of creatine supplements, [total plasma homocysteine] in [the experimental group] changed by an average of –0.9 micromol/L (range: –1.8 to 0.0), compared to an average change of +0.2 micromol/L in C (range: –0.6 to 0.9) during the same four weeks. The difference in the changes in [total plasma homocysteine] between the two groups was statistically significant (p < 0.01). CONCLUSION: Creatine supplements may be an effective adjunct to vitamin supplements for lowering [total plasma homocysteine].

Given that we must reduce the burden of superoxide, peroxynitrite, and homocysteine in order to ensure proper nitric oxide metabolism within the cardiovascular system, daily supplementation with pure creatine monohydrate probably represents one of the most physiologically sound ways to achieve all of these goals simultaneously – all the while supplying a vital energy substrate to the metabolically active cells of the muscle, brain, and heart.

In fact, it’s probably safe to say that although creatine is remarkably effective for increasing strength and energy production in athletes, those who have avoided taking creatine monohydrate because of its stigma as a mere “bodybuilding” supplement are likely to be missing out on one of the most remarkably health–promoting substances within the entire realm of nutritional supplementation.

And similarly, many people who currently think that they are taking creatine may not be. In recent years, many different types of creatine have been introduced to the nutritional supplement market, each claiming offer benefits above and beyond creatine monohydrate. In recent studies, however, two of the most heavily promoted “new” creatines, creatine ethyl ester, and a brand of “buffered” creatine, have both been shown to be vastly inferior to creatine monohydrate. In direct opposition to the marketing claims of these newfangled creatines, both creatine ethyl ester, and so–called buffered creatines have been shown to degrade into the useless byproduct creatinine much more readily than does creatine monohydrate.

Exposing the lies and misinformation surrounding creatine is best left for another newsletter, but for now, it’s important to realize that the full benefits of creatine, for cardiovascular health, or athletic improvement, can be obtained only through supplementation with pure creatine monohydrate.

The Cardiovascular System – Just The Beginning

Faulty nitric oxide metabolism has been implicated as a common denominator in various degenerative diseases including not only heart disease, but also such disorders as Alzheimer’s disease and cancer.

Fortunately, many of the dietary and supplement strategies we’ve covered here will also serve us well as we examine the effects of nitric oxide not directly related to the cardiovascular system. In the next Integrated Supplements Newsletter, we’ll take a look at nitric oxide’s role in other systems of the body, and in other disorders associated with aging. As we put all of the pieces of the nitric oxide puzzle together, the big picture of nitric oxide will begin to come into even clearer focus.

About Us: At Integrated Supplements, our goal is to bring you the wellness information and products you need to live your life to the fullest. We are dedicated to producing the highest–quality, all–natural nutritional supplements; and to educating the world on the health promoting power of proper nutrition. You can find out more by visiting: www.IntegratedSupplements.com


These statements have not been evaluated by the FDA. No Integrated Supplements product is intended to diagnose, treat, cure or prevent any disease.



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