Healthy Aging

Are You Nitric Oxide Deficient? Part 2 of 2

Dr. Nathan Bryan, Ph.D., on nitric oxide, the peroxynitrite issue, and nutritional tools that may help improve nitric oxide production

Biography: Nathan S. Bryan, Ph.D., is an international leader in molecular medicine and nitric oxide biochemistry. Specifically, Dr. Bryan was the first to describe nitrite and nitrate as indispensable nutrients required for optimal cardiovascular health. He was the first to demonstrate and discover an endocrine function of nitric oxide via the formation of S-nitrosoglutathione and inorganic nitrite.

Dr. Bryan has been involved in nitric oxide research for the past 18 years and has made many seminal discoveries in the field. Many of these discoveries and findings have transformed the development of new therapeutic agents for the treatment and prevention of human disease.

Dr. Bryan has published a number of highly cited papers and authored or edited five books. More about his work can be found at

Learn more about the common etiologies of inadequate nitric oxide production by starting with Part 1 of this interview.

NutritionInFocus: At one point, we saw nitric oxide as being a contributor to chronic inflammatory and neurodegenerative diseases. How is this viewpoint changing? Was there certain landmark research that prompted this change or has this just been a gradual paradigm shift?

Dr. Bryan: The consensus now, at least in the scientific community, is that nitric oxide (NO) is probably one of the most important molecules produced in the human body. With 160,000 published papers on NO and the 1998 Nobel Prize being awarded for its discovery, there is really no doubt of its importance for human physiology. The general acceptance is that NO is good, and when you can’t make it bad things happen.

Nitric oxide may be present in different diseases, but this doesn’t mean it is causing the pathology – it is the body’s way of policing or getting control of a situation.

Often times NO is present in disease-related processes. A good analogy is to cops always showing up at a crime scene. But being there doesn’t mean they caused the crime – they are there to clean it up. It’s the same thing with NO: it may be present, but this doesn’t mean it is causing the pathology – it is the body’s way of policing or getting control of a situation. A lot of other metabolites are detectable because NO itself is very reactive and hard to detect. For example, if you have a tyrosine residue in close proximity where you get superoxide and NO forming, you will nitrate the tyrosine residue.[1],[2] Then, when you look for these fingerprints of where NO was, you find things like nitrotyrosine, nitrite, nitrate, and a lot of other metabolites.[3] But again, this is a signature of NO being present, not that it is the cause of pathology.[4]

NutritionInFocus: Substantial amounts of clinical research have shown NO will interact with superoxide to form peroxynitrite, an aggressive oxidant. Is this a concern with promoting increased NO synthesis?

Dr. Bryan: This is a good question and comes up a lot. However, I really believe this is a non-issue under normal physiology and will tell you why.

Peroxynitrite is a strong oxidant that leads to irreversible protein oxidation or nitration and is a part of a lot of pathology in the medical literature.[5] But there is a very specific requirement for peroxynitrite to form: you must have NO and superoxide, which both are radicals, in close proximity to form peroxynitrite, or ONOO-. But ONOO- is also inorganic nitrate. So, when NO and superoxide react, it forms this cage-like molecule, and about 90 to 95% of this just rearranges to inorganic nitrate (NO3-),[6] which is inert.

The other thing is that if you are having a lot of superoxide being formed, that typically means you aren’t making a lot of NO because superoxide shuts down NO production. To the contrary, if you restore normal NO production, superoxide production goes down.[7],[8] We have looked at the three main sources of superoxide to identify how this works. First, we have nitric oxide synthase (NOS), the enzyme that makes NO. When it becomes uncoupled it only generates superoxide, not NO. So, in this case you are only generating one, and not the other, and therefor are not producing peroxynitrite. The second setting is the production of superoxide via NADPH oxidase. We know from the literature that if you restore NO production, or if you give NO or even nitrites, this inhibits the production of superoxide from NADPH oxidase.[9] The third main source of superoxide is uncoupled mitochondrial electron transport.[10] We know that both nitrite and NO recouple the electron transport chain and prevent electron leakage and prevent superoxide production. So, peroxynitrite becomes a non-issue if you do things to restore NO production, thereby shutting down superoxide production.

NutritionInFocus: What population or disease condition is most likely to benefit from increasing NO levels in the body?

Dr. Bryan: Really any condition with a vascular component to it – which to me is any dysfunctional tissue or disease. Raynaud’s is a microvascular disease in which the microvasculature in the periphery isn’t perfusing, so if you open up those small blood vessels by enhancing nitric oxide production Raynaud’s goes away.[11] In fact, we see complete symptom relief in ten minutes in Raynaud’s patients. Other forms of peripheral vascular disease such as intermittent claudication can also greatly benefit from improved NO production.

Raynaud’s is a microvascular disease in which the microvasculature in the periphery isn’t perfusing, so if you open up those small blood vessels by enhancing nitric oxide production Raynaud’s goes away.

Some of the overlaps between cardiovascular disease and diabetes actually occur because the NOS enzyme becomes glycosylated which shuts down NO production.[12] People don’t die from diabetes – they die from the vascular complications associated with diabetes because they can’t make NO. They have circulatory collapse: they develop diabetic retinopathy, macular degeneration, peripheral neuropathy, kidney disease, the arteries become inflamed, and they have a heart attack or stroke, or they lose limbs due to amputation. All of that is due to NO insufficiency.

As we consider conditions like diabetes and hypertension which have many microvasculature complications that affect organs like the eyes or kidneys, I really think the small vessel disease occurs before the large vessel disease. Glaucoma, diabetic retinopathy, macular degeneration – these are all small blood vessel diseases. If you restore NO production and you can regulate the flow of fluid into the eye then you can reduce the intraocular pressure of glaucoma, and you can get oxygen and nutrients in to cells that need them in these microenvironments, and things get better.

Improved NO production can also help with autoimmunity because it has very potent anti-inflammatory properties and inhibits oxidative stress – things that are basically hallmark features of any autoimmune disease. There also often is vascular dysfunction in autoimmunity[13] – multiple sclerosis, rheumatoid arthritis, or systemic lupus erythematosus are examples of this – and NO also helps with this aspect.

NutritionInFocus: How does exercise affect NO levels and conversely can support for NO production improve athletic performance?

Dr. Bryan: When you exercise, you need increased blood flow to the skeletal muscles to support the increased activity, and to get more oxygen and nutrients into the tissues and remove the waste products that are being generated. Exercise is a stimulus or activator of NO production because when you begin to exercise it causes shear stress in the lining of the blood vessels and this turns on NOS.[14]

In young healthy people with good endothelial function the enzyme works, so when you begin to exercise you see an increase in NO production, but in older people it often doesn’t. When people are put on a treadmill to assess their heart function this really is a test of their ability to produce NO – those that fail the test can’t make enough NO. The coronary arteries are unique in the fact that the only way to increase oxygen to the working heart muscle is to dilate the blood vessels. Other tissues and muscles can actually recruit more capillaries or increase oxygen extraction from the blood when under the demands of exercise. Even under resting conditions the heart extracts the maximum oxygen from the blood, so this aspect can’t be further increased. The only way to get more oxygen to the heart is to dilate the blood vessels by making NO.

Exercise is a stimulus or activator of NO production because when you begin to exercise it causes shear stress in the lining of the blood vessels and this turns on NOS.

When people become exercise intolerant like that, it can be dangerous to exercise without restoration of NO production because the body can’t make NO to increase blood flow to the heart. So, then they want to wheel you out and do bypass surgery to put metal in the arteries to open them up.

On the flipside, nutritional support for nitric oxide production can have an impact on exercise performance. You may recall in the 2012 Olympic games there was a lot of media around these athletes using beet root juice because it contains high levels of nitrate that reduces to NO. There are a number of beet products like this on the market that help do this.

NutritionInFocus: With the short half-life of NO, how can we consistently increase NO levels in the body? Is there a way we can improve NOS function?

Dr. Bryan: We have to understand what goes wrong in the body of people who can’t make NO and fix it – that’s really what my 20 years of research has been about. Although we can give the body the things it needs to make NO, to treat the cause of the problem, you need to fix the reason the body can’t make NO. And then, if you give the body what it needs, the body heals itself.

There are two pathways by which the body makes NO. One pathway that we have spoken about quite a bit already is through the reduction of nitrate to nitrite to NO (see Figure). That’s dependent on getting enough nitrates in the diet, the presence of oral nitrate-reducing bacteria, and stomach acidity. So, I tell people to eat a diet that is rich in green leafy vegetables and to avoid using mouthwash, antibiotics, and proton pump inhibitors to address this part of the system.

Pathways via which the human body produces nitric oxide

A challenge that many are not aware of is that the nitrate and nitrite levels in frequently consumed vegetables varies dramatically, potentially by a factor of ten or more.[15] We’ve done food surveys where we tested foods from five cities in the U.S., and just because you are eating celery, broccoli, kale, or spinach this doesn’t necessarily mean you are taking in enough nitrate to give your body what it needs to generate NO. So, you may need to supplement with some NO-supporting products with ingredients that are high in nitrates like beet root.

A challenge that many are not aware of is that the nitrate and nitrite levels in frequently consumed vegetables varies dramatically, potentially by a factor of ten or more.

The other pathway is via NOS – this enzyme converts L-arginine to NO (see Figure).[16] This was the first pathway discovered, and for that reason there are a lot of L-arginine products on the market. However, the body makes enough L-arginine through the urea cycle to make NO[17] – giving more isn’t going to do anything more. Giving an L-arginine product to an individual who is NO deficient is like putting gas in a car with a blown-out engine – these people aren’t out of fuel or L-arginine, they have just lost the ability to convert it. So, you have to fix the enzyme and recouple it, which is what we have figured out to do in our work. If you do this, then your body can make NO even if you are using an antiseptic mouthwash.

NOS is a homodimer – it is two twin molecules that come together to facilitate conversion of L-arginine to L-citrulline and NO in a complicated five electron multi-step oxidation reaction requiring numerous substrates and cofactors.[18] The rate limiting step in the formation of the NOS dimer is the oxidation of tetrahydrobiopterin (THB, also known as BH4). You need adequate amounts of THB, but you don’t want it oxidized.[19],[20] When the NOS enzyme becomes uncoupled, it disrupts the flow of electrons, and you reduce molecular oxygen to superoxide instead of NO.[21] If any of the cofactors become limiting, the production of NO also shuts down. But, if you can maintain a certain redox ratio of BH4 to dihydrobiopterin (BH2), you can maintain the coupled NOS structure and restore normal NOS production of NO.

What we focused on was that you have to recouple the enzyme, and basically prevent the oxidation of BH4.[22] Not just any antioxidant will do this, however. There is a proper redox potential for every reaction, the potential at which an electron can be transferred from one thing to another. The redox potential of this reaction and many of the antioxidants are coupled, and they are constantly transferring electrons from one to another, so it is really very tricky. We figured out how to recouple the NOS enzyme several years ago and make it functional even if people are taking things like PPIs.

Another thing worthy of note is how glutathione plays a role in the NO system. NO has a half-life of about one millisecond, so when its produced, it finds its target, it binds, and activates these second messenger systems and it does its job. But it also binds to sulfur residues or thiols.[23] So, things like glutathione, which contains a thiol within the cysteine residue in it, will bind NO forming S-nitrosoglutathione.[24] This molecule will transport and circulate NO, has a half-life of tens of minutes or hours, and is just as vasoactive as NO.[25] If you give S-nitrosoglutathione, it will dilate blood vessels and reduce blood pressure and does mostly what NO itself does.[26] In fact, there was some controversy when the Nobel prize was awarded for the discovery of NO – is it NO or a nitrosothiol that is responsible for these actions?

Glutathione binds NO forming S-nitrosoglutathione which transports and circulates NO and is just as vasoactive as NO. However, S-nitrosoglutathione has a half-life of tens of minutes or hours, making glutathione a very effective tool for increasing effectivity of other NO supportive supplements.

When NO binds to these thiols, whether they are on glutathione or proteins, it affects the structure and function of these proteins – that is how NO signals too. Once NO is bond to a thiol, antioxidants like ascorbate come in and can actually cleave or release that NO that was bound.[27] So, you are basically releasing NO from these preformed stores, and that is another way the body can generate NO – it is basically recapturing the activity of NO that has already been produced.

Other things also do this such as light therapy. Certain wavelengths of light like infrared or near infrared will release NO from metals, ultraviolet light will release NO from a thiol.[28],[29] A lot of the therapeutic benefits of low-level light therapy or infrared saunas these can be NO related effects – if your body makes sufficient NO you can basically activate these stored pools of NO and get its benefits. But, if you are NO deficient, these things may not work because you have to replete the stores first.

NutritionInFocus: Is there anything in closing that you’d like to say concerning the research you and others are doing surrounding NO?

Dr. Bryan: The fact that the oral bacteria can have such an impact on something systemic like blood pressure is a complete change in paradigm. We have 300 to 400 million people with hypertension, the number one modifiable risk factor for cardiovascular disease and related morality, many who are poorly managed on things like ACE inhibitors or other antihypertensives. What we know now is that they might not have problems with the systems we typically attribute high blood pressure; it may be a symptom of oral dysbiosis. If we can figure out how to harness these oral bacteria or generate NO in the oral cavity like the body is designed to do, we really can have an impact on non-responders to antihypertensive medications, lower their blood pressure, reduce all-cause mortality, and improve the quality of life for hundreds of millions of people. For me, this is exciting and really gives us a new target to come after for treating cardiovascular disease, dramatically impacting many other conditions as well.


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Are You Nitric Oxide Deficient? Part 1 of 2
Healthy Mouth, Healthy Body

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