By Nathan S. Bryan, PhD, on Nitric Oxide, the Peroxynitrite Issue, and Nutritional Tools That May Help Improve Nitric Oxide Production
Nathan S. Bryan, PhD, 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 he 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 www.drnathansbryan.com.
FOCUS: 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?
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 in Physiology or Medicine having been 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.
Often, NO is present in disease-related processes. A good analogy is that of 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 present because it’s 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 to where superoxide and NO are forming, you will nitrate the tyrosine residue.[1],[2] Then, when you look for these fingerprints of where NO was, you’ll find things like nitrotyrosine, nitrite, nitrate, and a lot of other metabolites.[3] But, again, this is a sign of NO being present, not the cause of pathology.[4]
FOCUS: 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?
BRYAN: This is a good question, and it comes up a lot. However, I really believe this is a nonissue under normal physiology, and I’ll tell you why.
Peroxynitrite is a strong oxidant that leads to irreversible protein oxidation or nitration, and it’s 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, both of which are radicals, in close proximity to form peroxynitrite (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-), 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. Similarly, if you restore normal NO production, superoxide production goes down.[6],[7] 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, not the other, and therefore 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 provide NO or even nitrites, it inhibits the production of superoxide from NADPH oxidase.[8] The third main source of superoxide is uncoupled mitochondrial electron transport.[9] We know that both nitrite and NO recouple the electron transport chain and prevent electron leakage and superoxide production. So, peroxynitrite becomes a nonissue if you do things to restore NO production, thereby shutting down superoxide production.
FOCUS: What population or disease condition is most likely to benefit from increasing NO levels in the body?
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.[10] In fact, we see complete symptom relief in 10 minutes in Raynaud’s patients. Other forms of peripheral vascular disease, such as intermittent claudication, can also greatly benefit from improved NO production.
Some of the overlaps between cardiovascular disease and diabetes actually occur because the NOS enzyme becomes glycosylated, which shuts down NO production.[11] 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; 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 into cells that need them in these microenvironments.
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 is also often vascular dysfunction in autoimmunity[12]—multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus are examples of this—and NO also helps with this aspect.
FOCUS: How does exercise affect NO levels, and, conversely, can support for NO production improve athletic performance?
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.[13]
In young, healthy people with good endothelial function, the NOS 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 is really a test of their ability to produce NO—those who 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. But, 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.
When people become exercise intolerant, such as those who fail exercise stress tests, 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 some doctors immediately want to wheel you out and do bypass surgery to put metal in the arteries to open them up.
On the flip side, nutritional support for nitric oxide production can have an impact on exercise performance. You may recall that, during the 2012 Summer Olympics, there was a lot of media around competing athletes using beetroot 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.
FOCUS: 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?
BRYAN: We have to understand what goes wrong in the body of people who can’t make NO and fix that—that’s really what my 20 years of research have been about. Although we can give the body the things it needs to make NO, to treat the cause of the problem, we 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 (in Part 1 of this article[14]) is through the reduction of nitrate to nitrite to NO (see Figure 1). 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.
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 10 or more.[15] We’ve done food surveys where we tested foods from five cities in the U.S., and we found that, 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 beetroot.
The other pathway is via NOS—this enzyme converts L-arginine to NO (see Figure 1).[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]—supplementing with 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 how 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 the conversion of L-arginine to L-citrulline and NO in a complicated, five-electron, multi-step oxidation reaction requiring numerous substrates and cofactors.8 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.[18],[19] When the NOS enzyme becomes uncoupled, it disrupts the flow of electrons, and you reduce molecular oxygen to superoxide instead of NO.[20] 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.
Our focus was on the fact that you have to recouple the enzyme, and basically prevent the oxidation of BH4.[21] Not just any antioxidant will do this, however. For every reaction, there is a proper redox potential, 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 it’s produced, it finds its target, binds, activates second messenger systems, and does its job. But it also binds to sulfur residues or thiols.[22] So, things like glutathione, which contains a thiol within the cysteine residue in it, will bind NO, forming S-nitrosoglutathione.[23] This molecule will transport and circulate NO, has a half-life of tens of minutes or hours, and is just as vasoactive as NO.[24] S-nitrosoglutathione will dilate blood vessels, reduce blood pressure, and do mostly what NO itself does.[25] 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?
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 bonded to a thiol, antioxidants like ascorbate come in and can actually cleave or release the NO that was bound.[26] So, you are basically releasing NO from these preformed stores, which is another way the body can generate NO—it is essentially recapturing the activity of NO that has already been produced.
Other things, such as light therapy, also do this. Certain wavelengths of light, like infrared or near infrared, will release NO from metals; ultraviolet light will release NO from a thiol.[27],[28] A lot of the therapeutic benefits of low-level light therapy or infrared saunas 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 replenish the stores first.
FOCUS: Is there anything in closing that you’d like to say concerning the research you and others are doing surrounding NO?
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 of whom who are poorly managed on things like ACE inhibitors or other antihypertensives. What we know now is that these individuals might not have problems with the systems we typically attribute high blood pressure to; 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 can really have an impact on nonresponders to antihypertensive medications—lowering their blood pressure, reducing all-cause mortality, and improving the quality of life for hundreds of millions of people. For me, this is exciting, and it gives us a new target for treating cardiovascular disease and dramatically impacting many other conditions as well.
References
[1] Kaminsky DA, et al. Nitrotyrosine formation in the airways and lung parenchyma of patients with asthma. J Allergy Clin Immunol. 1999 Oct;104(4 Pt 1):747-54.
[2] Nakazawa H, et al. Nitrotyrosine formation and its role in various pathological conditions. Free Radic Res. 2000 Dec;33(6):771-84.
[3] Feelisch M, et al. Concomitant S-, N-, and heme-nitros(yl)ation in biological tissues and fluids: implications for the fate of NO in vivo. FASEB J. 2002 Nov;16(13):1775-85.
[4] Dijkstra G, et al. Expression of nitric oxide synthases and formation of nitrotyrosine and reactive oxygen species in inflammatory bowel disease. J Pathol. 1998 Dec;186(4):416-21.
[5] Aicardo A, et al. Biochemistry of nitric oxide and peroxynitrite: sources, targets and biological implications. In: Gelpi RJ, et al., eds. Biochemistry of Oxidative Stress. Basel, Switzerland: Springer International Publishing; 2016:49-77.
[6] Tejero J, et al. Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation. Physiol Rev. 2019 Jan 1;99(1):311-79.
[7] Bryan NS, et al. Discovery of the nitric oxide signaling pathway and targets for drug development. Front Biosci (Landmark Ed). 2009 Jan 1;14:1-18.
[8] Clancy RM, et al. Nitric oxide, an endothelial cell relaxation factor, inhibits neutrophil superoxide anion production via a direct action on the NADPH oxidase. J Clin Invest. 1992 Sep;90(3):1116-21.
[9] Brand MD, et al. Mitochondrial superoxide: production, biological effects, and activation of uncoupling proteins. Free Radic Biol Med. 2004 Sep 15;37(6):755-67.
[10] Freedman RR, et al. Acute effect of nitric oxide on Raynaud’s phenomenon in scleroderma. Lancet. 1999 Aug 28;354(9180):739.
[11] Xu B, et al. Impairment of vascular endothelial nitric oxide synthase activity by advanced glycation end products. FASEB J. 2003 Jul;17(10):1289-91.
[12] Faccini A, et al. Coronary microvascular dysfunction in chronic inflammatory rheumatoid diseases. Eur Heart J. 2016 Jun 14;37(23):1799-806.
[13] Uematsu M, et al. Regulation of endothelial cell nitric oxide synthase mRNA expression by shear stress. Am J Physiol. 1995 Dec;269(6 Pt 1):C1371-8.
[14] Allergy Research Group. Are You Nitric Oxide Deficient? Part 1 of 2. FOCUS Newsletter. Fall 2019;12-15.
[15] Nuñez de González MT, et al. A survey of nitrate and nitrite concentrations in conventional and organic-labeled raw vegetables at retail. J Food Sci. 2015 May;80(5):C942-9.
[16] Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med. 1993 Dec 30;329(27):2002-12.
[17] Böger RH. Asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, explains the “L-arginine paradox” and acts as a novel cardiovascular risk factor. J Nutr. 2004 Oct;134(10 Suppl):2842S-2847S.
[18] Kuzkaya N, et al. Interactions of peroxynitrite, tetrahydrobiopterin, ascorbic acid, and thiols: implications for uncoupling endothelial nitric-oxide synthase. J Biol Chem. 2003 Jun 20;278(25):22546-54.
[19] Landmesser U, et al. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest. 2003 Apr;111(8):1201-9.
[20] Förstermann U, Münzel T. Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation. 2006 Apr 4;113(13):1708-14.
[21] Heller R, et al. L-ascorbic acid potentiates endothelial nitric oxide synthesis via a chemical stabilization of tetrahydrobiopterin. J Biol Chem. 2001 Jan 5;276(1):40-7.
[22] Bryan NS, et al. Cellular targets and mechanisms of nitros(yl)ation: an insight into their nature and kinetics in vivo. Proc Natl Acad Sci U S A. 2004 Mar 23;101(12):4308-13.
[23] Hornyák I, et al. Current developments in the therapeutic potential of S-nitrosoglutathione, an endogenous NO-donor molecule. Curr Pharm Biotechnol. 2011 Sep;12(9):1368-74.
[24] Singh SP, et al. The chemistry of the S-nitrosoglutathione/glutathione system. Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14428-33.
[25] Park JW, et al. Transnitrosation as a predominant mechanism in the hypotensive effect of S-nitrosoglutathione. Biochem Mol Biol Int. 1993 Aug;30(5):885-91.
[26] Smith JN, Dasgupta TP. Kinetics and mechanism of the decomposition of S-nitrosoglutathione by l-ascorbic acid and copper ions in aqueous solution to produce nitric oxide. Nitric Oxide. 2000 Feb;4(1):57-66.
[27] Sexton DJ, et al. Visible light photochemical release of nitric oxide from S-nitrosoglutathione: potential photochemotherapeutic applications. Photochem Photobiol. 1994 Apr;59(4):463-7.
[28] Zhelyaskov VR, et al. Control of NO concentration in solutions of nitrosothiol compounds by light. Photochem Photobiol. 1998 Mar;67(3):282-8.
