Nathan Bryan, PhDBy Nathan S. Bryan, Ph.D. 

In 2006, total healthcare expenditures in the U.S. exceeded $2 trillion, or $6,700 per person. This trend is expected to increase over the coming years, reaching $4 trillion in 2015.  Currently, costs associated with chronic diseases such as obesity, diabetes, hypertension, coronary artery disease account for 75% of the nation’s annual healthcare costs.  According to the American Heart Association, an estimated 81 million people had one or more forms of cardiovascular disease in the U.S. in 2006, including hypertension, coronary artery disease, myocardial infarction, angina pectoris, stroke and heart failure. Most, if not all, of the chronic conditions mentioned above are the result of a dysfunctional endothelium and inability to produce NO and/or maintain NO homeostasis and signaling.  Understanding and developing new strategies to restore NO homeostasis will have a profound impact on public health and on the healthcare system. Read More >> 


By Nathan Bryan, Ph.D.

Nitric oxide (NO) is a gas produced naturally in the body and serves as one of the most important signaling molecules in mammalian physiology.  Specifically, NO is the endothelium derived relaxing factor (EDRF) first described by Furchgott in 1980 (Furchgott and Zawadzki 1980; Ignarro, Buga et al. 1987).  NO produced or generated in the vasculature then diffuses into the underlying smooth muscle causing these muscles to relax.  This results in vasodilation and an increase in blood flow and oxygen delivery.  

The discovery of the NO pathway represented a critical advance in the understanding of cell signaling and resulted in major advancements in many clinical areas including, but not limited to, cardiovascular medicine.  Indeed, this discovery was viewed as so fundamentally important that NO was named “Molecule of the Year” by Science in 1992 (Koshland 1992) and the Nobel Prize in Physiology or Medicine was awarded to its discoverers, Drs.  Louis J.  Ignarro, Robert Furchgott, and Ferid Murad in 1998, a short 11 years after NO was identified.  Since the discovery of the importance of NO, an enormous amount of research has been devoted to unraveling the complex chemistry and biochemistry of this relatively simple molecule.  To date, there have been over 120,000 published papers on NO.  NO is also implicated in the pathophysiology of many diseases, whereby either there is decreased bioavailability or production of NO, or there is an enormous, prolonged, over-production of NO that exposes its toxic, noxious properties (Moncada and Higgs 1993).  As a result, maintaining NO homeostasis is critical for optimal health and disease prevention and developing drugs or therapeutics to accomplish this will have a profound effect on public health.   

It is well documented that as we age, our body naturally decreases NO production. For instance, Taddei et al. demonstrated a gradual decline in endothelial function due to aging with greater than 50% loss in endothelial function in the oldest age group tested (Taddei, Virdis et al. 2001).  Egashira et al. reported even more dramatic declines, reporting a 75% loss of endothelium-derived NO in 70-80 year old patients compared to young, healthy 20 year olds (Egashira, Inou et al. 1993). Similarly Gerhard et al. concluded that age is the most significant predictor of endothelium-dependent vasodilator responses (Gerhard, Roddy et al. 1996).  Collectively, these studies indicate that NO production and therefore endothelium-dependent vasodilation declines progressively with age, and this abnormality is present in healthy adults with no other cardiovascular risk factors. It is also well-documented that some of the most prevalent diseases result, at least in part, from decreased NO availability, including hypertension, atherosclerosis, diabetes mellitus and hypercholesterolemia (Torregrossa, Aranke et al. 2011).  Moreover, as we age, vessels do not dilate as efficiently and are not as flexible, resulting in decreased circulation, and further demonstrating the need for a way to increase NO production in the body (Vita, Treasure et al. 1990; Egashira, Inou et al. 1993; Gerhard, Roddy et al. 1996; Taddei, Virdis et al. 2001; Vita and Keaney 2002).   

The first endogenous pathway to be discovered involved the oxidation of L-arginine to nitrite (Hibbs, Taintor et al. 1987).  It was only later realized that NO is an intermediate in that cycle (Marletta, Yoon et al. 1988).  Nitric oxide synthase (NOS) enzymes produce NO by catalyzing a five electron oxidation of the guanidino nitrogen of L-arginine (L-Arg).  This is a very complex reaction involving a number of co-factors and substrates including oxygen, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), nicotinamide adenine dinucleotide phosphate (NADPH), heme, tetrahydrobiopterin (BH4) and Ca2+-calmodulin (CaM).  Under healthy normal conditions, NO production typically proceeds quite efficiently.  However, if there is a problem with L-arginine uptake or L-arginine production from L-citrulline, or if any of the co-factors become limiting due to oxidative stress, NOS uncoupling or conditions of hypoxia where oxygen is limited, NO production from NOS shuts down and, in many cases, NOS produces superoxide instead (Vasquez-Vivar, Kalyanaraman et al. 2003).  For years scientists and physicians have investigated L-arginine supplementation as a means to enhance NO production.  This strategy has been shown to sometimes 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.  Patients with endothelial dysfunction, however, by definition, are unable to convert L-arginine to NO and, therefore, this strategy will not and cannot work.  In fact, L-arginine therapy in acute myocardial infarction: the Vascular Interaction With Age in Myocardial Infarction (VINTAGE MI), concluded that L-arginine, when added to standard postinfarction therapies, did not improve vascular stiffness measurements or ejection fraction and was associated with higher postinfarction mortality (Schulman, Becker et al. 2006).  L-arginine should not be recommended following acute myocardial infarction (MI).  Similarly, chronic dosing with L-arginine in peripheral artery disease (PAD) does not provide benefit and may do harm (Wilson, Harada et al. 2007).

A more recently described pathway utilizes nitrite and nitrate, rather than L-arginine, as the precursor to produce the same biologically active NO (Lundberg, Gladwin et al. 2009).  Specifically, research performed over the past decade made clear that nitrate and nitrite are physiologically recycled in blood and tissues to form NO and other bioactive nitrogen oxides (Bryan and Loscalzo 2011).  As a result, nitrate and nitrite are now viewed as storage pools to be acted upon when enzymatic NO production from NOS is insufficient.  Nitrite is derived both from a byproduct of the endogenous oxidation of NO described above, and both nitrite and nitrate are derived from the dietary sources like meat, vegetables and drinking water.  Nitrite and nitrate is reduced to NO in one of two ways.  In the first, nitrate from the diet is rapidly absorbed in the upper gastrointestinal tract.  In the blood, it mixes with the nitrate formed from the oxidation of endogenous NO produced from the NOS enzymes.  Although much of the nitrate is eventually excreted in the urine, up to 25% is actively taken up by the salivary glands and is concentrated up to 20-fold in saliva (Spiegelhalder, Eisenbrand et al. 1976; Lundberg and Govoni 2004).  Once in the mouth, commensal facultative anaerobic bacteria reduce nitrate to nitrite (Duncan, Dougall et al. 1995; Lundberg, Weitzberg et al. 2004).  When saliva enters the acidic stomach (1–1.5 L per day), much of the nitrite is rapidly protonated to form nitrous acid (HNO2; pKa 3.3), which decomposes further to form NO and other nitrogen oxides (Benjamin, O’Driscoll et al. 1994; Lundberg, Weitzberg et al. 1994).  A second method, the one-electron nitrite reduction to NO, can occur in a much simpler mechanism than the two-electron reduction of nitrate by bacteria.  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-. 

Understanding the endogenous pathway for reducing nitrate and nitrite back to NO provides a new paradigm for restoring NO homeostasis.  However, the stepwise reduction of nitrate to nitrite to nitric oxide can be an inefficient process, which may be further hindered by oxygen (Feelisch, Fernandez et al. 2008; Lundberg, Weitzberg et al. 2008).  The reservoir of nitrite and nitrate is further reduced in humans with known cardiovascular risk factors and endothelial dysfunction (Kleinbongard, Dejam et al. 2006).  Recognizing this limitation, we sought to identify natural products that enhance the efficiency of the nitrate/nitrite pathway.  To do so, we assessed over 400 food and natural products and screened over 100 herbs that have been used for many years in traditional medical practices in Europe and Asia.  This research was conducted over 12 years at the University of Texas Health Science Center in Houston.  The results of this research identified a number of NO active herbs and natural products food products that are enriched in nitrate and provide the substrate nitrite in the pathway, as well as a number of herbs with very robust nitrite reductase activity that are unaffected by oxygen (Tang, Garg et al. 2009).  Additional experiments revealed that hawthorn berry has the most robust and effective nitrite reductase activity of any herb tested to date and is also unaffected by oxygen.   

Based on these discoveries and observations, several patents were filed and issued to Dr. Nathan S, Bryan and the University of Texas Board of Regents (US patents 8,298,589  8,303,995 & 8,435,570).  This technology was commercialized by Neogenis Labs, Inc. and manufactured into an orally disintegrating tablet that utilizes the patented composition of matter for generating NO.  Neogenis Laboratories licensed this technology and developed NEO40™, a GMP certified, over the counter, all natural formulation that provides a system for generating NO in an endothelium-dependent and independent manner.  Clinical studies using Neo40 found that it could modify cardiovascular risk factors in patients over the age of 40, significantly reduce triglycerides, and reduce blood pressure (Zand, Lanza et al. 2011).  This same lozenge was used in a pediatric patient with argininosuccinic aciduria and significantly reduced his blood pressure when prescription medications were ineffective (Nagamani, Campeau et al. 2012).  These new innovations in NO therapeutics offer significant advancements in our understanding on how to safely and effectively deliver NO in humans to combat chronic disease.   

Please join Nathan Bryan, PhD at the 2014 Integrative Healthcare Symposium for the following presentation:
Revolutionary Advancement in Nitric Oxide(NO): Renewal beyond L-Arginine. The Premise for Health, Cellular Anti-Aging, Prevention & Disease Management
Friday, February 21, 2014 from 7:45pm- 8:30pm
**Please note: Continuing Medical Education credits and Continuing Education credits are not available for this session

For further information or research, please visit: and/or call 512-732-2240.

Neogenis Labs is dedicated to groundbreaking scientific research and continuous product innovations, bringing the power of nitric oxide (NO) to the medical community, athletes, and the world. We are fast becoming recognized as the definitive experts in nitric oxide, on the leading edge, but supported with ironclad patented science. Our scientists, have spent more than 16 years studying how to effectively restore natural nitric oxide production in the human body. With the highest standards in scientific innovation and product development, we lead the market’s adoption of nitric oxide as a powerful positive force in human health.  This research has led to the development of superior and safe patented products , backed by several Neogenis clinical trials. Our pledge to excellence is validated by two recently awarded N-O patents and 5 additional patents-pending. 

*Visit Neogenis Labs at the 2014 Integrative Healthcare Symposium from February 21-22 at
Booth #237


Benjamin, N., F. O’Driscoll, et al. (1994). “Stomach NO synthesis.” Nature 368(6471): 502. 

Bryan, N. S. and J. Loscalzo, Eds. (2011). Nitrite and Nitrate in Human Health and Disease. Nutrition and Health. New York, Humana Press. 

Duncan, C., H. Dougall, et al. (1995). “Chemical generation of nitric oxide in the mouth from the enterosalivary circulation of dietary nitrate.” Nat Med 1(6): 546-551. 

Egashira, K., T. Inou, et al. (1993). “Effects of age on endothelium-dependent vasodilation of resistance coronary artery by acetylcholine in humans.” Circulation 88(1): 77-81. 

Feelisch, M., B. O. Fernandez, et al. (2008). “Tissue Processing of Nitrite in Hypoxia: An Intricate Interplay of Nitric Oxide-Generating and -Scavenging Systems.” J Biol Chem 283(49): 33927-33934. 

Furchgott, R. F. and J. V. Zawadzki (1980). “The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetycholine.” Nature 288(5789): 373-376. 

Gerhard, M., M. A. Roddy, et al. (1996). “Aging progressively impairs endothelium-dependent vasodilation in forearm resistance vessels of humans.” Hypertension 27(4): 849-853. 

Hibbs, J. B., Jr., R. R. Taintor, et al. (1987). “Macrophage cytotoxicity: role for L-arginine deiminase and imino nitrogen oxidation to nitrite.” Science 235(4787): 473-476. 

Ignarro, L. J., G. M. Buga, et al. (1987). “Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide.” Proc. Natl. Acad Sci. USA 84: 9265-9269. 

Kleinbongard, P., A. Dejam, et al. (2006). “Plasma nitrite concentrations reflect the degree of endothelial dysfunction in humans.” Free Radic Biol Med 40(2): 295-302. 

Koshland, D. E., Jr. (1992). “The molecule of the year.” Science 258(5090): 1861. 

Lundberg, J. O., M. T. Gladwin, et al. (2009). “Nitrate and nitrite in biology, nutrition and therapeutics.” Nat Chem Biol 5(12): 865-869. 

Lundberg, J. O. and M. Govoni (2004). “Inorganic nitrate is a possible source for systemic generation of nitric oxide.” Free Radic Biol Med 37(3): 395-400. 

Lundberg, J. O., E. Weitzberg, et al. (2004). “Nitrate, bacteria and human health.” Nat Rev Microbiol 2(7): 593-602. 

Lundberg, J. O., E. Weitzberg, et al. (2008). “The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics.” Nat Rev Drug Discov 7(8): 156-167 

Lundberg, J. O., E. Weitzberg, et al. (1994). “Intragastric nitric oxide production in humans: measurements in expelled air.” Gut 35(11): 1543-1546. 

Marletta, M. A., P. S. Yoon, et al. (1988). “Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate.” Biochemistry 27(24): 8706-8711. 

Moncada, S. and A. Higgs (1993). “The L-arginine-nitric oxide pathway.” N Engl J Med 329(27): 2002-2012. 

Nagamani, S. C., P. M. Campeau, et al. (2012). “Nitric-oxide supplementation for treatment of long-term complications in argininosuccinic aciduria.” Am J Hum Genet 90(5): 836-846. 

Schulman, S. P., L. C. Becker, et al. (2006). “L-arginine therapy in acute myocardial infarction: the Vascular Interaction With Age in Myocardial Infarction (VINTAGE MI) randomized clinical trial.” Jama 295(1): 58-64. 

Spiegelhalder, B., G. Eisenbrand, et al. (1976). “Influence of dietary nitrate on nitrite content of human saliva: possible relevance to in vivo formation of N-nitroso compounds.” Food Cosmet Toxicol 14(6): 545-548. 

Taddei, S., A. Virdis, et al. (2001). “Age-related reduction of NO availability and oxidative stress in humans.” Hypertension 38(2): 274-279. 

Tang, Y., H. Garg, et al. (2009). “Nitric oxide bioactivity of traditional Chinese medicines used for cardiovascular indications.” Free Radic Biol Med 47(6): 835-840. 

Torregrossa, A. C., M. Aranke, et al. (2011). “Nitric oxide and geriatrics:  Implications in diagnostics and treatment of the elderly.” Journal of Geriatric Cardiology 8: 230-242. 

Vasquez-Vivar, J., B. Kalyanaraman, et al. (2003). “The role of tetrahydrobiopterin in superoxide generation from eNOS: enzymology and physiological implications.” Free Radic Res 37(2): 121-127. 

Vita, J. A. and J. F. Keaney, Jr. (2002). “Endothelial function: a barometer for cardiovascular risk?” Circulation 106(6): 640-642. 

Vita, J. A., C. B. Treasure, et al. (1990). “Coronary vasomotor response to acetylcholine relates to risk factors for coronary artery disease.” Circulation 81(2): 491-497. 

Wilson, A. M., R. Harada, et al. (2007). “L-arginine supplementation in peripheral arterial disease: no benefit and possible harm.” Circulation 116(2): 188-195. 

Zand, J., F. Lanza, et al. (2011). “All-natural nitrite and nitrate containing dietary supplement promotes nitric oxide production and reduces triglycerides in humans.” Nutr Res 31(4): 262-269.