Nitric oxide (NO) is a messenger molecule that plays a role in nearly every organ system in the body. Nitric oxide regulates hormone secretion, nutrient metabolism, cardiac contractility, smooth muscle relaxation, immune function, and cognitive health, and supports a healthy microbial balance. Healthy NO levels may promote athletic performance, as clinical trials have shown that NO increases blood flow and helps with healthy muscle contraction, oxygen supply to muscles, glucose uptake, muscle fatigue, mitochondrial respiration, and oxidative phosphorylation. 

One of NO’s primary functions is to serve as a vasodilator to promote increased blood supply to tissues and to relax the inner muscles of blood vessels. As a result, NO helps to promote normal blood pressure and blood flow, increasing the delivery of oxygen and nutrients to tissues and facilitating the clearance of metabolic by-products. Clinical studies suggest impaired NO production, activity, or bioavailability is associated with endothelial dysfunction in cardiovascular disease, hypertension, hyperlipidemia, and diabetes mellitus. 

Despite its vital functions for overall health, NO status naturally declines with age or in cases of certain metabolic conditions. Fortunately, throughout the aging process, the body utilizes two major pathways that work in tandem to maintain healthy NO homeostasis: the endogenous pathway and the enterosalivary pathway. The steps and required nutrients for the dual pathways of NO are explained below. 

The Endogenous Pathway – Requires L-arginine and healthy vascular endothelial structure and function. 

Nitric oxide is endogenously produced as a by-product of the oxidation of the amino acid L-arginine (Arg), catalyzed by endothelial nitric oxide synthase (eNOS) – an isoform of NO synthase (NOS) enzymes. Once produced, NO can form nitrosothiols or become oxidized to nitrite and nitrate, which can be recycled to regenerate NO, thus promoting sustained and healthy NO homeostasis. 

As Arg is a precursor to NO, it may increase NO bioavailability. Arg can be obtained from dietary sources or endogenous metabolism. Human studies suggest that Arg may help promote healthy blood pressure and may support athletic performance through its role in NO production. 

L-citrulline (Cit) can serve as an endogenous precursor to Arg, thereby increasing NO production. Some research suggests that Cit supplementation may be more effective than Arg supplementation in promoting healthy NO levels due to different metabolic pathways. Arginine undergoes gastrointestinal and hepatic extraction. Unlike Arg, Cit bypasses the metabolism by the intestine and liver, where it reaches the kidneys and gets converted into Arg. On the other hand, human and animal studies suggest that the combined effect of Cit and Arg supplementation may be more beneficial than each individual amino acid alone. 

The NOS enzymes require multiple nutrient cofactors – relying on the adequate nutritional status of riboflavin (vitamin B2), glutathione, niacin (vitamin B3), folate (promotes the cofactor tetrahydrobiopterin [BH4] bioavailability), and calcium. Thiamine may also modulate eNOS-dependent NO production. In rodent models, a thiamine deficiency led to reduced NO production and increased vascular dysfunction. 

Under healthy conditions, the endogenous pathway proceeds normally and can support proper NO status. However, during the aging process or in the presence of metabolic conditions, several problems can disrupt this pathway. One is insufficient nutrient cofactor availability. Two, a reduction in the expression of total eNOS. Lastly, limited L-arginine availability, transport, and conversion to NO due to eNOS enzyme uncoupling. 

Uncoupling of eNOS from electron transfer occurs as a result of cellular damage from oxidative stress or insufficient cofactor availability. If uncoupling occurs, eNOS switches from preferentially producing NO to, instead, generating superoxide, which contributes to oxidative stress and endothelial dysfunction. For instance, inadequate levels of BH4 (a cofactor for eNOS) may generate superoxide radicals from eNOS rather than NO. NOS-derived superoxide reacts with NO to produce highly reactive peroxynitrite radicals, which rapidly oxidize BH4 and trigger the uncoupling of NOS. Depletion of BH4 and uncoupling of NOS are associated with cardiovascular disease. 

Consequently, it is vital to support antioxidant status during the endogenous production of NO. Research suggests that supplementing with L-arginine and antioxidants, such as vitamin C and glutathione, may be more effective in promoting NO production and sustained NO release in the body than supplementing with Arg and Cit alone. In vivo and in vitro, the combination of glutathione and L-citrulline was shown to help promote plasma levels of NO. 

Due to the potential limitations of the endogenous pathway of NO production, the body simultaneously utilizes the second pathway, the enterosalivary pathway. 

The Enterosalivary Pathway – Requires dietary nitrates and a healthy oral microbiome. 

The enterosalivary pathway produces the same biologically active NO produced through the endogenous pathway but with dietary nitrite as the precursor rather than L-arginine. The enterosalivary pathway is associated with elevations in plasma nitrite and cyclic guanosine monophosphate (cGMP), a sensitive marker of bioactive NO production. This pathway may be a protective measure to compensate for insufficient eNOS activity or NO insufficiency. Additionally, this route is not impacted by age. Instead, it requires two necessary elements: one, the dietary intake of nitrates through food or supplements, and two, a healthy oral microbiome. 

The enterosalivary pathway starts when an exogenous source of inorganic dietary nitrate (NO3^-) is digested through nitrate-rich foods, such as beets and green, leafy vegetables. The gastrointestinal tract rapidly absorbs nitrate, where it enters circulation and is either excreted through the kidneys (~70%) or transported to the saliva glands (~25%), forming salivary nitrate. This explains why salivary nitrate levels are ten to twentyfold higher than in the blood. 

Next, salivary NO3^- is reduced to nitrite (NO2^-) by the action of nitrate reductases found in certain commensal oral bacteria, such as Rothia mucilaginosa, Haemophilus parainfluenzae, Neisseria flavescens, and Neisseria subflava. Therefore, healthy oral microbial balance is vital for proper NO conversion through the enterosalivary pathway. 

The saliva containing NO2^- is swallowed into the stomach, where it is reduced to NO by the acidic environment and in the presence of endogenous reductants, such as vitamin C. In addition to vitamin C, human studies suggest that berry polyphenolic compounds may promote NO production in the stomach. Interestingly, nitrite-derived NO may support the host immune defense and regulate gastric mucosal integrity. It is important to note that the conversion of nitrite into NO in the stomach is pH-dependent, where insufficient stomach acid may disrupt this process. 

Various clinical trials have investigated the general clinical benefits of dietary nitrates to promote healthy blood pressure (BP). A meta-analysis of 16 randomized, placebo-controlled trials with 254 participants examined the effects of dietary nitrate (between 157 mg and 1,488 mg per day) on BP. The researchers concluded that inorganic nitrate and beetroot juice consumption was associated with a statistically significant 4.4 mmHg reduction in systolic BP, with a non-significant, modest 1.1 mmHg decrease in diastolic BP. 

Supportive Nutrients for Both Pathways 

Recent literature has explored the role of hydrogen sulfide (H2S), a gaseous transmitter, to promote healthy NO homeostasis and vasodilation. H2S can be produced endogenously or obtained dietarily. Compounds in garlic, including allicin and S-allyl-L-cysteine, act as H2S donors. H2S may promote NO synthesis through eNOS-dependent and enterosalivary pathways to maintain elevated cGMP levels in several ways. First, it may promote eNOS function and the bioavailability of NO-derived cGMP. Second, H2S may promote the conversion of nitrite into NO by shifting the activity of xanthine oxidoreductase (XOR) enzymes to nitrite reductases to form NO. Third, H2S may also interact with oxidized forms of NO to produce stable intermediates that serve as reservoirs for NO, such as in the muscles. 

Vitamin C is necessary for collagen synthesis, endothelial health, and promoting antioxidant status. As a potent antioxidant, vitamin C may help attenuate the oxidative stress and inflammation associated with vascular dysfunction. Moreover, vitamin C helps the conversion of nitrite to NO in the enterosalivary pathway in the stomach. Vitamin C, alone or in combination with nitrate consumption, has been shown in clinical trials to support NO production, healthy BP, and endothelial health in older adults and individuals with hypertension, type 2 diabetes mellitus, or hypercholesterolemia. 

A small open-label clinical trial with 12 hypertensive participants aged 52 to 73 examined the effects of a supplement that contained dietary nitrates, black garlic extract, and vitamin C on NO bioavailability and blood pressure. After four weeks, the subjects exhibited a decrease in systolic BP by ~11 mmHg (p < 0.001) at two weeks, which persisted until the end of the trial. Diastolic BP decreased up to 11 mmHg (p < 0.01) in the patients with elevated diastolic BP, but no decrease was observed in the participants with normotensive diastolic BP. All 12 participants had consistent, rapid increases of salivary NO2^- concentrations (from fourfold to forty-fourfold times higher than baseline) at two hours post-administration, which slowly decayed but remained above baseline in all participants after 24 hours. This suggests combining these nutrients may promote sustained, healthy NO status, potentially up to 24 hours daily, for longer-lasting clinical benefits. However, these are preliminary results, and further research is required on this subject. 

Conclusion 

Promoting sustained NO production and bioavailability is vital for overall health, particularly for cardiovascular health, brain health, immune health, and supporting athletic performance. The body utilizes two complex yet important pathways to help ensure proper NO status. However, both pathways require specific precursors and nutrient cofactors for enhanced and sustained production. Supporting antioxidant status, promoting a healthy oral microbial environment, and consuming nitrate-rich food or supplements can help maintain healthy NO levels. 

By Danielle Moyer Male, MS, CNS, LDN