The Noncanonical Pathway for In Vivo Nitric Oxide Generation: The Nitrate-Nitrite-Nitric Oxide Pathway

1. Dietary Intake

The predominant dietary source of nitrate comes from vegetable intake. Using the World Health Organization (WHO) recommended intake of mixed vegetables of 400 g daily as a guide would lead to a total daily nitrate intake of ∼2.5 mmol, although in reality, most people consume less than this, with estimates at 1.5–2 mmol (or 93–124 mg) nitrate daily (World Cancer Research Fund/American Institute for Cancer Research, 2007; European Food Safety Authority, 2008). However, in vegetable-rich diets, such as the proposed diet based upon the Dietary Approaches to Stop Hypertension (DASH) study, estimates suggest that intake levels can easily be increased to somewhere in the region of 6–20 mmol daily (Hord et al., 2009). Other dietary patterns, such as the traditional Japanese diet, may also have significantly higher nitrate intakes (Sobko et al., 2010) compared with European or United States diets. This variation in intake is driven by the differences in nitrate levels found in vegetables. In general, large green leafy vegetables contain the largest amounts of nitrate per gram (European Food Safety Authority, 2008), and this source is thought to be responsible for the elevated amounts consumed within the traditional Japanese diet. Importantly, because of concerns associated with toxicity (see later), the WHO has set an acceptable daily intake (ADI) recommendation for dietary nitrate at 3.7 mg/kg daily, which equates to ∼4 mmol nitrate daily for an average 70-kg person (European Food Safety Authority, 2008).

2. Water Supplies

Because of historical concerns regarding toxicity from nitrate in drinking water supplies, mostly concerning the risk of infantile methemoglobinemia (see later), there exists regulatory control of the nitrate level in water at <50 mg/l (U.S. Public Health Service, 1962). The levels of nitrate in drinking water derived from surface water do not exceed 10 mg/l in most countries. In such conditions, the contribution of drinking water to nitrate intake is usually less than 14% (World Health Organization, 2011), and therefore, vegetables are the main source of nitrate intake (Chilvers et al., 1984; European Centre for Ecotoxicology and Toxicology of Chemicals, 1988).

In some particularly agricultural areas, however, concentrations of nitrate in drinking water are higher because of runoff and the discharge of sewage effluent and certain industrial wastes. In 15 European countries, evidence was shown indicating that the percentage of the population exposed to nitrate levels in drinking water above 50 mg/l ranged from 0.5% to 10% (European Centre for Ecotoxicology and Toxicology of Chemicals, 1988). Individual wells in agricultural areas throughout the world often contain drinking water with nitrate levels exceeding 50 mg/l (World Health Organization, 2011), and in such circumstances, drinking water will be the major source of total nitrate intake, especially for bottle-fed infants (World Health Organization, 2011).

1. Prokaryotic Nitrate Reductases

The nitrogen cycle details the chemical transformations that recycle nitrogen between the atmosphere, biosphere, and hydrosphere. The most reduced form of nitrogen is ammonia, the oxidized form of nitrogen, whereas the most oxidized form is nitrate. Microbes drive the nitrogen cycle, as they use an assortment of redox reactions to metabolize nitrogen for energy transduction, detoxification, or assimilation (Stein and Klotz, 2016). These microbial reactions are essential to most Terran life, as they are essential to maintain bioavailability of nitrogen.

Many bacteria readily reduce nitrate to nitrite via nitrate reductases. All prokaryotic nitrate reductases are molybdenum-dependent enzymes. They are traditionally separated into three major groups: membrane-bound periplasmic nitrate reductase (Nap), membrane-bound respiratory nitrate reductase (Nar), and the cytoplasmic, assimilatory nitrate reductase (Nas) (Sparacino-Watkins et al., 2014). Nitrate is retained in soils, sediments, and water, providing a biologically useful pool for many bacteria. Enzymatic reduction to nitrite generates enormous energy (Moreno-Vivián et al., 1999), which the organisms use to maintain homeostatic processes and life.

2. Mammalian Nitrate Reductase Activity

Despite the fact that it is accepted that mammals, in the main, lack the enzymes capable of undertaking nitrate reduction to nitrite and are therefore reliant on functioning oral microflora, there is some evidence that mammalian nitrate reductases may have a role. Although antibacterial mouthwash treatment in humans totally abrogated ex vivo nitrate reduction in saliva incubated with 1 mmol/l nitrate solution and prevented any increase in saliva nitrite levels in vivo, there was still a small increase in plasma nitrite level after an inorganic nitrate load (Govoni et al., 2008). Although there could be nitrate reduction from lower GI commensals, an alternative explanation of mammalian nitrate reduction has also been postulated that is particularly focused on XOR.

XOR demonstrates nitrate-reduction activity in vitro under anoxic conditions that can be abrogated by XOR inhibition (Li et al., 2003). Furthermore, in normoxic wild-type rats, increases in plasma nitrite are partially abrogated after nitrate supplementation by XOR inhibition (Jansson et al., 2008). Similar results were demonstrated in endothelial ·NO synthase (eNOS)-deficient and germ-free mice, thereby excluding bacterial nitrate reduction and vascular ·NO synthase (NOS) activation as the source of nitrite (Jansson et al., 2008). Germ-free mice exhibited greater tissue levels of XOR, suggesting that this may represent a functional compensatory response to uphold nitrate production in the absence of a commensal microflora (Huang et al., 2010). These studies seem to confirm much earlier studies on ex vivo mammalian liver/muscle homogenates suggesting that XOR may exhibit nitrate-reducing capability (Bernheim and Dixon, 1928; Ward et al., 1985, 1986). In contrast, very recently, Moretti et al. (2019) have published findings suggesting a lack of mammalian nitrate reductase activity in germ-free mice in a separate study, indicating some heterogeneity in studies with animals rendered “germ-free.” Irrespective, the presence and importance of putative nitrate reductases in human physiology is yet to be firmly established.

1. Molybdenum-Containing Oxidase Enzymes

In mammals, there are four known molybdenum-containing enzymes: XOR, sulfite oxidase (SO), aldehyde oxidase (AO), and mitochondrial amidoxime reductase (mARC) (Fig. 6). Although all have been shown to function as a nitrite reductase to various degrees, XOR has been identified as perhaps the major mammalian nitrite reductase and certainly is the most prominent molybdenum-containing enzyme responsible for nitrite bioactivation.

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Fig. 6.

The primary sequence structure of the four known mammalian molybdenum-containing enzymes. Numbers denote the first and last residues of the domains highlighted (data obtained from NCBI/Protein). aa, amino acid; Cyt, cytochrome b₅; FE-S, iron-sulfur; Moco, molybdenum cofactor; NCBI, National Center for Biotechnology Information; XO, xanthine oxidase.

2. Mitochondrial Respiratory Chain Enzymes

mARC is not the only mitochondrial component capable of reducing nitrite to ·NO. Experiments with rat liver homogenates demonstrate that inhibition of complex I or II leads to an attenuation of nitrite-derived ·NO (NaNO₂ 50 µmol/l) at pH 7.25 and under anoxic conditions (Kozlov et al., 1999). It was suggested that inhibition of complex I or II will prevent electron flow to complex III, which has been implicated as the site of mitochondrial nitrite reduction. As such, inhibition of complex III (also known as cytochrome bc₁ complex/cytochrome c reductase) completely abolished nitrite reduction (Kozlov et al., 1999; Nohl et al., 2000). There is also evidence to suggest that complex IV (cytochrome c oxidase) may reduce nitrite to ·NO. Treatment of rat liver homogenate with either antimycin A or myxothiazol (both complex III inhibitors) in the presence of the electron donor N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride, which feeds electrons downstream of complex III of the respiratory chain, did not block nitrite reduction (Castello et al., 2006). Furthermore, carbon monoxide, a complex IV inhibitor, inhibited nitrite reduction (Castello et al., 2006). However, NaNO₂ (1 mmol/l) reduction in this in vitro setting was only evident when experiments were conducted at O₂ concentrations below 2% being maximal under anoxic conditions (Castello et al., 2006). The physiologic relevance of this phenomenon likely pertains only to the ischemic setting, and further investigation assessing the impact of this pathway upon biologic function is needed to better understand significance.

3. Globins

The globins are a family of globular heme-containing proteins that are involved in the transport of O₂; however, an abundance of literature supports a role for these proteins in facilitating nitrite reduction. In particular, we summarize below the evidence for Hb, Mb, neuroglobin, and cytoglobin as nitrite reductases.

4. Indoleamine 2,3-Dioxygenase 1

Very recently, it has been demonstrated that indoleamine 2,3-dioxygenase 1 (IDO1), a cytosolic heme enzyme involved in the initial and rate-limiting step of l-tryptophan metabolism in the kynurenine pathway, is capable of reducing nitrite to ·NO under anaerobic conditions (Lim et al., 2019). Similar to Mb and Hb, the heme in IDO1 is in a penta-coordinate state, which may account for the increased rates, as discussed above. A contrasting feature, in comparison with other heme-containing nitrite reductases, is that the nitrite reductase activity of IDO1 is enhanced in alkaline conditions (pH 8), which Lim and colleagues speculate may be due to the close proximity of histidine residues potentially capable of donating a proton (Lim et al., 2019). These initial observations have been made in recombinant human IDO1, and further studies are required to assess the in vivo relevance of these findings.

5. Nitric Oxide Synthase

Under anoxic conditions, it has been demonstrated that endothelial NOS can function as a nitrite reductase (Gautier et al., 2006; Vanin et al., 2007). Recombinant eNOS in the presence of anoxia, with NADPH (100 μmol/l) and NaNO₂ (500 μmol/l) at pH 7.6, resulted in ferrous-nitrosyl eNOS formation and then, consequently, ·NO generation (Gautier et al., 2006). This proposal was supported by studies using ¹⁵N-labeled NaNO₂ with EPR measurements of ¹⁵·NO (Gautier et al., 2006). Subsequently, studies with recombinant iNOS and neuronal NOS have suggested that this activity is unique to the eNOS isoform, since neither isoform generated ·NO from relatively high concentrations of NaNO₂ (500 µmol/l) under anoxic conditions generated with argon (Mikula et al., 2009). In addition, we have shown that erythrocytes, collected from healthy individuals, when incubated with physiologic concentrations of NaNO₂ (10–100 μmol/l) under hypoxic conditions, generate ·NO; a response that is attenuated by the eNOS inhibitors N(γ)-nitro-l-arginine methyl ester (L-NAME) and NG-monomethyl-l-arginine acetate (300 μmol/l) (Webb et al., 2008a). The identification of eNOS as a nitrite reductase under hypoxia and anoxia is an interesting finding and suggests that this enzyme, utilizing distinct substrates dependent upon the environmental conditions, may be equipped to produce ·NO under the full spectrum of oxygen tension that is experienced in vivo.

6. Carbonic Anhydrase

More recently it has been suggested that carbonic anhydrase, a key component involved in acid-base regulation in the body through the hydration of CO₂ to produce protons and thus H₂O and bicarbonate (HCO₃⁻) (eq. 1.8), also acts as a nitrite reductase, particularly in metabolically active tissue. Hydration of CO₂ is as follows:(1.8)In 2004 work from Innocenti et al. (2004) demonstrated that anions, including nitrite, could bind to the active site of carbonic anhydrase and, in this way, inhibit CO₂ hydration (Innocenti et al., 2004). However, it was not until 2009 that the possibility that this interaction of nitrite with the enzyme might actually yield ·NO too was considered. Addition of NaNO₂ (100 μmol/l) to purified carbonic anhydrase at pH 7.2 resulted in ·NO generation, an effect that was potentiated at pH 5.9 (Aamand et al., 2009). Addition of dorzolamide (250 μmol/l), an inhibitor of the hydration of CO_2, resulted in a significant elevation of ·NO generation (Aamand et al., 2009). The explanation for the inhibitory activity for CO₂ hydration but not nitrite reduction was suggested by the authors to imply that distinct binding sites for the two exist, with the inhibitors only binding to the active Zn²⁺ binding site for CO₂. The reactions proposed to underlie this are shown previously (eqs. 1.4–1.6) with a theoretical N₂O₃ intermediate formed (the levels of which are increased with reducing pH) that rapidly dissociates to give ·NO (along with an equivalent of NO₂).

This nitrite-reducing activity of carbonic anhydrase was tested for functional importance using rat aorta segments treated with NaNO₂ (10 μmol/l) with and without dorzolamide. The studies showed, as with the purified enzyme, an increase (∼50%) in the functional relaxation response to NaNO₂ (Aamand et al., 2009). However, using EPR and measurement of nitrosyl Hb as a readout, inhibition of carbonic anhydrase using dorzolamide did not affect erythrocyte nitrite reduction (Liu et al., 2015). Recently, it has also been shown that human platelets treated with NaNO₂ (100 μmol/l) showed no change in cGMP or phospho-vasodilator–stimulated phosphoprotein (phospho-VASP)^S239, a marker of protein kinase G activity; however, with the addition of recombinant bovine carbonic anhydrase, there was a significant elevation in both markers (Hanff et al., 2016). In healthy volunteers, treatment with dorzolamide enhanced NaNO₂-induced vasodilation of the radial artery (Omar et al., 2015) and a similar BP-lowering effect was seen in healthy volunteers treated with acetazolamide and NaNO₂ (Rosenbaek et al., 2018). In contrast, more recently, a re-evaluation of the nitrite reductase activity of carbonic anhydrase has suggested little activity. Incubation of purified bovine carbonic anhydrase at pH 5.9 with 100 μmol/l NaNO₂ demonstrated neither N₂O₃ nor ·NO generation (Andring et al., 2018).

Although it is clear that there are numerous mammalian nitrite reductases that have been identified in cell-free and cellular preparations, in WT and transgenic animals, and in humans, the importance of these discoveries lies within the translational potential for enhanced ·NO delivery into real benefits in health and disease. Below we discuss the major therapeutic areas in which targeting and translation of the noncanonical pathway for ·NO in humans and patients has been investigated.

1. Vasodilation

Although it had been long since demonstrated that once-acidified supraphysiologic concentrations of inorganic nitrite exerted significant vasodilator responses (Furchgott and Bhadrakom, 1953), it was only more recently in 2001 that the view that nitrite was inert at physiologic levels within the CV system was finally eroded. In rat aortic rings, application of low micromoles per liter NaNO₂ (2.5 μmol/l), although inactive under physiologic pH, was shown to cause significant relaxation of precontracted rat aorta under acidic (pH 6.6) conditions (Modin et al., 2001). The importance of this observation was finally appreciated with demonstration by Gladwin and colleagues that infusion of NaNO₂ into the forearm of healthy volunteers causes vasodilatation with concomitant increased forearm blood flow, a phenomenon augmented when the volunteers were asked to exercise by conducting handgrips (Cosby et al., 2003). The authors proposed that this effect of exercise to increase nitrite-induced vasodilation related to the fact that conversion of nitrite to ·NO is enhanced under reduced O₂ conditions. This observation simply and elegantly indicated that nitrite exerts significant biologic effects in the relative hypoxia created by conditions relevant to the normal and typical ranges in O₂ tension that occur in day-to-day activity. These findings intimated a potentially important role of this anion in sustaining/regulating normal vasodilator tone in the healthy individual, particularly since, in the normal passage of blood through the circulation, O₂ tension will drop from 100 mm Hg as it exits the heart to 40 mm Hg when it returns via the venous circulation.

The thesis that O₂ tension determines the contribution that nitrite plays in regulating vascular tone was further developed by studies conducted by Frenneaux and colleagues, who used radiolabeled, autologous blood together with standard forearm plethysmographic techniques, thus enabling the separate determination of arterial and venous vasodilation. Vascular reactivity was measured over a 20-minute infusion period. Under normoxic conditions, intra-arterial delivery of increasing doses of NaNO₂ (314 nmol/min to 7.84 μmol/min) decreased venous tone in a dose-dependent manner by up to 20%–35% (Maher et al., 2008). However, in contrast to Gladwin and colleagues’ earlier works (Cosby et al., 2003), low-dose NaNO₂ infusions (314 nmol/min), associated with ∼30% increases in forearm blood flow, did not dilate the arterial side of the circulation (Maher et al., 2008). Indeed, arterial dilation was only apparent (increasing forearm blood flow by 60%–80%) at much higher doses (3.14–7.84 μmol/min) (Maher et al., 2008). These findings replicate much earlier work that used detailed tilt-table testing to elucidate the role of dilation of the venous circulation (and not arterial circulation) as the cause of nitrite-induced CV collapse (Weiss et al., 1937; Wilkins et al., 1937). However, to make the arterial side of the circulation “hypoxic,” subjects were put through the protocol again, but only after arterial oxygen saturations were maintained at 83%–88% by breathing 12% O₂. In this situation, although there was no change in the magnitude or duration of the effects on the venous side of the circulation, infusion of 314 nmol/min NaNO₂ (which had no effect in normoxic conditions) increased forearm blood flow by ∼40% (Maher et al., 2008). Such a finding is consistent with the concept that the vasodilator potential of nitrite is not solely limited to anoxia or extreme ischemia but is proportional to the extent of “hypoxia” in the tissues and blood as it deoxygenates from arterial to venous sides.

More recently, the effect of nitrite on muscular, conduit blood vessels has been shown. Infusion of a single dose of NaNO₂ (8.7 µmol/min) for 60 minutes into the brachial artery caused an ∼30% increase in radial artery diameter (Omar et al., 2015). Comparison of the dose-response relationship of NaNO₂ to GTN demonstrated comparable effects. For example, with an infusion of NaNO₂ of 26 μmol/min (1.8 mg/min) for 20 minutes, a statistically significant ∼30% increase in diameter was evident, a magnitude of response similar to that produced in the same healthy volunteers by GTN 1.3 nmol/min, which is equivalent to 0.3 μg/min (Omar et al., 2015). Furthermore, similar effects have been seen in the coronary circulation (O’Gallagher et al., 2018). Paradoxically the effect in the peripheral circulation was most pronounced in normoxia and inhibited by hypoxia or hyperoxia (Omar et al., 2015), an observation that conflicts with the findings previously discussed. Exactly why this is the case is uncertain.

Physiologically, the role of nitrite as an endocrine storage form of ·NO that is released after a decrease in O₂ tension and is thus responsible for hypoxic vasodilatation has been investigated. Hypoxic vasodilation is responsible for matching O₂ demand from respiring tissues to O₂ delivery; thus, when metabolic demand rises, O₂ consumption increases, and with this, a mild hypoxia develops that then triggers vasodilation to increase O₂ availability to the organ. Erythrocytes have been identified as a key hypoxic sensor acting as a source of ·NO delivery that underlies the biologic effect (Segal and Duling, 1986; Stamler et al., 1997). However, two competing theories for the form of ·NO that is transported and stored ready to deliver ·NO on demand have been proposed: nitrite and S-nitrosothiols. Arterial-venous gradients of nitrite (Gladwin et al., 2000) and nitrosothiols (Stamler et al., 1992), and the formation of iron-nitrosylHb or S-nitroso-Hb as markers of consumption and ·NO delivery have been measured in the peripheral and cerebral circulation, with exercise and under hypoxia. These studies demonstrate clear evidence of arterial-venous gradients for nitrite and formation of iron-nitrosylHb only, suggesting a role for nitrite as an endocrine reservoir of ·NO activity (Gladwin et al., 2000; Bailey et al., 2017).

It is likely that nitrite-induced vasodilatation is dependent upon activation of the canonical ·NO-dependent GC-1 pathway, since the GC-1 inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) attenuates responses to nitrite in blood vessels of rodents and pigs (Botden et al., 2012; Ghosh et al., 2013). It is noteworthy that these observations recapitulate much earlier studies using drugs such as methylene blue to block GC-1 activity (Mittal et al., 1978; Ignarro and Gruetter, 1980; Gruetter et al., 1981); however, such observations have not been observed in all vascular beds tested. Recent studies in the renal microvasculature of the mouse demonstrate nitrite is two orders of magnitude more potent than in other vascular beds (Gao et al., 2015). Interestingly, ·NO scavenging abolished nitrite-induced vasodilatation, but treatment with ODQ did not, indicating a GC-1-independent effect. In the same study, inorganic nitrate supplementation reduced NADPH oxidase activity, a prime source of oxidative stress, and the actions of NADPH oxidase inhibition were synonymous and not additive. These results suggest that NADPH oxidase, at least in the renal microvasculature, could be a mechanistic target for nitrite-derived ·NO and associated BP lowering through reduction in oxidative stress (Gao et al., 2015).

Furthermore, there may be additional effects on vascular tone that are affected by nitrite-derived ·NO that are centrally driven. Elevation of systemic nitrite levels reduces renal sympathetic nerve signaling in rats under L-NAME or angiotensin II–driven hypertension. This treatment also reduces muscle sympathetic nerve activity in humans, a gold-standard measure of efferent sympathetic signaling (Notay et al., 2017; Guimarães et al., 2019). The precise mechanism of nitrite-derived ·NO sympatholysis is not known but may relate to alterations in expression of angiotensin II receptors in the rostral ventral lateral medulla, the brainstem sympathetic control center (Guimarães et al., 2019), or to changes in peripheral chemoreflex activity. The peripheral chemoreflex is primarily responsible for augmenting ventilation in response to hypoxia but is known to be augmented in human hypertension in the absence of hypoxia and responsible for elevated BP through efferent central sympathetic signaling (Marshall, 1994), and this mechanism is attenuated after inorganic nitrate supplementation in older adult humans (Bock et al., 2018b).

Irrespective of the precise mechanism, these reported effects of nitrite on vascular tone raise the possibility that systemic nitrite administration or dietary nitrate supplementation, as a means to elevate circulating levels of nitrite, may be useful in the treatment of hypertension.

2. Blood Pressure

These demonstrations of the vasodilator potential of nitrite are also concordant with the effects of nitrite supplementation in normotensive animals, as well as in animal models of hypertension. In anesthetized rats, administration of NaNO₂ (10–1000 μmol/kg, i.v.) was associated with dose-dependent reductions in MAP for up to 30 minutes (Vleeming et al., 1997), observations we ourselves have reproduced in conscious tethered rats (Ghosh et al., 2013). In free-moving rats, supplementation of drinking water with extremely high concentrations of NaNO₂ (36 mmol/l equivalent to 2.48 g/l) reduced BP measured by telemetry (Vleeming et al., 1997). If we consider that a typical Sprague-Dawley or Wistar averaged-sized adult rat (∼250 g) will drink 30–50 ml of water daily, this would give a maximum daily dose of NaNO₂ of 124 mg, which in turn, would approximate to 500 mg/kg, a level exceeding the ADI by 135 times. Nevertheless, these early studies clearly demonstrate BP-lowering potential.

Similarly, in spontaneously hypertensive rats, acute bolus doses of KNO₂ (1–30,000 nmol/kg, equivalent to ∼10 nmol to 0.3 mmol/l circulating concentrations) (Ghosh et al., 2013), as well as prolonged oral administration (up to 1 year) of extremely high concentrations of NaNO₂ (50–100 mmol/l equating to 3.5–6.9 g/l in drinking water), were associated with dose-dependent reductions in BP (Beier et al., 1995; Haas et al., 1999). These observations indicate two important issues: firstly, that the efficacy of nitrite was still evident in the setting of raised BP [indeed, our studies indicated a substantially enhanced potency of nitrite in this setting (Ghosh et al., 2013)], and secondly, that significant BP lowering was evident even after 1 year of ingestion. This latter observation hints at a key characteristic of nitrite, and potentially nitrate, that might endow these anions with a superior potential for therapeutics over the organic nitrates, i.e., no development of tolerance, which is the key limiting factor in the use of organic nitrates clinically (Münzel et al., 2005). This was tested more formally by Dejam et al. (2007), in which NaNO₂ (12.5 μg/kg per minute) was infused in primates continuously over a 2-week period and was also followed by a daily bolus of NaNO₂ at a dose of 12,000 μg/kg per minute. This bolus dose lowered MAP by 18 mm Hg, with no diminution of effect over time, indicating lack of tolerance. The utility of nitrite as an antihypertensive either by acute or chronic daily oral gavage or supplementation in water has been confirmed in other models of hypertension, such as the deoxycorticosterone acetate (DOCA)-salt, two-kidney/one-clip, and L-NAME–induced hypertension (Tsuchiya et al., 2005; Montenegro et al., 2011, 2012; Amaral et al., 2015).

There is also evidence that nitrite lowers BP in humans. Infusion of NaNO₂ into the brachial artery was associated with local nitrite concentrations of ∼200 μmol/l and systemic elevation of nitrite to 16 μmol/l and was associated with a reduction in MAP of 7 mm Hg (Cosby et al., 2003). Further studies from the same group using stepped brachial infusions of NaNO₂ achieving local venous plasma nitrite concentrations of 30 μmol/l were associated with a drop in MAP of 10 mm Hg, an effect that persisted for up to 3 hours (Dejam et al., 2007). Studies assessing the feasibility of long-term infusion of NaNO₂ in humans has been assessed in healthy volunteers. Pluta et al. (2011) examined the duration of action of increasing doses of NaNO₂ (4.2–445.7 μg/kg per hour) in healthy volunteers, measuring circulating levels of nitrite, together with assessment of metHB levels and BP. This approach was taken because nitrite has a short half-life (∼30 minutes), and the authors wished to identify whether continuous infusion might provide a mechanism to exert persistent effects upon blood flow, particularly focusing upon the potential in conditions such as cerebral vasospasm. In this study, they showed that the BP-lowering effects of NaNO₂ were maintained over 48 hours and were dose-dependent but that ceasing of infusion resulted in a prompt restoration of BP to baseline levels in association with circulating levels of nitrite (Pluta et al., 2011).

The authors of some of the earlier animal studies assessing responses to NaNO₂ hypothesized that elevation of systemic nitrite levels from nitrate via bacterial reduction would reduce BP, although they did not explicitly test this (Classen et al., 1990; Vleeming et al., 1997). However, there is now a substantial body of evidence in both animal models and humans to support this proposal.

In rats supplemented with 10 mmol/l NaNO₃ in drinking water over a week, BP was reduced when measured by cannulation under anesthesia or in free-moving animals using telemetric methods compared with matched control. This effect of NaNO₃ was abolished by use of antibacterial mouthwash twice daily. Notably, this oral treatment did not affect BP lowering in response to NaNO₂ supplementation (Petersson et al., 2009). In uninephrectomized rats fed a high-salt diet as a model of hypertension, supplementation with NaNO₃ (0.1–1 mmol/kg per day) lowered BP and ameliorated cardiac and renal fibrosis, suggesting beneficial effects beyond BP lowering (Carlström et al., 2011).

A key feature in human hypertension and CVD is that eNOS is dysfunctional, resulting in a generalized ·NO deficiency. Several research groups have sought to explore the potential utility of nitrite and/or nitrate⁻ to restore ·NO homeostasis and lower BP in hypertensive animal models driven by reduced eNOS activity. Using the eNOS inhibitor L-NAME to induce hypertension, administration of NaNO₂ in drinking water (20–200 μmol/l) or by oral gavage (0.2 mmol/kg per day) for 3 to 4 weeks prevented the development of hypertension (Tsuchiya et al., 2005; Montenegro et al., 2014). Similarly, oral gavage with 0.1 mmol/kg per day NaNO₃ reduced BP in both eNOS KO mice and L-NAME–treated rats (Carlström et al., 2010, 2015). These results suggest that elevating systemic nitrite levels, whether directly or via dietary nitrate supplementation, can compensate for diminished eNOS-derived ·NO, whether it is provided as a pretreatment or used to reverse pre-existing hypertension driven by reduced ·NO levels.

The above studies and concepts have been translated into a range of clinical studies. Although much of the preclinical data suggests that inorganic nitrite and nitrate are efficacious approaches to lower BP, whatever the cause of the hypertension, in the clinical setting, the majority of the studies to date have tested dietary nitrate rather than nitrite, and this relates to a number of reasons that include issues related to half-life and potential toxicity (discussed later).

Given the pharmacokinetic disadvantages of nitrite as a long-term therapy, only one major human study has explored inorganic nitrite therapy and BP control specifically. In this study, individual adults aged 50–79, who were free of all cardiometabolic disease and were normotensive at the start of the study (but with diminished endothelial function at baseline), were randomized in three parallel groups to receive either 40 mg (0.6 mmol) or 80 mg (1.2 mmol) NaNO₂ or placebo, all twice daily for 10 weeks (n = 10–11). Trough plasma nitrite levels were significantly elevated (∼2-fold) in both intervention groups after 10 weeks, but this was not associated with any change in BP (DeVan et al., 2016). The lack of any BP lowering in subjects with optimal/normal BP is not surprising and may be considered ideal, identifying an approach that does not lead to symptomatic hypotension and/or hypotension-related syncope. The lack of any BP effect likely reflects the accepted dogma that the magnitude of BP reduction expected from any single dose of antihypertensive agent is proportional to basal BP level (Law et al., 2003). However, this result is at odds with the majority of studies that demonstrate BP lowering in normotensive persons after systemic nitrite elevation after inorganic nitrate supplementation (Table 1) (Larsen et al., 2006; Webb et al., 2008b; Bailey et al., 2009, 2010; Kapil et al., 2010; Sobko et al., 2010; Vanhatalo et al., 2010; Lansley et al., 2011a,b; Bahra et al., 2012; Bondonno et al., 2012, 2014b; Cermak et al., 2012a; Coles and Clifton, 2012; Bond et al., 2013, 2014; Joris and Mensink, 2013; Kelly et al., 2013; Liu et al., 2013; Wylie et al., 2013; Jajja et al., 2014; Rammos et al., 2014; Ashor et al., 2015; Ashworth et al., 2015; Bourdillon et al., 2015; Jovanovski et al., 2015; Lee et al., 2015; Choi et al., 2016; Flueck et al., 2016; Jonvik et al., 2016; Raubenheimer et al., 2017; Jones et al., 2019). The first contemporary demonstration of BP-lowering effect of inorganic nitrate via conversion to nitrite used NaNO₃ [0.1 mmol/kg (6.2 mg/kg)] daily for 3 days compared with matched NaCl control in 17 healthy subjects. Plasma nitrite levels increased ∼1.5-fold after nitrate supplementation only, together with a significant reduction in diastolic BP (DBP; Δ3.7 mm Hg) (Larsen et al., 2006).

Nitrate is also available in dietary form from vegetables, so the equivalency of providing nitrate in salt form (as KNO₃) and dietary form (as beetroot juice) was established by us in 2008. Acute, single administration of 22.5 mmol (1395 mg) dietary nitrate as beetroot juice (compared with water control) and an approximate equivalent of KNO₃ (24 mmol; 1488 mg nitrate) compared with matched KCl control in separate cohorts of healthy subjects demonstrated similar time courses of systemic nitrite elevation and BP reduction over 24 hours. Peak nitrite levels were contemporaneous to peak BP reduction at 2.5–3 hours post–inorganic nitrate supplementation. Additionally, peak BP reduction from baseline was similar for both forms of inorganic nitrate supplementation (∼10/7 mm Hg), with no changes in either placebo group (Webb et al., 2008b; Kapil et al., 2010) (Fig. 7A). Furthermore, change in SBP was significantly inversely correlated to changes in plasma nitrite and the determining effect on BP (Webb et al., 2008b; Kapil et al., 2010). These studies additionally demonstrated a dose-dependent effect of inorganic nitrate supplementation on BP reduction and provided the first evidence of bioactive ·NO production after nitrate supplementation by determining significant elevation of circulating cGMP concentrations (Kapil et al., 2010); cGMP is a sensitive marker that confirms bioactive ·NO generation (Batchelor et al., 2010). An unintended but positive outcome of our work has been the introduction of beetroot juice, with known and measurable amounts of nitrate, as a mechanism to explore the biologic efficacy of the enterosalivary circuit of nitrate. The impact of this is shown in a PubMed search demonstrating the uptake of this approach after the 2008 publication (Fig. 8).

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Fig. 7.

Effects of changing nitrite concentration on BP in humans. (A) Changes in plasma nitrite (red) and nitrate (blue) and BP (green) after acute nitrate supplementation. (B) Effect of antiseptic mouthwash on plasma nitrite levels. (C) Correlation of changes in BP with changes in plasma nitrite levels after antiseptic mouthwash. (D) Changes in home BP after 4 weeks of dietary nitrate (black circles) or placebo (open circles) ingestion in patients with hypertension. ^##P < 0.01 and ^###P < 0.001 for Bonferroni post hoc test, following 2-way ANOVA.

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Fig. 8.

Number of publications using beetroot juice.

The importance of enterosalivary generation of nitrite was confirmed in our studies by asking subjects to refrain from swallowing saliva post–nitrate ingestion, thereby interrupting oral nitrate reduction but not interfering with nitrate absorption itself (Webb et al., 2008b). Elevation of circulating nitrate levels were unaffected by this intervention, but it did prevent the rise in circulating plasma nitrite level as had been demonstrated by others previously (Lundberg and Govoni, 2004). However, in addition to this, we demonstrated that this effect was associated with an absence of the BP reduction effect, thus confirming nitrite as the bioactive moiety after nitrate supplementation (Webb et al., 2008b).

Although the acute (i.e., <24 hour) effects of inorganic and dietary nitrate supplementation on BP are important, for translation to patients with hypertension, interventions need to provide sustained BP reduction in the long term. Therefore, studies over prolonged time periods (>7 days) in healthy subjects were required. This was first tested in humans indirectly by making a comparison of the effects of a traditional, nitrate-rich Japanese diet [0.3 mmol/kg (18.6 mg/kg) nitrate daily], with a low-nitrate–containing control diet in a crossover study (n = 25, 10 days per dietary intervention). In this study, the high-nitrate diet was associated with significantly lower BP (DBP −4.5 mm Hg) (Sobko et al., 2010). Since this observation, several studies have tested dietary nitrate supplementation over longer periods. Using beetroot juice to deliver dietary nitrate [5.2 (322 mg) or 6.4 (397 mg) mmol/day nitrate] compared with low-nitrate control juice (n = 8–14 for 15 days per intervention), sustained lowering of DBP (∼5–6.5 mm Hg) was evident in healthy volunteers (Vanhatalo et al., 2010; Lee et al., 2015). In healthy older persons with prehypertension (baseline BP 137/80 mm Hg, n = 10 to 11), 4 weeks of daily 150 μmol/kg (9.3 mg nitrate/kg) NaNO₃ (∼10 mmol nitrate for a 70-kg subject) lowered SBP by 8 mm Hg compared with a matched NaCl control (Rammos et al., 2014).

The first study in patients with hypertension followed a single dose over 24 hours and used water as a control in a crossover design. In all, 15 drug-naïve patients with hypertension (baseline daytime ambulatory BP; ABP 142/85 mm Hg) were given a single dose of dietary nitrate [3.3 mmol (205 mg) as beetroot juice]. Plasma nitrate and nitrite profiles were similar to responses seen in normotensive persons, confirming that the enterosalivary circulation was intact in hypertension. Additionally, a peak BP reduction of ∼11/10 mm Hg (Ghosh et al., 2013) was observed, similar to the peak reduction in healthy volunteers given 22.5 mmol despite having a fraction of the nitrate dose (Webb et al., 2008b; Kapil et al., 2010). Importantly, in healthy persons given a comparable 4 mmol (248 mg) nitrate dose, BP reduction was not significant (Kapil et al., 2010). The enhanced BP response in subjects with hypertension after nitrate supplementation could simply reflect higher baseline BP evident in the hypertensive cohort, as BP lowering is proportional to pretreatment BP (Law et al., 2003). However, it could also reflect increased potency of nitrate/nitrite. In Wistar Kyoto rats treated with phenylephrine to titrate BP to match the levels evident in the strain-matched SHR, there is a diminished BP response to acute bolus doses of KNO₂, suggesting pretreatment BP is not solely responsible for the augmented response seen in hypertension (Ghosh et al., 2013). There is some evidence to suggest that, in the scenario of reduced ·NO levels, the downstream signaling target GC-1 or the components of the pathway further downstream from GC-1 become sensitized to ·NO. This has been demonstrated best with respect to vasoactivity, in which an enhanced vasorelaxant response to ·NO donors was shown in mice treated with NOS inhibitors (Moncada et al., 1991) and in eNOS KO mice (Brandes et al., 2000). However, whether this is similar in human hypertension, a situation in which eNOS activity is similarly reduced (Linder et al., 1990; Panza et al., 1993), is not known.

In our single dietary dosing study in patients with hypertension (Ghosh et al., 2013), SBP was reduced 24 hours postsupplementation by ∼8 mm Hg, which is at a level comparable to the expected BP-lowering effect of antihypertensive medicines when used at standard dose in mild hypertension (∼9.1 mm Hg) (Law et al., 2009). This dose also represented a peak-to-trough ratio of 60% for dietary nitrate in this study and is a profile of activity consistent with an effect size suitable (>50%) to consider as a once-daily dosage for an antihypertensive medication (Lipicky, 1994; Meredith, 1994). This initial study was been taken forward into a prolonged study assessing whether the acute effects are sustained in the longer term (Kapil et al., 2015). In this double blind, randomized, placebo-controlled, parallel study (n = 32 in each limb) of drug-naïve and treated patients with hypertension and uncontrolled BP, 4 weeks of daily dietary nitrate [6.4 mmol (397 mg) as beetroot juice] was compared with a nitrate-free beetroot juice [originally developed to provide a suitable placebo (Gilchrist et al., 2014)] as a control intervention to maintain patient and investigator blindness to achieve optimal study design. BP, measured by three separate methods, was significantly reduced over 4 weeks in the active intervention limb only: clinic (−8/2 mm Hg), home (−8/4 mm Hg), and 24-hour ABP (−8/5 mm Hg), with no pharmacodynamic tolerance demonstrated by weekly home BP (Fig. 7D) (Kapil et al., 2015).

There are now several studies that have assessed the effects of dietary nitrate in patients with hypertension. The individuals in these studies in general have baseline BPs above 130/70 mm Hg (as some have controlled BP on medication) and on balance indicate a lowering of BP with dietary nitrate in patients with uncontrolled BP at baseline (see Table 2) (Ghosh et al., 2013; Kapil et al., 2015; Kerley et al., 2018; Broxterman et al., 2019; Zafeiridis et al., 2019). Conversely, there are studies that show BP-lowering benefits in treated, controlled patients on medication. As an example, in a smaller, shorter duration (n = 27, 1 week) in treated, controlled patients with hypertensions (baseline BP 133/76 mm Hg), 7 mmol (434 mg) dietary nitrate daily as beetroot juice compared with control demonstrated no effect on home or ambulatory BP, with means in low- versus high-nitrate groups being 127–128/73–74 (Bondonno et al., 2015b). In a similar study to the Kapil et al. (2015) study, in a mixed population of both patients with prehypertension and untreated hypertension, 2.5 mmol/day nitrate compared with <0.5 mmol/day nitrate, both as blended vegetable juice (4 weeks, n = 30), in a crossover study showed no difference in ABP (Blekkenhorst et al., 2018). The reasons for these differing data are uncertain; however, it is possible that these studies were underpowered (n = 27–30) and, also, that at least some of the volunteers had BP in the normotensive range and certainly substantially lower than the screening BPs; this fact may underlie the neutral effects. In addition, biochemical analyses suggest that at baseline, this group of participants had unusual and very high circulating levels of nitrite of 2 μmol/l (Bondonno et al., 2015b; Blekkenhorst et al., 2018). Although the authors suggested that this was likely due to contamination of samples through the analytical procedures (although no measure of this was made), it is also possible that these levels relate to dietary intake. Dietary habits were recorded in this study; however, they were not reported, and thus it is not possible to assess this. Finally, 63% of the recruited patients in the first study were women (Bondonno et al., 2015b). Recent evidence from our laboratory has shown that although women appear to have greater baseline oral nitrate reductase activity and thus greater circulating nitrite levels, this seems to result in a reduced response to additional dietary nitrate loading, possibly due to the fact that the pathway is already being maximally activated (Kapil et al., 2018). It is possible that the high number of women in this cohort have therefore skewed the effect.

Although all these studies measured peripheral BP, recent evidence suggests that elevation of systemic nitrite levels may have a greater effect on central hemodynamics. Systemic intravenous NaNO₂ nitrite (8.7 μmol/min) in healthy volunteers selectively reduced central BP rather than peripheral BP (Omar et al., 2015), and 6 months of dietary nitrate in patients with or at risk of diabetes had no effect on peripheral BP compared with placebo but did have a small, yet significant, effect on central BP (Mills et al., 2017). The explanation for the lack of peripheral BP reductions in these studies in contrast to the large body of evidence above is not clear, but it is worthy of further exploration. Certainly, previous studies have failed to demonstrate BP lowering in type 2 diabetes after increases in systemic nitrite levels (Gilchrist et al., 2013; Mohler et al., 2014; Shepherd et al., 2015a).

Overall, two recent independent meta-analyses of BP-lowering studies suggest a meaningful pooled effect size of ∼5/2 mm Hg reduction on resting clinic BP (Ashor et al., 2017; Jackson et al., 2018), although the studies included ranges in duration from a few hours to a few weeks, as indicated in Tables 1 and 2. For a BP-lowering intervention to be adopted clinically, support for sustained efficacy over several months is clearly needed. Further studies are underway, including a randomized, double blind, placebo-controlled evaluation of 4 months of dietary nitrate in patients with hypertensive target organ damage to assess possible reversal (clinicaltrials.gov: NCT03088514).

3. Enterosalivary Generation of Nitrite Regulates Basal BP

Nitrite is bioactive in the circulation, leading to vasodilatation and BP lowering (Cosby et al., 2003; Dejam et al., 2007), and further, the inverse correlation between baseline plasma nitrite levels and BP in healthy subjects (Kapil et al., 2010) intimates that basal plasma nitrite levels (arising from endogenous sources, i.e., eNOS) regulate BP of healthy subjects. It was thus hypothesized that interruption of the enterosalivary circulation of nitrate to nitrite under basal conditions would lower basal plasma nitrite levels and increase BP. To test this hypothesis, oral microbiome–related nitrate reduction (i.e., nitrite synthesis) was disrupted using a validated antiseptic mouthwash protocol (Govoni et al., 2008). In a 2-week crossover study (n = 19), twice daily use of antiseptic mouthwash for 7 days nearly abolished oral nitrate reduction to nitrite. More importantly, this effect was accompanied by a decrease in plasma nitrite levels by ∼25% (Fig. 7B), similar to that predicted by studies using eNOS inhibition in humans (Lauer et al., 2001; Kleinbongard et al., 2003). These studies indicate that, when one excludes the influence of diet, 25% of circulating nitrite levels arise from the enterosalivary circuit of nitrate derived from the oxidative metabolism of endogenously generated ·NO. In addition, this effect was associated with a concomitant increase in BP (2–3.5 mm Hg) that was measured by three distinct methods: clinic, home, and ambulatory BP (Kapil et al., 2013). Evaluation of home BP in this study revealed that the BP effect was apparent within 1 day of mouthwash use, with no evidence of tachyphylaxis over the week-long period of intervention (Kapil et al., 2013). This effect of increasing BP with disruption of the enterosalivary circulation has been confirmed in further studies, including in healthy volunteers (n = 27) with confirmed disease-free oral health status at baseline using chlorhexidine twice daily for 7 days (Tribble et al., 2019). Importantly, this effect is also seen in patients with CVD. In treated, controlled patients with hypertension (n = 15, baseline home BP ∼134/79 mm Hg) randomized to receive antiseptic mouthwash as above or water control, mouthwash use led to significant increase in home SBP (2.3 mm Hg) compared with control (Bondonno et al., 2015a). More recently, however, in 17 healthy females randomized to 3 days of antiseptic mouthwash or non-antibacterial placebo mouthwash, despite abrogation during the intervention period of both oral nitrate reduction and salivary nitrite levels, there was no change in plasma nitrite levels, and thus, ambulatory BP was not different (Sundqvist et al., 2016).

Further studies have shown that other oral hygiene treatments, in addition to chlorhexidine mouthwashes, impact nitrate reduction and that this effect is dependent upon the severity of the impact on oral microbiome function (McDonagh et al., 2015; Woessner et al., 2016). Support for the hypothesis that the changes in BP were related to lesser nitrite generated from the oral microbiome comes from robust correlation of changes in plasma nitrite concentration and changes in BP after 7 days of antiseptic mouthwash intervention (Fig. 7C) (Kapil et al., 2013). It is unlikely that these effects relate to other mechanisms, such as increased stress due to instillation of mouthwash, since the effects on SBP were evident in the averaged nighttime BP mean as well (Kapil et al., 2013). Although these increases in BP were small (∼ΔSBP 2–3.5 mm Hg), such effects at a population level are known to lead to a significantly increased risk of stroke and ischemic heart disease over a life course (Lewington et al., 2002). Importantly, it appears that recovery from acute use of antibacterial mouthwash (twice daily for 1 week) is apparent both in microbiological ecology and in vascular responses within 3–7 days (Tribble et al., 2019).

4. Endothelial Dysfunction

Endothelial dysfunction is synonymous with reduced bioavailable ·NO, is an early marker common to all CVD risk factors, and is predictive of future CVD events (Brunner et al., 2005). Long-term dietary nitrite and nitrate deficiency worsens ex vivo–assessed endothelial function in C57BL6 mice and was associated with CV death (Kina-Tanada et al., 2017). Additionally, nitrite supplementation improves ex vivo endothelial function in several different models associated with cardiometabolic disease (Sindler et al., 2015; Ling et al., 2016), whereas dietary nitrate has been shown to improve endothelial function in models of atherosclerosis (Bakker et al., 2016) as well as to prevent the endothelial dysfunction that occurs in mice subjected to long-term low dietary nitrate and nitrite (Kina-Tanada et al., 2017) or aging (Rammos et al., 2015).

There are several studies that have explored whether such findings in mice translate to humans. With twice daily NaNO₂ (80 or 160 mg total daily for 10 weeks) in older adults, there was a trend toward improved FMD (DeVan et al., 2016). However, conflicting results have been published for supplementation with inorganic nitrate in healthy persons with normal baseline endothelial function. Some studies have shown a positive effect of dietary nitrate (Bondonno et al., 2012; Heiss et al., 2012; Rammos et al., 2014), whereas others did not (Bahra et al., 2012). In addition, in healthy volunteers in which a transient endothelial dysfunction is induced by a brief exposure to an ischemic insult in the forearm, prior (3 hour) acute nitrate supplementation in the form of beetroot juice or KNO₃ capsules prevented this dysfunction (Webb et al., 2008b; Kapil et al., 2010). In patients with pre-existing endothelial dysfunction, a somewhat mixed picture has developed. In scenarios in which endothelial function is impaired, and this impairment is associated with risk factors for CVD, chronic oral nitrate supplementation improves FMD, i.e., in patients with hypertension and hypercholesterolemia (Kapil et al., 2015; Velmurugan et al., 2016). However, FMD was not improved after chronic supplementation in patients with diabetes and after acute supplementation in patients with peripheral arterial disease (PAD) (Kenjale et al., 2011; Gilchrist et al., 2013).

5. Arterial Stiffness

Central, large-artery stiffening caused by aging-related arteriosclerosis is an important independent predictive marker of CV risk (Vlachopoulos et al., 2010b), with the gold-standard measurement of this phenomenon being aortic pulse wave velocity (PWV) (Laurent et al., 2006). The evidence indicates that in those with stiffened central arteries, a rapid reflection of incident pressure waves leads to greater augmentation of central SBP (Safar, 2010), which is more predictive of CV events than brachial SBP (Vlachopoulos et al., 2010a). It has been demonstrated that increased arterial stiffness precedes incident hypertension in large, prospective cohorts (Kaess et al., 2012; Zheng et al., 2015). Moreover, the importance of stiffening in driving hypertension comes from observations that large-artery stiffening alone explains age-related increases in BP and failure of normal renal and baroreflex-mediated regulatory mechanisms to prevent this (Pettersen et al., 2014).

Studies in healthy volunteers have shown that acute inhibition of eNOS activity increases measures of elastic artery stiffness in vivo (Wilkinson et al., 2002; Schmitt et al., 2005; Bellien et al., 2010). Hence, increasing bioavailable ·NO, via increment of systemic nitrite, should decrease PWV. In this respect, nitrite supplementation in aged rodents (0.8 mmol/l NaNO₂ in drinking water for 3 weeks) and adults (1.2 or 2.4 mmol NaNO₂ orally for 10 weeks) reduced PWV (Sindler et al., 2011; Fleenor et al., 2012; DeVan et al., 2016). Infusion of NaNO₂ (8.7 µmol/min i.v.) for 60 minutes into the brachial artery resulted in significant, large reductions in central SBP (Omar et al., 2015), with no change in brachial SBP, similar to the effects of oral nitrite dosing (DeVan et al., 2016). In healthy subjects, acute (8 mmol KNO₃) or chronic [4 weeks of daily 150 μmol/kg NaNO₃ (∼10 mmol nitrate for a 70-kg subject)] inorganic nitrate supplementation reduced PWV (Bahra et al., 2012; Rammos et al., 2014), with elevation of plasma nitrite and cGMP levels, confirming the production of bioactive ·NO (Bahra et al., 2012).

In patients with hypertension, acute (3.3 mmol nitrate as beetroot juice) or chronic (daily 6.4 mmol nitrate as beetroot juice for 4 weeks) inorganic nitrate supplementation also resulted in significant reductions in PWV (Ghosh et al., 2013; Kapil et al., 2015). This result intimate, therefore, that benefits accrued from nitrate supplementation observed in hypertensive cohorts are not only due to reductions of elevated arterial tone but likely also reflect modulation of elastic distensibility of the arterial tree by nitrite⁻-derived ·NO. More recently, despite reduction of central BP after 6 months of dietary nitrate supplementation in patients with or at risk of diabetes, there was no effect on PWV (Mills et al., 2017). Hutchinson-Gilford progeria syndrome is a rare genetic disease associated with multiple hallmarks of accelerated aging, including arterial stiffness and premature CVD despite a relative lack of traditional CV risk factors, leading to an average age of death <15 years old (Hennekam, 2006). Mutant LMNA (the gene encoding lamin A that is defective in the syndrome) mice supplemented with NaNO₂ (500 mg/l in drinking water) had improved aortic stiffness parameters and remodeling, suggesting a potential therapeutic approach in this orphan disease (Del Campo et al., 2019).

Further studies are now needed to determine the relative contributions of the reduction of distending BP in the aorta and other elastic arteries, and specific direct effects on arterial destiffening in mediating the effects of nitrite-derived ·NO.

6. Cerebral Blood Flow

The cerebral vasculature is a vascular bed in which the potential effects of the noncanonical pathway have been particularly assessed. In part, this is due to the fact that ·NO is known to regulate cerebral blood flow (Buchanan and Phillis, 1993) and critically regulates neurovascular coupling of neural activity to blood flow (Attwell et al., 2010). Since elevation of systemic nitrite concentration delivers ·NO and improves blood flow in the peripheral circulation, whether similar effects can be seen within the cerebral circulation, which is normally tightly autoregulated for flow in health, is particularly intriguing.

Nitrite infusion (1 μmol/kg per minute) in rats has been shown to reverse L-NAME–induced cerebral vasoconstriction and the consequent reduction in cerebral blood flow (Rifkind et al., 2007). Raising systemic levels of nitrite via acute dietary inorganic nitrate supplementation in older adults is also associated with improved blood flow to the frontal cortex, which is implicated in cognitive function and may be particularly vulnerable during aging (Presley et al., 2011), and reduces cerebral vascular resistance in the middle cerebral artery (Bond et al., 2013). The effect of inorganic nitrate supplementation on cognitive function has also been studied, although results are equivocal, with some smaller studies showing neutral results (Kelly et al., 2013; Bondonno et al., 2014; Thompson et al., 2014), and one study demonstrating improvement on some tasks that activate the frontal cortex in young adults (Wightman et al., 2015).

Recently, there has been some controversial work suggesting that migraneurs express an oral microbiome that is likely to lead to increased generation of ·NO, which might contribute to the cerebral vasodilatation that is thought to underlie headaches in this setting (Gonzalez et al., 2016). The authors used analyses of the 16S rRNA Illumina sequencing of a cohort of migraneurs and nonmigraneurs from The American Gut Microbiome Project, matching to known genomes for their analyses. The authors made predictions regarding the expression of nitrate, nitrite, and ·NO-reducing genes and suggested that there were higher levels of bacteria expressing these genes in the migraneurs (Gonzalez et al., 2016). However, the authors did not actually measure these genes, and unfortunately, detail regarding the individuals from whom the samples were collected was not made available. Further studies are needed to determine the importance, or not, of such a pathway in migraine.

Subarachnoid hemorrhage (SAH) is associated with significant morbidity and mortality related to delayed cerebral vasospasm that has been linked to diminished ·NO levels (Pluta et al., 2001; Sadamitsu et al., 2001). Infusion of NaNO₂, whether at the time of experimental SAH formation or delayed by 7 days, was associated with reduced arteriographic vasospasm in primates (Pluta et al., 2005; Fathi et al., 2011), suggesting nitrite could be a useful adjunct to existing therapies, and a small pilot study has recruited six patients for a 7-day nitrite infusion after a diagnosis of SAH with vasospasm, although results have not been published to date (clinicaltrials.gov NCT02176837). Interestingly, electroencephalographic responses to intravenous nitrite infusion (NaNO₂ 10 μg/kg per minute for 1 hour) predicted the occurrence of delayed cerebral ischemia, suggesting such a test could be used to prognosticate and stratify treatment (Garry et al., 2016).

1. Nitrate and Exercise Performance on Moderately Trained Athletes

The first study to observe benefits of dietary nitrate ingestion on exercise was a randomized, double blind, placebo-controlled crossover study involving nine well trained healthy men of ∼28 years (Larsen et al., 2007). In this study, the subjects consumed 0.1 mmol/kg per day of NaNO₃ or a NaCl placebo for 3 days before completing a continuous incremental cycle ergometer test. Nitrate supplementation caused a significant decrease in maximal oxygen consumption (VO_2max) at submaximal work rates, with a mean reduction in VO_2max of 5%. This reduction was surprisingly associated with significant improvements in muscle efficiency, from 19.7% ± 1.6% to 21.1% ± 1.3% (calculated as the work output per unit energy expended), and occurred without changes in blood (lactate), intimating that there was no increase in energy production. In addition, there were no differences in heart rate, ventilation, or respiratory exchange ratio between nitrate and placebo for any of the submaximal work rates (corresponding to 45%–80% VO_2max). These results were particularly surprising since a basic principle of human exercise physiology is that the O₂ cost of submaximal exercise at a given work rate is fixed, irrespective of other factors such as health, fitness status, and age and indifferent to known nutritional, physical, or pharmacological interventions (Suhr et al., 2013). From these observations, the authors suggested that endurance exercise performance is a function of VO_2max, the fractional utilization of VO_2max, and exercise efficiency and that if all other factors are constant, an improvement in muscle efficiency would be expected to enable a greater work output for the same energy cost. This, in turn, translates into improved exercise performance (Larsen et al., 2007). These seminal findings caught the imagination of the sports and exercise world and led to a surge of interest to assess whether inorganic nitrate may be useful in terms of placing the body in an optimal condition to use O₂ most efficiently to gain advantage in numerous forms of exercise, particularly for the elite endurance athlete.

The first independent study to corroborate the work of the Karolinska group came from Jones and coworkers in the UK (Bailey et al., 2009). This group took advantage of the recent discovery at that time that beetroot juice could be used as a simple and effective nitrate delivery method (Webb et al., 2008b). As per the study of Webb and colleagues, they gave 500 ml of beetroot juice (containing 11.2 ± 0.6 mmol/l nitrate) in a placebo-controlled crossover study of eight men (aged 19–38 years) in which black currant cordial (containing negligible nitrate) was used as the placebo for six consecutive days. The study was described as double blind; however, it is likely that the differences in the two liquids was evident to all. In this study the volunteers, as in the 2007 study of Larsen et al., completed a series of “step” moderate-intensity and severe-intensity exercise tests on the last 3 days (Bailey et al., 2009). As expected, plasma nitrate concentration was significantly elevated compared with placebo (beetroot juice: 273 ± 44 nmol/l vs. placebo: 140 ± 50 nmol/l), and this was associated with statistically significant decreases in SBP, demonstrating efficacy of the intervention. However, the key observation documented was that, in agreement with the Karolinska group, dietary nitrate reduced the “O₂ cost” of cycling at a fixed submaximal work rate. The authors show that muscle fractional O₂ extraction during modest exercise and gain in pulmonary O₂ uptake after onset of moderate exercise were reduced by 19%. However, in addition to this benefit, the authors found that during severe exercise, the O₂ uptake “slow component” was reduced. This slow component is thought to represent a progressive loss of muscle efficiency as high-intensity exercise continues (Jones et al., 2011). The peak oxygen uptake (VO₂) attained during high-intensity exercise was not different between treatments, but attainment of the peak VO₂ was delayed with nitrate treatment, resulting in a longer time to exhaustion. These observations were repeated by the group in another study, which used a different form of exercise (i.e., knee extensors) (Bailey et al., 2010) and was again reproduced by Larsen et al. (2010) using combined arm and leg cranking as the form of exercise. Interestingly, in this study, the group found that the peak VO₂ was reduced with dietary nitrate (Larsen et al., 2010) during maximal exercise using a large active muscle mass and that it was associated with a trend toward increased time to exhaustion. The authors suggested that this implied involvement of two separate mechanisms in the benefits of inorganic nitrate: one that reduces VO_2max and another that improves the energetic function of the working muscles.

Although these early observations have fueled interest in assessing the mechanisms and whether the effects can be sustained in the long term, there has been some controversy regarding effective dose. Using a similar exercise test of submaximal cycle ergometry, VO_2max was shown to be reduced (by ∼3%–5%) after the acute ingestion of 0.033 mol/kg nitrate, equating to ∼2.5 mmol nitrate in a 70-kg individual (Larsen et al., 2010), 5.2 (Vanhatalo et al., 2010), 6 (Wylie et al., 2016), and 16.8 mmol nitrate (Wylie et al., 2013), but conversely not after the acute ingestion of 3 (Wylie et al., 2016), 4.2 (Wylie et al., 2013) and ∼8 mmol nitrate (Betteridge et al., 2016) with no effect upon exercise tolerance (Vanhatalo et al., 2010; Wylie et al., 2013). However, in the study of Wylie and colleagues, increasing the dose substantially (8.4 and 16.8 mmol) did result in significant improvements (Wylie et al., 2013). These results seem to suggest that the acute ingestion of nitrate from 2.5 to 6 mmol of nitrate may be sufficient to enhance exercise economy during submaximal cycling but that higher doses are required to demonstrate any improvement in exercise tolerance.

Importantly, these effects on cycling economy after short-term nitrate supplementation have been shown to be maintained when dosing is extended over 15 days (although not at 5 days in this study) (Vanhatalo et al., 2010) or 6 mmol/day for 28 days (Wylie et al., 2016). These findings indicate that, as with the effects upon BP, there is no development of tolerance as occurs with organic nitrates (Münzel et al., 2005). It is also worth noting that there are dissenting studies in which, despite elevation in plasma nitrite concentration after supplementation with nitrate in both recreationally active and moderately trained endurance athletes, no improvement in cycling exercise economy or performance was evidenced (Breese et al., 2013; Kelly et al., 2014).

Similar improvements in exercise economy and tolerance after nitrate supplementation have been shown during running (Lansley et al., 2011b; Murphy et al., 2012; Porcelli et al., 2015), knee extension (Bailey et al., 2009), walking (Lansley et al., 2011b), desert marching (Kuennen et al., 2015), kayaking (Muggeridge et al., 2013; Peeling et al., 2015), and rowing (Bond et al., 2012; Hoon et al., 2014). However, whether improved exercise tolerance (i.e., time to exhaustion in a constant work rate task) in turn then translates into an associated improvement in exercise performance (i.e., a set distance or amount of work is completed in a shorter time frame) is controversial. In one study, no such benefit is seen with acute administration of nitrate (Cermak et al., 2012b), but chronic (up to 6 days) nitrate supplementation did enhance cycling performance (Cermak et al., 2012a) in moderately trained endurance, suggesting that chronic nitrate supplementation may have more possibilities as an ergogenic aid than acute nitrate ingestion.

2. Nitrate and Exercise Performance in Patient Populations

The above evidence supports the view that during the hypoxemia created by exercise in which type II muscle fibers are recruited to drive muscle contraction, inorganic nitrate/nitrite acts to improve function. Thus, in individuals with a greater proportion of type II muscle (Hernandez et al., 2012; Ferguson et al., 2013a; Kelly et al., 2014), such as that found in patients with metabolic, respiratory, and CVD (Schaufelberger et al., 1995; Gosker et al., 2000; Mador and Bozkanat, 2001; Raguso et al., 2004; Askew et al., 2005; Oberbach et al., 2006), inorganic nitrate/nitrite may be therapeutically useful. Indeed, several studies have now shown improved exercise capacity in patients with chronic obstructive pulmonary disease (COPD) (Berry et al., 2015; Kerley et al., 2015; Leong et al., 2015; Shepherd et al., 2015b), PAD (Kenjale et al., 2011) and heart failure (HF) (Coggan et al., 2015; Zamani et al., 2015; Eggebeen et al., 2016). However, healthy older adults (Kelly et al., 2013) or patients with type II diabetes did not benefit from dietary nitrate intervention (Shepherd et al., 2015a). These studies are discussed more fully below. Finally, in addition to improving exercise performance, there is also preliminary evidence to suggest that nitrate supplementation can aid recovery postexercise (Clifford et al., 2016).

There are now numerous studies that have explored the potential advantage that inorganic nitrate might provide to endurance athletes. The evidence to date is mixed, with some reports identifying small but competitively important benefits, and others showing no effect (see Table 3). This discord has in part been attributed to the fact that in well trained endurance athletes, physiologic remodeling of the skeletal muscle occurs during chronic endurance training. Moreover, this remodeling is associated with an increase in NOS expression in both skeletal muscle [neuronal NOS (McConell et al., 2007)] and the vasculature supplying the muscles [eNOS (Green et al., 2004)], and also to an increase in the content of Ca²⁺-handling proteins in type II muscle (Kinnunen and Manttari, 2012) to promote both a lower percentage of type II skeletal muscle (Wilson et al., 2012) and lower mitochondrial UCP3 content (Fernstrom et al., 2004), which is an important determinant of exercise efficiency (Mogensen et al., 2006). Hence, the differing vascular physiology and skeletal muscle of well trained endurance athletes compared with lesser trained athletes may explain a diminished advantage of nitrate supplementation in well trained endurance athletes.

Since there is a greater proportion of type II muscle used during high-intensity exercise (Krustrup et al., 2004), several research groups have focused on the possibility that this form of exercise may preferentially benefit from nitrate supplementation. Again, there are studies demonstrating benefit, whereas others show no advantage (see Table 4). This apparent discord may be due to the enormous heterogeneity in the studies with respect to the exercise modality, training status of the athletes/participants, the nitrate supplementation protocol, and/or the intermittent nature of the exercise regimes. On balance, however, it does seem that nitrate supplementation has ergogenic potential for athletes participating in sports in which speed, power, and repeated sprint ability are important determinants of success.

3. Cardiac Muscle Function and Heart Failure

Dysfunction of the classic pathways that underlie ·NO production are thought to play a major role in the pathogenesis of both HF with reduced ejection fraction (HFrEF, commonly referred to as systolic HF) and preserved ejection fraction (HFpEF, commonly referred to as diastolic HF) (Drexler, 1999; Cai and Harrison, 2000; van Heerebeek et al., 2012). Left ventricular ejection fraction (LVEF) for humans with HFrEF is typically <40%, HFpEF LVEF >50%, with an intermediate group of HF with mildly reduced ejection fraction for those with LVEF 40%–49% (Ponikowski et al., 2016). Patients are high risk for HF as the cause of their symptoms with elevated NT-pro-brain natriuretic peptide (NT-proBNP) (sinus rhythm >1000 pg/ml and atrial fibrillation >1600 pg/ml) and brain natriuretic peptide (BNP) (sinus rhythm >300 pg/ml and atrial fibrillation >500 pg/ml) levels, although lower levels remain compatible with the diagnosis depending on the acuity of the clinical setting (Ponikowski et al., 2016). Standard prognostic therapy in HF with angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, statins, and the novel β-adrenoceptor antagonists all increase ·NO bioavailability through reduced oxidative stress and increased NOS activity (Omar et al., 2016). Although the organic nitrates provide an efficacious treatment option in the setting of acute HF and have shown some benefit in chronic HF (Cohn et al., 1986; Taylor et al., 2004), they have proven difficult to use as a long-term option because of the rapid development of tolerance, reflex tachycardia, and the induction of endothelial dysfunction (Münzel et al., 2011, 2013). As the global prevalence of HF increases, with the associated high levels of morbidity and mortality (Meta-analysis Global Group in Chronic Heart Failure (MAGGIC), 2012; Ponikowski et al., 2016), it has become increasingly important to identify and exploit novel methods by which to treat the HF syndrome. The role of inorganic nitrite and nitrate to restore diminished ·NO is one such possibility (Paulus and Tschöpe, 2013).

1. Thrombosis

Platelets are critically important in the formation of clinically relevant thrombosis (Davì and Patrono, 2007) and are exquisitely sensitive to local generation of ·NO (Azuma et al., 1986), so much so that in the early days of ·NO biology, this response was used as a bioassay for ·NO production (Radomski et al., 1990). ·NO has numerous effects on platelet biology, including reduction in adhesion, aggregation, recruitment, and formation of platelet-leukocyte aggregates (Radomski et al., 1987, 1990; Freedman et al., 1997; Chung et al., 2004). Accordingly, a deficiency of ·NO is associated with a state of platelet activation in relation to the actions above (Cadwgan and Benjamin, 1993; Stagliano et al., 1997; Freedman et al., 1998, 1999; Gkaliagkousi et al., 2009), and organic nitrovasodilators are known to have modest effects on platelet function (Hampton et al., 1967; Schafer et al., 1980).

Platelet inhibition by ·NO is mediated by GC-1/cGMP/PKG canonical signaling (Massberg et al., 1999), and this PKG activation, in turn, through phosphorylation of specific targets, leads to attenuation of platelet reactivity by triggering downstream inhibition of P-selectin expression (Murohara et al., 1995), phosphorylation of the thromboxane receptor (Wang et al., 1998), and VASP (Massberg et al., 2004), which further inhibits glycoprotein IIb/IIIa. Whatever the specific molecular pathways involved, it is clear that ·NO influences all of the major components of platelet function, including Ca²⁺ mobilization, shape change, secretion, and both integrin activation and outside-in signaling and thus, in this way, plays a key role in ensuring a coordinated regulation of platelet activity (Naseem and Roberts, 2011; Makhoul et al., 2018). However, the exact cellular source and localization of ·NO generation that reduces platelet activity is more controversial. There is evidence that supports the endothelium (Moore et al., 2010); however, there are also several observations identifying the platelet itself as the site for NOS-derived ·NO (Radomski et al., 1990), particularly eNOS (Freedman et al., 1999; Webb et al., 2008a; Radziwon-Balicka et al., 2017), although others have failed to localize eNOS within platelets (Gambaryan et al., 2008; Gambaryan and Tsikas, 2015). Irrespective of localization, it is clear that basal ·NO generation plays a critical role in repressing platelet reactivity under normal conditions. Thus, in scenarios in which there is a deficiency of bioavailable ·NO, restoration of ·NO levels, through ·NO delivery, should recover normal platelet function. Several research groups have assessed whether provision of inorganic nitrate and nitrite might achieve this outcome.

Ex vivo incubation of platelet-rich plasma, collected from healthy volunteers with supraphysiologic nitrite concentrations (60 μmol/l NaNO₂) was shown many years ago to reduce aggregatory responses to a wide range of platelet stimuli (ADP, arachidonic acid, and collagen), whereas similar concentrations of NaNO₃ had no effects (Schafer et al., 1980). This finding has been supported more recently, when physiologically relevant nitrite concentrations (no more than 1 μmol/l) achieved through in vivo treatment with dietary inorganic nitrate (Webb et al., 2008b) resulted in a suppression of ex vivo–assessed platelet aggregation responses. However, further work has shown convincingly that ex vivo treatment with nitrite salts does not directly alter platelet reactivity at physiologic concentrations. Rather, the evidence suggests that to reveal the antiplatelet effects of nitrite, reactivity must be assessed in the whole blood environment wherein platelets remain in close contact with the circulating elements of the blood.

In platelet-rich plasma, 0.1 μmol/l NaNO₂ inhibited stimulus-induced platelet aggregation only when in the presence of erythrocytes (Srihirun et al., 2012). Similar findings were separately published by Velmurugan et al. (2013), in which KNO₂ (0.1–0.3 μmol/l) caused concentration-dependent suppression of platelet aggregation only when incubated with whole blood and not when incubated with platelet-rich plasma alone. Interestingly, the antiplatelet effects of nitrite are enhanced by deoxygenation of erythrocytes and blocked by ·NO scavenging (Srihirun et al., 2012), as well as being associated with elevations in cGMP (Velmurugan et al., 2013). These findings fit well with the view that nitrite reduction in blood is in part dependent upon the reductase activity of erythrocytic Hb and requires partial deoxygenation (Cosby et al., 2003).

As one would suspect, the elevation of cGMP in the platelet with nitrite treatment results in the antiplatelet effects typical of ·NO-induced GC-1 activation and is associated with suppression not only of P-selectin expression (Akrawinthawong et al., 2014) but also gpIIb/IIIa expression, both key pathways regulating platelet activity and known to be influenced by ·NO (Murohara et al., 1995; Keh et al., 1996). Further studies with inhaled NaNO₂ (40 mg) have also demonstrated increased platelet phospho-VASP, confirming NO/GC-1/cGMP/PKG signaling and diminution of stimulus-induced platelet aggregation (Parakaw et al., 2017). These results together suggest that nitrite reduction does not occur at the level of the platelet itself but rather occurs at the level of the erythrocyte and that the ·NO generated acts on the neighboring platelet to elevate cGMP, resulting in downstream inhibition of key platelet activating pathways.

Platelets themselves contain nitrite (Apostoli et al., 2014), the levels of which are substantially reduced by treatment with ascorbic acid ex vivo [a nonspecific reducing agent, as per previous evidence shown in erythrocytes (Sibmooh et al., 2008)], which also simultaneously results in inhibition of platelet aggregation that is associated with elevations in cGMP and phospho-VASP and effects that are enhanced by inhibition of PDE-V and inhibited if treated with ODQ or C-PTIO. However, in this paper, the authors also show nitrate in platelets, the levels of which likewise are decreased with ascorbate. This observation is curious, since ascorbate provides a one-electron reduction; however, for nitrate reduction, two electrons are needed. The authors also show that treatment with NaNO₂ causes inhibition of platelet aggregation that is also enhanced by treatment with ascorbate or PDE-V inhibitors; and that is prevented by treatment with ODQ or C-PTIO. The authors propose a number of controversial explanations for their observations. Since coincubation with NOS inhibitors had no impact upon any of the responses and since the authors previously have failed to show eNOS expression in platelets, they reason that the source of the widely reported capacity of platelets to generate ·NO is in fact due to nitrite reduction and not NOS catalytic activity. Moreover, unlike several other publications, since the authors conducted all of their ex vivo experiments in the absence of erythrocytes, they suggest that the platelet itself has the capacity to reduce nitrite directly (Apostoli et al., 2014), although the exact nitrite reductase was not identified.

As mentioned briefly, the beneficial effect of nitrite upon platelet reactivity can be exposed after elevation of circulating nitrite levels through oral administration of inorganic nitrate. Oral supplementation of healthy volunteers with either 2–8 mmol KNO₂ (Richardson et al., 2002; Velmurugan et al., 2013) or 3.1–22.5 mmol dietary nitrate (192–1395 mg nitrate as beetroot juice) (Webb et al., 2008b; Velmurugan et al., 2013) prior to venepuncture, followed by examination of ex vivo platelet aggregation, results in a modest reduction in the platelet aggregatory response to stimulatory agonists such as ADP and collagen, with associated increases in platelet cGMP and suppression of P-selectin expression (Velmurugan et al., 2013). The modulating effects of dietary nitrate supplementation are additionally seen in healthy older adults after acute nitrate supplementation (13 mmol nitrate, n = 12) with reduction in platelet P-selectin expression and clotting time in whole blood, relating to both intrinsic and extrinsic pathways in whole blood but not plasma coagulation tests post–dietary nitrate supplementation (Raubenheimer et al., 2017).

Interruption of the enterosalivary circulation by spitting negated the anti-platelet effects of dietary nitrate, reconfirming the requirement for bioactivation of nitrate to nitrite within the oral cavity (Webb et al., 2008b). Similar effects are seen in mice—whilst treating mice for 1 week with NaNO₂ (0.1 g/l) or NaNO₃ (1 g/l) reduced ex vivo platelet aggregation and prolonged bleeding time, when mice were fed with a low-NOx diet, the opposite occurred, with enhanced platelet aggregation and shorter bleeding time (Park et al., 2013). Finally, this beneficial profile of activity of nitrite and nitrate upon platelets evidenced in these acute healthy volunteer and preclinical studies does translate into the patient setting. In patients with endothelial dysfunction and modest hypercholesterolemia, 6 weeks of a once-per-day dietary nitrate supplementation (∼6 mmol) was associated with lower basal and stimulus-induced platelet P-selectin expression and leukocyte-platelet aggregates (Velmurugan et al., 2016). Again, these studies show a modest but significant effect. Such a profile of activity, we argue, lends itself as an alternative approach in primary and secondary prevention. Perhaps one of the key difficulties associated with antiplatelet therapy in the secondary prevention setting is the negative side effect profile of bleeding complications. Similarly, it is these very same bleeding complications that have resulted in cessation of the use of aspirin as a primary prevention strategy. We speculate that with such a modest but important antiplatelet profile, dietary nitrate offers a relatively safe and easy-to-administer option.

2. Peripheral Arterial Disease

The positive effects of inorganic nitrate/nitrite against inflammation and platelet function, in addition to the benefits in terms of skeletal muscle function, suggest that in diseases with chronic tissue, ischemia such as PAD associated with impaired blood flow, enhanced inflammation, and platelet activation, inorganic nitrate may be of value. In addition, since ·NO has a positive impact upon angiogenesis (Papapetropoulos et al., 1997; Cooke and Losordo, 2002), effective ·NO delivery through inorganic nitrate/nitrite might offer a mechanism for therapeutic angiogenesis.

In a mouse hindlimb ischemia model, both chronic nitrite and nitrate therapy increase ischemic tissue blood flow with associated endothelial cell proliferation and angiogenesis (Kumar et al., 2008; Hendgen-Cotta et al., 2012). In addition, evidence in humans suggests that dietary nitrate supplementation is associated with mobilization of circulating angiogenic cells (Heiss et al., 2012), strongly supporting the suggestion that nitrate therapeutics may work in PAD. However, again there is controversy. Major optimism was generated by the first study in patients with PAD demonstrating benefit. After a 3-month supervised exercise program in patients with PAD, significant improvements in exercise capacity were seen and correlated to increases in plasma nitrite concentration (Allen et al., 2010). But more importantly, acute dietary nitrate supplementation (18 mmol nitrate in beetroot) was associated with ∼20% improvement in claudication onset time and peak walk time (Kenjale et al., 2011). However, divergent results have been apparent with chronic administration. After 10 weeks of twice-daily supplementation with NaNO₂ (40 or 80 mg) versus placebo in diabetic patients with PAD, there was an improvement in endothelial function in these patients with nitrite treatment, but there was no effect on 6-minute walk time (Mohler et al., 2014). Conversely, 8 weeks of NaNO₃ (8.5 mmol/day nitrate) was associated with a ∼40 m improvement in the 6-minute walk test (Bock et al., 2018a), although there was only a trend for similar effect (∼30 m) after 12 weeks of 4.2 mmol nitrate with a structured exercise program compared with exercise and placebo (Woessner et al., 2018). However, these effects may have been explained, despite randomization and blinding, by a slightly worse baseline in the nitrate-treated group and thus greater apparent improvement. Further definitive studies are awaited, but at present, whether dietary nitrate might be useful for PAD is uncertain.

3. Atherosclerosis

There is a wealth of evidence demonstrating that vascular ·NO is critical in sustaining vascular patency and that a deficiency in bioavailable ·NO is a pivotal phenomenon involved in triggering atherogenesis (Böger et al., 1997; Antoniades et al., 2007). As discussed above, we know that ·NO reduces leukocyte adhesion and transmigration, reduces low density lipoprotein cholesterol oxidation, and inhibits vascular smooth muscle proliferation [for review, see Moncada and Higgs (1993)]. Moreover, from studies using NOS inhibitors and eNOS KO mice, we know that eNOS is the primary source of the vascular ·NO that underlies protection against atherogenesis in health (Kauser et al., 2000; Kuhlencordt et al., 2001). Furthermore, the uncoupling of eNOS in the disease setting is thought to be responsible for the reduced bioavailable ·NO and endothelial dysfunction, which is implicated in the atherosclerotic process (Libby, 2002; Napoli et al., 2006). As a result, numerous studies have sought to restore ·NO levels utilizing ·NO donors, particularly the organic nitrates, l-arginine (the substrate for conventional ·NO synthesis), or the essential cofactor for NOS, tetrahydrobiopterin. Many of these studies have shown positive outcomes, including improvements in vascular function, systemic inflammatory profile, and local inflammation at lesional sites and positive effects upon platelet reactivity (Clarkson et al., 1996; Theilmeier et al., 1997; Wolf et al., 1997). However, translation of these findings to the clinical setting has not gone as well as hoped (e.g., Nakamura et al., 1999; Blum et al., 2000; Cunnington et al., 2012). The reasons for these discordant findings are variously attributed to eNOS uncoupling for l-arginine (Förstermann and Sessa, 2012), tolerance and induction per se of vascular dysfunction with the organic nitrates (Münzel et al., 2013), and/or oxidation of tetrahydrobiopterin (Cunnington et al., 2012). Although all of these approaches are distinct, they all aim to provide ·NO delivery to the atherogenic blood vessel. The failure of translation of all of these approaches supports the contention that alternative, but more effective, strategies to improve bioavailable ·NO levels are warranted. It is this need that underlies the testing of inorganic nitrate and nitrite in atherosclerosis.

As mentioned earlier in the review, there have been some studies that have begun to assess the potential of nitrate/nitrite as an ·NO delivery method in atherosclerosis. Inorganic nitrite and nitrate supplementation has been shown to reduce leukocyte-endothelial cell interactions in hypercholesterolemia (Stokes et al., 2009), and a 6-week, once-per-day dietary nitrate treatment in patients with hypercholesterolemia improved endothelial function together with some evidence (although the study was not powered for some of these indices) suggesting that this improved vascular function was associated with a reduced systemic inflammation and reduced platelet reactivity (Velmurugan et al., 2016).

These positive mechanistic findings have been followed up by testing of the impact of inorganic nitrate salt supplementation upon plaque formation and size in mouse models of atherosclerosis. The first study assessing this reported in 2016 and, disappointingly, was negative. In this study, low density lipoprotein cholesterol receptor KO mice were fed a Western diet for 14 weeks and treated concomitantly with NaNO₃ (1 g/l) or equimolar NaCl in drinking water. At the end of this treatment period, there were no differences between the two groups in plaque size, histologic plaque scores, and macrophage, collagen, and smooth muscle content. It is noteworthy, however, that although there have been numerous studies demonstrating efficacy of such a dosing schedule, in this study, nitrate supplementation was not associated with an increase in systemic nitrite levels, and there was no change in BP. These results suggest that, at least in these animals, this dosing regimen was insufficient to evoke modulation of the nitrate-nitrite-·NO pathway (Marsch et al., 2016). This failure may underlie the absence of any effects seen.

Interestingly, in ApoE KO mice, supplementation with 15 mmol/l KNO₃ for 12 weeks was associated with increases in systemic and organ nitrite levels in comparison with mice treated with equimolar KCl. This rise was associated with reduced leukocyte rolling and adherence, assessed using intravital microscopy of the cremaster microcirculation, reduced circulating neutrophil numbers and monocyte activation, and upregulation of IL-10–dependent anti-inflammatory pathways. This multitude of anti-inflammatory effects resulted in a reduction in the inflammatory load within the plaque, while having no effect upon plaque size (Khambata et al., 2017). Intriguingly, in this study, we showed that tissues of the ApoE KO expressed greater nitrite reductase activity assessed ex vivo and that this activity, at least in part, was driven by elevated XOR activity, observations fitting with studies in humans indicating that individuals with coronary artery disease have increased vascular XOR expression and activity (Spiekermann et al., 2003). In a separate study, low to moderate dosing with KNO₃ (0.1 and 1 mmol/kg per day) but not high-dose (10 mmol/kg per day) supplementation for 10 weeks in ApoE KO mice was associated with increased collagen expression and reduced lipid deposition and also, similarly, no differences in plaque size (Bakker et al., 2016). These two latter studies support the contention that reduced inflammation within the atherosclerotic plaques coupled with increased smooth muscle and collagen content, although not influencing plaque size, likely provides a more stable plaque phenotype (Stefanadis et al., 2017).

Interestingly, and perhaps contradicting the above work, is evidence suggesting that nitrite has important activity limiting intimal hyperplasia. Rats pretreated with NaNO₂ (administered intraperitoneally, orally, or nebulized) inhibited pathologic intimal hyperplasia, caused in response to vascular wall injury through insertion of a balloon and inflation in the carotid artery. Importantly, treatment of rats after injury (15 days later) also resulted in a statistically significant reduction in hyperplasia assessed at 28 days. In addition, the authors demonstrated that this beneficial effect of nitrite was attenuated if rats were pretreated allopurinol, implicating XOR as the nitrite reductase in this setting (Alef et al., 2011).

Although there does seem to be some contrast in pathways, the evidence does support the view that the nitrate-nitrite-·NO pathway may offer opportunities for limiting atherosclerosis and that such an approach may have utility not only in terms of dietary lifestyle interventions that limit disease from occurring in the first place but also as an additional strategy that may prove useful in the secondary prevention setting.

1. Pulmonary Hypertension

Pulmonary arterial hypertension (PAH) is a rare condition characterized by increased pulmonary vascular resistance and associated elevated pulmonary arterial pressure (PAP), leading to right HF. Patients with PAH experience significant dyspnea, fatigue, and accelerated mortality. Pathophysiological alterations in PAH include abnormal pulmonary vasoconstriction and endothelial dysfunction, smooth muscle cell proliferation, and adverse remodeling of the pulmonary circulation. It has been known for some time that defects in ·NO-mediated vasodilatation is one of the hallmarks of disease (Adnot et al., 1991), and this has led to the exploration of drugs that target the ·NO-GC-1-cGMP pathway for the reduction of PAP.

Inhaled NO (iNO), by virtue of its scavenging in the circulation by Hb, results in selective delivery of ·NO to the pulmonary circulation without the potential adverse effects of systemic ·NO delivery, such as systemic arterial hypotension. Inhaled ·NO was demonstrated to be a selective pulmonary vasodilator more than 30 years ago (Frostell et al., 1991; Pepke-Zaba et al., 1991), but the need for continuous nebulization restricts its utility in ambulant patients with PAH. This limitation has, however, been overcome by taking the strategy of using selective inhibitors of cGMP breakdown by blocking the activity of PDE-V, thus augmenting the pulmonary vasodilatory effects of endogenously produced ·NO. This approach is associated with clear improvements in patient-related and hemodynamic outcomes (Palmieri et al., 2004; Galiè et al., 2005, 2009; Pepke-Zaba et al., 2008) and is now commonly used clinically for this indication. Since it is widely accepted that there is a reduction in ·NO bioavailability in PAH (Hu et al., 2010), there has been considerable focus on bypassing this step of the pathway through generation of direct activators/stimulators of GC-1. Riociguat is the first in class of these drugs to have received widespread marketing authorization for PAH after the positive results of the PATENT studies, demonstrating improvements in the 6-minute walking test (the most commonly used intermediate endpoint) and dyspnea scores in patients with PAH (Ghofrani et al., 2013).

Given the recent evidence of the ability of nitrate-nitrite-·NO reduction to produce bioactive ·NO and the fact that this pathway appears to be augmented in hypoxia, this knowledge has spurred interest in whether inorganic nitrite and nitrate could ameliorate pulmonary hypertension. Recent preclinical studies have explored the utility of this pathway in pulmonary hypertension. In ovine models of PAH induced by hypoxia, thromboxane, and hemolysis, both iNO and inhaled NaNO₂ reduced pulmonary vasoconstriction and ameliorated PAP. An important distinction between the two treatment forms was that nitrite therapy was associated with less systemic hypotensive effects (Hunter et al., 2004; Blood et al., 2011). The effects of inhaled nitrite were kinetically slower than those for iNO and associated with less rebound vasoconstriction on termination of intervention (Hunter et al., 2004). Similarly, in both monocrotaline and hypoxia-induced PAH in rats, repeated inhaled nitrite application prevents but also reverses established effects of PAH on PAP, vascular smooth muscle proliferation, and right ventricular function (Zuckerbraun et al., 2010). Assessment of nitrite reductase ex vivo suggested that this effect was largely XOR-mediated, and in vivo, the effects of nitrite were abrogated by both XOR inhibition and dietary tungsten replacement of molybdenum in XOR (Zuckerbraun et al., 2010).

The potential beneficial effects of elevation of systemic levels of nitrite have been explored by both injection of inorganic nitrite and through dietary nitrate (via the enterosalivary circulation) and nitrite provision in drinking water. Acute intravenous and chronic intraperitoneal inorganic nitrite application are associated with improvements in PAP and right ventricular hypertrophy in monocrotaline- and hypoxia-induced PAH in rats (Casey et al., 2009; Pankey et al., 2012). However, for effective translation to human therapeutics, a more convenient form of systemic nitrite elevation would be desirable. In mice exposed to 3 weeks of hypoxia to induce typical pathologic changes of PAH, both dietary nitrite (0.6 mmol/l) and nitrate (45 mmol/l) supplementation via drinking water reversed elevation of right ventricular pressure and hypertrophy. Dietary nitrate supplementation was also effective in a pathologically distinct model of PAH induced using bleomycin (Baliga et al., 2012) and monocrotaline (Tawa et al., 2019). Further work utilizing eNOS KO mice and XOR inhibitors suggested that both enzyme systems were important for the beneficial effects of elevated nitrite levels in these models, with evidence also implicating ·NO-mediated activation of GC-1-cGMP pathways (Baliga et al., 2012).

Whether local or systemic elevation of nitrite will translate to effective treatments in human disease is currently unknown. Earlier clinical studies in healthy volunteers given i.v. NaNO₂ (1 μmol/min) suggested acute improvements in hypoxia (12%)-induced pulmonary artery pressures (measured by transthoracic echocardiography) (Ingram et al., 2010). Furthermore, a series of dose-ranging phase I pharmacokinetic studies of inhaled NaNO₂ in healthy persons have suggested suitable safety parameters supporting a thrice daily maximal tolerated dose of 90 mg of NaNO₂ (Rix et al., 2015) to allow further exploration of inhaled nitrite in patients with PAH. In patients with PAH due to HFpEF and chronic lung disease, acute inhaled nitrite lowered PAP, pulmonary capillary wedge pressures, and pulmonary vascular resistance (Simon et al., 2016). Recently, a report in five patients with echocardiographic evidence of PAH associated with β-thalassemia has suggested that acute inhaled nitrite rapidly decreases PAP, and this reverts shortly after inhalation terminates (Yingchoncharoen et al., 2018). In a pilot crossover study in 15 patients with PAH, randomized to two 7-day treatment periods (16 mmol/day nitrate vs. placebo), ingestion of a fixed dose of dietary nitrate was associated with increases in exhaled ·NO and plasma and salivary NOx and was not associated with any adverse systemic hemodynamic changes. There were no significant changes in pulmonary hemodynamic parameters assessed by echocardiography or cardiopulmonary exercise testing, or of function tests, such as the 6-minute walk test, although there were trends for improvements in right ventricular function and peak power per liter oxygen consumed, particularly in those patients in whom plasma nitrite flux was >30% post–nitrate ingestion (Henrohn et al., 2018). Although these data are encouraging, it remains for larger, adequately powered trials to determine whether inorganic nitrate supplementation has benefit in PAH, with or without other drugs that modulate the ·NO-GC-1-cGMP system.

2. Chronic Obstructive Pulmonary Disease

COPD describes a progressive respiratory disease affecting the airways and lung parenchyma, resulting in irreversible airway obstruction. Patients with COPD describe dyspnea and reduced exercise tolerance due to a combination of ventilatory/gas-exchange impairment leading to hypoxia and skeletal muscle deconditioning. Exercise and pharmacological management improve the exercise capacity and physical function of patients with COPD (Barnes et al., 2015).

Given the effects of inorganic nitrate supplementation, via elevation of systemic nitrite levels, on various measures of exercise capacity in healthy persons described above and hypoxia-related augmentation of the nitrite-derived ·NO production, the utility of inorganic nitrate supplementation in COPD has been studied. Two separate studies have shown that acute supplementation of dietary nitrate (7.6–12.9 mmol nitrate) improves 75% submaximal exercise time by 30 seconds and incremental shuttle walk test by 40 m compared with placebo (Berry et al., 2015; Kerley et al., 2015); in contrast, however, further studies using 13.5 mmol nitrate daily for 2.5 days (Shepherd et al., 2015b) or 20 mmol nitrate for 6 days (Friis et al., 2017) showed no benefit on the O₂ cost of exercise or 6-minute walk test (Shepherd et al., 2015b; Friis et al., 2017). Importantly, in these studies, plasma nitrite levels were increased, and BP was robustly reduced, suggesting that both the chemical and functional effects of the nitrate-nitrite-·NO pathway are present in patients with COPD. The difference between the responses in the two sets of studies is uncertain but may relate to differences in dosing schedules and warrants further investigation. Further evaluation of dietary nitrate supplementation (6.5 mmol/day nitrate for 8 days) in patients with COPD showed no improvement in gas diffusion or peak exercise capacity, despite robust increases in exhaled ·NO, but was associated with statistically significant (but small in magnitude) improvements in respiratory-focused quality of life (St. George’s Respiratory Questionnaire) scores (Behnia et al., 2018). Similar to the position with PAH, large adequately powered studies are awaited to definitively explore the therapeutic potential of inorganic nitrate in COPD.

3. Sleep-Disordered Breathing

Obstructive sleep apnea syndrome (OSA) is the most common form of sleep-disordered breathing problem and results from recurrent occlusion of the upper airway during sleep. Numerous epidemiologic studies have implicated OSA as a causative, independent factor for CVD (Arzt et al., 2005; Yaggi et al., 2005; Somers et al., 2008). The pathophysiological links between OSA and CVD are not fully understood, but several mechanisms, such as elevated sympathetic drive, increased oxidative stress, and vascular inflammation resulting from repeated nocturnal hypoxia/reoxygenation cycles, have been suggested (Somers et al., 2008). Importantly, ·NO levels are diminished in OSA and are partially restored with the most common, effective form of therapy, continuous positive airway pressure (Ip et al., 2000; Schulz et al., 2000; Alonso-Fernández et al., 2009), suggesting that ·NO delivery in this setting may be of value.

The only study exploring the nitrate-nitrite-·NO pathway in patients with OSA is restricted to a small (n = 3) 14-day, controlled crossover study. Nitrate supplementation (12.9 mmol nightly) was associated with increased plasma nitrate levels (nitrite was not measured) and with improvement in fatigue and visual attention scores, although no change in sleep quality or Epworth sleepiness scores, and changes in apnea/hypopnea index were not assessed. All patients had controlled BP levels on ambulatory monitoring at index, but there was a trend toward reduced nocturnal BP after nitrate supplementation (Kerley et al., 2016), providing confidence in the dosing schedule efficacy in this study.

Sleep-disordered breathing is also an occurrence in the acclimatization to hypobaric hypoxia at altitude. Given the reductions in O₂ consumption associated with nitrate supplementation seen at sea level (see earlier), Patrician et al. (2018) investigated the acute effects of acute nocturnal dietary nitrate supplementation (10 mmol/day nitrate) in lowlanders after ascent to 3700–4900 m, in comparison with a peri–sea level (300 m) on cardiorespiratory responses during sleep. Acute dietary nitrate supplementation had no effect on various measures of sleep-disordered breathing. Similar longer-term studies in lowland volunteers ascending to high altitude (4600 m) demonstrated that 7 days of dietary nitrate supplementation (3.7 mmol nitrate three times daily) had no effect on respiratory parameters, such as O₂ saturation, or acute mountain sickness, despite robust increase in fractional exhaled ·NO (Cumpstey et al., 2017).

Further studies are required to delineate the potential benefit of nitrite-derived ·NO in patients with sleep-disordered breathing, not only on the respiratory abnormalities but also on the elevated CV risk profile that such patients exhibit.

4. Toxic Lung Injury

Chlorine gas has been implicated in mass casualties due to accidental or deliberate exposure in civilian and military settings. The pathophysiology of chlorine gas–related lung injury includes pulmonary and systemic increased oxidative and nitrosative stress and hypoxemia, with persistence of airway remodeling and airway hyperreactivity over time (White and Martin, 2010). Since nitrite-derived ·NO has beneficial effects in I/R lung injury (Sugimoto et al., 2012; Okamoto et al., 2013), this has spurred interest as to whether it may be beneficial in other forms of lung injury. Rats exposed to 400 ppm chlorine gas for 30 minutes were given NaNO₂ (1 mg/g, i.p.) repeatedly for up to 6 hours and then euthanized. Treatment with nitrite was associated with reduced bronchoalveolar lavage protein levels and reduced epithelial cell apoptosis (Yadav et al., 2011), with some evidence that intramuscular delivery was more effective than intraperitoneal delivery (Samal et al., 2012), which is more translatable to the management of mass casualty disaster scenarios by first responders. In an adaptation of this methodology, in rats exposed to a higher exposure of chlorine (600 ppm for 30 minutes) that induces 80% fatality, a single intramuscular injection of nitrite (10 mg/kg) 30–60 minutes postexposure reduced mortality to 50%.

These findings have been repeated in a nonrodent model using New Zealand white rabbits. Chlorine gas (600 ppm, 45 minutes) is 35% lethal after 18 hours and is associated with airway protein and neutrophil accumulation. Nitrite (1 or 10 mg/kg, via i.m. injection) administered 30 minutes post–chlorine gas exposure prevented death in all rabbits, and this was associated with attenuated indices of acute lung injury (Honavar et al., 2017). Further mechanistic evaluation revealed that neutrophil depletion reduced the lethality of chlorine exposure but also abrogated any further improvement by nitrite, suggesting a neutrophil-dependent mechanism for nitrite-derived protection in this model (Honavar et al., 2014).

Taken together, these data provide further rationale for developing nitrite as a postexposure treatment to ameliorate chlorine gas exposure–related lung injury, although the pathway for further development of this approach is awaited given the complexities of trying to perform a clinical trial in this area.

1. Cytoprotection

There is a significant body of evidence demonstrating that nitrite operates as a major storage form of ·NO in blood and tissues (Bryan et al., 2005) that can be reduced readily to ·NO to initiate cytoprotective signaling during pathologic states, particularly during I/R injury (van Faassen et al., 2009; Calvert and Lefer, 2010). The mechanisms by which this process occurs have been extensively studied, with increasing evidence suggestive of changes in mitochondrial function and anti-inflammatory, antiplatelet, and vasodilatory effects.

2. Potential Mechanisms for Nitrite-Induced Reduction in I/R Injury

A number of distinct molecular pathways have been proposed to underlie the reduction of nitrite in the setting of I/R injury. There is strong evidence demonstrating that the benefits of nitrite in I/R injury are due, at least in part, to its conversion to ·NO. Several studies have shown that nitrite-induced protection is blocked by the ·NO scavenger C-PTIO (Webb et al., 2004; Jung et al., 2006; Tripatara et al., 2007). This effect is also independent of NOS activity, as cytoprotection induced by nitrite is not prevented by NOS inhibition (Webb et al., 2004; Lu et al., 2005) or affected in eNOS KO models (Duranski et al., 2005; Milsom et al., 2010). We now also understand that nitrite reduction during I/R is not just a phenomenon precipitated by acidic disproportionation, as suggested by Zweier et al. (1995), who identified nitrite-derived ·NO generation in the ischemic heart in their seminal findings in 1995. Over the past decade, a number of different nitrite reductases have been implicated in nitrite-derived ·NO formation during I/R injury, with the role of each catalytic pathway identified being influenced both by the site/organ of injury and the severity of hypoxia. Much of the research focused on identifying the key nitrite reductase involved in the I/R injury setting has been conducted within the heart, where it appears that the two key nitrite reductases are XOR and Mb.

XOR has been implicated in the reduction of nitrite to ·NO in the myocardium in both animal and human heart tissue, with the first studies being conducted in the isolated rat heart and atrial appendage collected from patients undergoing cardiac surgery, respectively. In tissue supernatants, nitrite-derived ·NO generation was profoundly attenuated (50%) after incubation with the XOR inhibitor allopurinol (Webb et al., 2004), observations that have been replicated by others (Baker et al., 2007; Li et al., 2008). Similarly, XOR has been implicated as the nitrite reductase in other organs, including when delivered topically in renal I/R injury in vivo in both rat and mouse models (Tripatara et al., 2007; Milsom et al., 2010). XOR also contributes to nitrite bioactivation in other hypoxic settings, including pulmonary hypertension (Baliga et al., 2012) and vascular remodeling after balloon injury (Alef et al., 2011), thus identifying a widespread function of XOR as a key nitrite reductase in hypoxic environments. This profile of activity is not unexpected, since studies with purified bovine enzyme demonstrate increasing XOR-dependent nitrite reduction with reducing O₂ tension (Li et al., 2001). This bioactivity, as mentioned earlier, occurs at the molybdenum site of the enzyme and is a reaction facilitated by at least three different reducing substrates, including xanthine, NADH, and aldehydes (Khambata et al., 2015). Perhaps of particular note in this respect is NADH, the levels of which rise in ischemic environments (Sahlin, 1983), possibly suggesting why XOR-dependent nitrite reduction predominates in such reduced O₂ tension environments and plays a much lesser role under physiologic conditions (Dejam et al., 2007).

Mb represents the other key nitrite reductase implicated in the bioactivation of nitrite during I/R injury and, again, particularly in the heart (Rassaf et al., 2007; Shiva et al., 2007a). Mb must be partially deoxygenated to act as a nitrite reductase, only occurring when O₂ levels fall below the p₅₀ of Mb, as for Hb (Cosby et al., 2003; Crawford et al., 2006). Studies in the heart suggest that 60%–70% of nitrite-derived ·NO is due to deoxyMb-dependent conversion, with the balance due to XOR activity (Rassaf et al., 2007; Shiva et al., 2007a). Accordingly, nitrite reduction was abolished in the Mb KO mouse (Hendgen-Cotta et al., 2008), and the beneficial effects of nitrite after coronary artery occlusion were lost, including improved postischemic LV developed pressure. These studies suggest that Mb plays an important role in nitrite-induced regulation of cardiac energetics and oxygen utilization under conditions of hypoxia. To date, whether Mb is also critical in nitrite reduction in the human heart has not been elucidated.

3. Potential Mechanisms for Nitrite-Induced Cytoprotection in I/R Injury

The activity of nitrite in I/R injury has been attributed to both direct and ·NO-dependent actions as a consequence of chemical modifications of both cellular proteins or lipids, mitigating the injury occurring at reperfusion. Potential molecular mechanisms for cytoprotection of the former include the direct S-nitrosation of critical regulatory thiols on proteins such as caspase-3, a pivotal protein of the apoptotic pathway, leading to its inactivation and preventing apoptotic cell death (Maejima et al., 2005), or the L-type calcium channel, which results in reduced cytosolic Ca²⁺ and subsequent I/R injury (Sun et al., 2006). There are several functional consequences of these chemical interactions that underlie the benefits of nitrite, with the key ones summarized below.

4. Nitrite as a Mediator of Remote Ischemic Preconditioning

In the early 1990s, Przyklenk et al. (1993) proposed the existence of signal transduction between the local site of remote ischemia and the myocardium. This link has been identified as underlying the efficacy of remote ischemic preconditioning (rIPC), although the exact nature of this endocrine communication has remained uncertain. In 2014, Rassaf and coworkers conducted a series of experiments suggesting that nitrite may be the elusive endocrine factor responsible for the effects of rIPC (Rassaf et al., 2014). Their studies demonstrated that plasma nitrite concentration increases during reactive hyperemia in the brachial artery of healthy volunteers as a consequence of shear-stress triggering eNOS activation (Rassaf et al., 2006). In mouse models of rIPC, the authors demonstrated a similar elevation in circulating nitrite levels and that the benefits of rIPC were blocked with C-PTIO and absent in eNOS KO mice. In the final experiment, the authors infused human plasma collected from individuals subjected to, or not, rIPC into isolated mouse heart Langendorff preparations. In these experiments, they demonstrated that nitrite accounted for the effects of rIPC, since treatment of the transferred plasma with acidified sulfanilamide eliminated the transfer of protection (Rassaf et al., 2014). Prior to this work, Elrod et al. (2008), who used a transgenic mouse with cardiac restricted overexpression of eNOS, also provided evidence in support of an endocrine action of nitrite. These mice are characterized by increased levels of nitrite and other ·NO metabolites in the circulation, resulting in subsequent transport and storage in organs throughout the body, including the liver. Elrod et al. (2008) demonstrated that this elevation of tissue nitrite provided protection against the damaging effects of a consequent hepatic I/R injury.

An important caveat of the above findings is that the levels of nitrite generated in these models are approximately 10-fold lower than those typically shown to be cytoprotective in preclinical models of I/R injury (Webb et al., 2004; Duranski et al., 2005). This discrepancy in efficacy implies potential differences in bioactivity/potency of endogenously derived versus exogenously delivered nitrite and is an issue that is worthy of further interrogation.

5. Translational Aspects