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Inorganic nitrate: a major player in the cardiovascular health benefits of vegetables?

Ajay Machha, Alan N Schechter
DOI: http://dx.doi.org/10.1111/j.1753-4887.2012.00477.x 367-372 First published online: 1 June 2012

Abstract

Epidemiological evidence suggests a higher consumption of vegetables confers a protective effect against the risk of cardiovascular disease. Impaired bioavailability of nitric oxide (NO), which is a critical regulator of vascular homeostasis, in the vasculature is thought to be a major problem in cardiovascular disease. Classically, vascular endothelium is suggested to be the sole source of bioactive NO in the vasculature. Emerging literature, however, associates the nitrate-nitrite-NO pathway, in which endogenous nitrate undergoes reduction to nitrite and then to NO in various tissues, including blood, with the production of bioactive NO. Indeed, NO generated from the nitrate-nitrite-NO pathway has recently been associated with the maintenance of NO homeostasis in the body. Endogenous nitrate originates mostly from NO oxidation in the biological milieu and from exposure to dietary nitrate. Consumption of vegetables accounts for approximately 80–85% of daily nitrate exposure in humans, thereby establishing inorganic nitrate as a promising factor in the cardiovascular health benefits of vegetables. At this point in time, however, the benefit : hazard ratio of inorganic nitrate and its active metabolite nitrite remains less clear and must be studied in prospective controlled studies. This brief review discusses the potential role of inorganic dietary nitrate in the cardiovascular health benefits of vegetables.

  • cardiovascular
  • diet
  • nitrate
  • nitric oxide
  • vegetables

INTRODUCTION

Epidemiological evidence suggests that increased consumption of vegetables reduces the risk of cardiovascular disease, which is the leading cause of mortality in the Western world.15 Although this benefit was traditionally postulated to be linked to the antioxidant factors in vegetables, studies over the past two decades have proposed many nonantioxidant factors as likely candidates as well. Of late, one such factor that has garnered much scientific and medical interest is inorganic nitrate. Vegetables are a rich source of inorganic nitrate and account for approximately 80–85% of daily dietary nitrate exposure in the average population.69 Several recent experimental and clinical studies show that dietary nitrate supplementation at doses commonly found in vegetable-rich diets exerts beneficial effects on the cardiovascular system.1016 These beneficial effects of nitrate are largely thought to be mediated by the reduction of nitrate to nitric oxide (NO) in the body, since NO is a critical regulator of vascular homeostasis.1016 Current data suggest that nitrate undergoes reduction to nitrite and then to NO through a nitrate-nitrite-NO pathway, which recently was appreciated as an alternative pathway to the classical L-arginine-nitric oxide synthase (NOS) pathway for NO production in the body.1721 In the present short review, a potential role for inorganic nitrate in the cardiovascular health benefits of vegetables is discussed. Nutritional epidemiologists often join vegetables with fruits when discussing cardiovascular health benefits because these foods, in general, share similar nutrients, phytochemicals, and functional aspects. However, in the context of the present review, it is unwise to join these foods together since the inorganic nitrate content of fruits and, accordingly, the contribution of fruits to endogenous nitrate levels is severalfold lower (or negligible) in comparison with vegetables.6,7

NITRIC OXIDE SYNTHESIS FROM THE CLASSICAL L-ARGININE-NITRIC OXIDE SYNTHASE PATHWAY

NO synthesis from the classical L-arginine-nitric oxide (NOS) pathway involves L-arginine oxidation by three different isoforms of the NOS enzyme (i.e., endothelial [eNOS], neuronal [nNOS], and inducible [iNOS]).2224 The three NOS isoforms exhibit different characteristics and produce NO at different rates. The eNOS is associated with the plasma membrane, whereas nNOS and iNOS are found predominantly in the cytosol.2224 In addition, eNOS and nNOS are expressed constitutively as latent enzymes in healthy cells and require a higher calcium concentration for their enzymatic activity, whereas iNOS is expressed mainly in the presence of infection or inflammation, and its activity is calcium independent.2224 Although all three NOS isoforms play distinct roles, eNOS is thought to be involved mainly in the regulation of the cardiovascular system because it is the major source of basal and stimulated NO synthesis in the vasculature.2224 The eNOS has a bidomain (oxygenase and reductase) structure and functions as a dimer.2224 The catalysis of L-arginine oxidation by eNOS requires the availability of molecular oxygen and several cofactors (nicotinamide adenine dinucleotide phosphate [NADPH], flavin mononucleotide [FMN], flavin adenine dinucleotide [FAD], tetrahydrobiopterin [BH4], and calcium/calmodulin). The catalytic mechanisms of eNOS involve the stepwise transfer of electrons from NADPH to FAD to FMN to a heme-containing oxygenase domain where oxygen is reduced and incorporated into the guanidine group of L-arginine, leading to NO production. The eNOS can be stimulated by a variety of mechanical forces (e.g., shear stress, hypoxia), humoral factors ranging from growth factors to peptide hormones (e.g., acetylcholine, vascular endothelial growth factor, bradykinin, estrogen), and calcium-mobilizing agents (e.g., angiotensin-II, epinephrine).2224 For example, acetylcholine leads to an increase in intracellular calcium in endothelial cells, activation of calmodulin, alignment of the oxygenase and reductase domains of the eNOS, and, ultimately, to efficient NO synthesis.25

NITRIC OXIDE SYNTHESIS FROM THE NITRATE-NITRITE-NITRIC OXIDE PATHWAY

NO generation from endogenous nitrate was recently discovered as an alternative pathway to the classical L-arginine-eNOS pathway for the production of bioactive NO in the body (Figure 1).1721 Interestingly, endogenous nitrite and nitrate originate predominantly from the oxidation of endogenously produced NO in the biological fluids.19 In addition, a significant portion of body nitrate also originates from the diet, particularly from the consumption of plant foods.19 Current data suggest that nitrate reduction to NO in the body involves the initial reduction of nitrate to nitrite and then to NO (Figure 1).1721 Nitrate reduction to nitrite has been largely thought to be carried out by commensal bacteria present on the dorsal surface of the tongue and possibly in the gastrointestinal tract.1721 The bacteria use nitrate as an alternative electron acceptor to produce energy, thereby effectively reducing nitrate to nitrite. Indeed, the use of antibacterial mouthwash11 or the spitting of saliva12 after dietary nitrate load has been reported to attenuate the expected rise in systemic nitrite levels. In addition, more recently, allopurinol-sensitive nitrate reductase enzymes that reduce nitrate to nitrite have been suggested to contribute to the reduction of nitrate to nitrite in the body, but the magnitude of this pathway is not clear.13 Once formed, nitrite can be further reduced to NO by a variety of sources in the body, including but not limited to deoxyhemoglobin and xanthine oxidoreductase.1721 It is important to note that, at present, the relative contribution of these pathways to NO production from the nitrate-nitrite-NO pathway and to vascular NO levels remains unknown. Interestingly, unlike NO production from the eNOS, whose activity is oxygen dependent, NO production from the nitrate-nitrite-NO pathway has been suggested to be increased with diminished oxygen.1721 In this context, NO generation from the nitrate-nitrite-NO pathway should be viewed as an alternative source of NOS-dependent NO production in the body.1721 The nitrate and nitrite reduction mechanisms are the subject of detailed discussions in earlier reviews1726 and are not discussed further here.

Figure 1

Schematic presentation of inorganic nitrate (NO3-) mediation of cardiovascular protection by vegetable-rich diets. After vegetables are ingested, NO3- is absorbed into the circulation, where it undergoes reduction to nitrite (NO2-) and then to nitric oxide (NO), leading to possible cardiovascular protection. NO3- and NO2- reduction pathways and classical L-arginine-endothelial nitric oxide synthase (eNOS) pathway for NO production in the body are depicted.

NITRIC OXIDE, VASCULAR HOMEOSTASIS, AND CARDIOVASCULAR DISEASE

NO influences vascular homeostasis in many ways.2730 For example, NO enters vascular smooth muscle cells, where it increases production of cyclic guanosine 3′,5′-monophosphate by activating soluble guanylyl cyclase enzyme, leading to smooth muscle relaxation (vasodilation). In addition, NO inhibits platelet aggregation and platelet adhesion to the endothelium. Moreover, NO suppresses the proliferation of vascular smooth muscle cells, the adhesion and migration of leukocytes/monocytes into the arterial wall, the activity of inflammatory factors, and the expression of certain adhesion molecules. Therefore, NO represents the most important regulator of vascular homeostasis, and its loss of function has been implicated in a variety of cardiovascular diseases, including hypertension and atherosclerosis.3133 Diminished synthesis and/or increased depletion of NO, as from increased levels of superoxide anions, has been implicated predominantly in the reduction in NO bioavailability in the vasculature.3133 Superoxide anions react with NO in a near-diffusion-controlled reaction to form peroxynitrite, leading subsequently to the depletion of NO in the vasculature.3133

CARDIOVASCULAR PROTECTION BY DIETARY NITRATE

Nitrate has been used to treat cardiovascular disease conditions (angina and digital ischemia) since medieval times, as evidenced by a translation of medieval Buddhist manuscripts.19 However, it is only in the past several years that knowledge regarding the potential cardiovascular beneficial effects of nitrate, particularly the nitrate present in vegetables, has advanced enormously. Current data suggest that dietary nitrate influences the cardiovascular system by increasing the vascular bioavailability of NO via the nitrate-nitrite-NO pathway1721 (Figure 1). In this context, studies over the past two decades demonstrated that antioxidants and other interventions that can improve NO bioavailability in the vasculature, presumably through their interaction with the vascular endothelium, can lead to improved clinical outcome in patients with cardiovascular disease.32,33 Dietary nitrate deficiency,10 dietary nitrate load,12 and/or supplementation with inorganic nitrate salts (e.g., sodium nitrate, potassium nitrate)1416 have been used to investigate the cardiovascular effects of dietary nitrate in animals and humans.

Animal studies

Bryan et al.10 demonstrated that mice fed a diet low in nitrite and nitrate (i.e., dietary nitrite and nitrate deficiency) displayed exacerbated myocardial injury (up by 59%) and postmyocardial mortality rates (up by 13%) compared with a control group of mice that received a standard diet. This study also demonstrated that nitrate supplementation for 7 days (1 g/L in drinking water) afforded significant protection against myocardial ischemic injury in mice fed a standard diet as well as in mice fed a diet low in nitrite and nitrate.10 In another study, Jansson et al.13 demonstrated that intravenous administration of nitrate (sodium nitrate, 10 mg/kg) reduced mean arterial blood pressure (∼10% versus placebo group, which received sodium chloride) in control rats as well as in rats pretreated with Nω-nitro-L-arginine methyl ester (L-NAME, a nonspecific NOS inhibitor that inhibits endogenous NO production). In this study, nitrate administration also improved the aortic blood flow in rats, albeit by a nonsignificant level.13 More recently, Carlstrom et al.16 demonstrated that supplementation with sodium nitrate (85 mg/L, 1 mM), at a dose readily achievable through diet, reduced blood pressure and circulating levels of triglycerides and improved glucose homeostasis (i.e., features of metabolic syndrome) in eNOS-deficient mice. Overall, the results from these animal studies show that dietary nitrate exposure may reverse or improve pathological changes that are paralleled by the loss of endothelium-derived NO bioavailability, leading to improved cardiovascular parameters.

Human studies

Few studies in humans have investigated the cardiovascular effects of dietary nitrate, and the results from these studies are consistent with those of the animal studies. Larsen et al.14 demonstrated that dietary supplementation with sodium nitrate (0.1 mmol/kg of body weight/day, for 3 days) in doses equivalent to a diet rich in vegetables reduces blood pressure (reduction in diastolic blood pressure, ∼3.5 mmHg; reduction in mean arterial pressure, ∼3.2 mmHg; and no change in systolic blood pressure) in healthy humans. In addition, in another study, Webb et al.12 demonstrated that ingestion of a dietary nitrate load (500 mL of beetroot juice, containing ∼45 mmol/L or ∼2.79 g/L of inorganic nitrate) lowers blood pressure (3 h postingestion, reductions in mmHg: systolic ∼10.4 ± 3, diastolic ∼8.1 ± 2.1, mean arterial pressure ∼8.0 ± 2.1), inhibits platelet aggregation (2.5 h postingestion, adenosine diphosphate 30 µM-induced aggregation was reduced by ∼20%), and improves ischemia-induced endothelial dysfunction (∼30% increase in flow-mediated dilation compared with control subjects after acute ischemia) in healthy volunteers. Moreover, using a randomized crossover study design, Kapil et al.15 recently demonstrated that nitrate ingestion by means of either supplementation (i.e., potassium nitrate capsules) or elevation of dietary intake (i.e., 250 mL of beetroot juice, 5.5 mmol of nitrate) reduces blood pressure in healthy volunteers.

DIETARY NITRATE AND RISK OF CANCER

A major health concern with dietary nitrate intake is the risk of development of cancer because of the proposed association between nitrate and the in vivo formation of N-nitrosamines, a class of carcinogenic substances.34 However, experimental studies and epidemiological studies failed to consistently show either increased formation of N-nitrosamines or increased risk of cancer with increasing consumption of dietary nitrate.638 For example, Pannala et al.38 demonstrated no significant change in plasma 3-nitrotyrosine concentrations in healthy volunteers following high dietary intake of nitrate (<3.65 mg/kg body weight or ∼250 mg nitrate). Indeed, in 2003, the Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives reviewed studies that investigated a possible association between nitrate intake and cancer risk and concluded there was no evidence that nitrate was carcinogenic to humans.39 Most importantly, epidemiological evidence mostly indicates that abundant consumption of vegetables reduces the risk of cancer.40,41 Collectively, these studies suggest that dietary nitrate does not exert carcinogenic activity in humans and would not be harmful to human health via this mechanism.

VEGETABLES AS A SOURCE OF DIETARY NITRATE INTAKE

Vegetables are the main source of dietary nitrate exposure and represent about 80–85% of the nitrates consumed per day (see Table 1 for the classification of vegetables on the basis of their nitrate content).69 In the United States, the average intake of dietary nitrate was found to be approximately 40–100 mg/day, whereas reported international estimates of daily nitrate intake range between 53 mg/day and 350 mg/day.9,39 This variability could be due to several factors, including number of servings, species, fertilizer application, maturity, and storage conditions. For example, the average nitrate content of spinach collected from different markets in Delhi, India, varied from 710 mg/kg to 4,293 mg/kg fresh weight.42 Following ingestion of vegetables, nitrate is rapidly absorbed in the small intestine, enters circulation, mixes with NO-derived nitrate, and readily distributes throughout the body.43,44 In one study, the ingestion of beetroot juice (500 mL, mean nitrate concentration ∼45 mmol/L) by healthy volunteers rapidly increased plasma nitrate levels (approximately 16-fold).12 The half-life of nitrate after ingestion is about 5–7 h, and approximately 60–70% of the ingested nitrate is rapidly excreted unchanged in urine.43,44 Unlike nitrate content, the nitrite content of vegetables is very low (<10 mg per kg) and rarely exceeds 100 mg/kg.69 However, nitrite levels of up to 400 mg/kg have been found in vegetables that have been damaged, poorly stored, or stored for extended periods as well as pickled or fermented.45

View this table:
Table 1

Classification of vegetables according to inorganic nitrate content.

NITRATE VERSUS OTHER CONSTITUENTS OF VEGETABLES IN CARDIOVASCULAR PROTECTION

Studies over the past two decades have demonstrated that, apart from inorganic nitrate, several constituents of vegetables, particularly polyphenols and vitamin C, can also improve cardiovascular health.4648 The effects of such constituents are attributed to improvement in endothelial function, achieved mainly through increased endothelial NO production and/or bioavailability.4648 It is now clear that endothelial NO production is oxygen dependent, meaning that endothelial NO-driven processes decline with depletion of normal oxygen levels. In addition, cardiovascular diseases share a common pathophysiology involving depletion of normal oxygen levels, coupled with diminished blood supply due to atherosclerosis (i.e., thickening or narrowing of the arteries due to the development of plaques in the arterial wall) and/or thrombosis (i.e., arterial blood clotting).4952 For example, thickening of coronary arteries can restrict blood supply to the myocardium, leading to myocardial infarction or acute heart attack. Moreover, as discussed above, NO production from the nitrate-nitrite-NO pathway increases with a decrease in oxygen. In this regard, indeed, a growing number of studies demonstrate that NO generation from the nitrate-nitrite-NO pathway may contribute to hypoxic vasodilation.5355 Furthermore, emerging data indicates that polyphenols and vitamin C are able to reduce nitrite to NO, which, in turn, may exert cardiovascular protection (Figure 1).56,57 For example, Rocha et al.57 recently demonstrated that quercetin, a polyphenolic antioxidant flavonoid widely available in vegetables, potentiated vasodilating effects of nitrite by enhancing NO production from nitrite under acidic conditions. Putting these findings together suggests an intriguing possibility that inorganic nitrate may play a major role in the apparent protective effects of vegetables against cardiovascular disease. Indeed, this would explain the findings from recent large cohort studies that demonstrated that green leafy vegetables, which are a rich source of inorganic nitrate, and vitamin-C-rich fruits and vegetables contribute most to the cardiovascular protective effects of total fruit and vegetable intake.2,3

CONCLUSION

From the studies presented above, it appears that inorganic nitrate may play a major role in the cardiovascular health benefits of vegetables, presumably through enhancing NO bioavailability in the vasculature. At this point in time, however, the contribution of this pathway relative to the total cardiovascular benefit of vegetable-rich diets as compared with other potential mechanisms remains unknown.

Acknowledgments

All the authors contributed equally to the manuscript. Dr Barbora Piknova (Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases) is thanked for her valuable input.

Declaration of interest.  ANS is a co-inventor of a patent held by the National Institutes of Health for the use of nitrite salts for the treatment of cardiovascular diseases.

REFERENCES

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