Production of nitric oxide in the body

Sources of dietary inorganic nitrate and nitrite

N is vital to life on Earth and can undergo many chemical and biological changes in order to be amalgamated into living and non-living material. An essential form of environmental N includes inorganic nitrate, as an adequate nitrate supply in the soil is essential for plant growth⁽ Reference Bryan and Loscalzo ^{43 ,} Reference Crawford ^{78 )}.

The two major determining factors of the nitrate content of vegetables and fruit include their species and the amount of available nitrate in the soil⁽ Reference Bryan and Loscalzo ^{43 )}. Some species of vegetables such as green leafy vegetables (mean nitrate about 975–3624 mg/kg) and beetroot (mean nitrate about 1992 mg/kg) are naturally high in nitrate; however, environmental factors can lead to great variation among samples⁽ Reference Hord, Tang and Bryan ^{22 )}. These factors include seasonal differences and disruption to normal plant growth, leading to nitrate accumulation in the plant leaves, stems and stalks, due to changes in the photosynthetic conversion of plant nitrate to amino acids⁽ Reference Crawford ^{78 –} Reference Kaiser and Brendle-Behnisch ^{80 )}. Therefore, established factors shown to effect the normal growth of plants include drought conditions, high temperatures, shady and cloudy conditions, deficiency of soil nutrients, and excessive soil N⁽ Reference Bryan and Loscalzo ^{43 )}. Additionally, farming practices leading to damaged produce, early harvest, storage and transport conditions, processing and cooking practices will also result in significant variation in vegetable and fruit nitrate content⁽ Reference Bryan and Loscalzo ^{43 )}.

European-based studies have demonstrated that organically grown vegetables have a lower nitrate content than conventionally grown crops, despite the fact that organic fertilisers may cause high nitrate levels in vegetables, depending on the types and amount of organic fertilisers applied⁽ Reference Muramoto ^{81 )}. A California-based study by Muramoto⁽ Reference Muramoto ^{81 )} reiterated this notion, as it found that spinach grown and harvested during the same season and under the same farming practices had a wide range of nitrate contents. This range appeared greatest in organic spinach, in which the maximum nitrate content measured was 3000 mg/kg, which was five times higher than the minimum (600 mg/kg)⁽ Reference Muramoto ^{81 )}. However, this study also demonstrated that conventionally grown spinach contained on average 30 % more nitrate than spinach grown organically, a result most probably explained due to the wide use of N-containing fertilisers in conventional farming⁽ Reference Muramoto ^{81 )}.

Muramoto⁽ Reference Muramoto ^{81 )} also found a statistically significant seasonal difference in the nitrate content of iceberg lettuce, as winter samples were found to have on average 52 % more nitrate than summer samples⁽ Reference Muramoto ^{81 )}. This finding is consistent with Ekart et al. ⁽ Reference Ekart, Gorenjal and Madorran ^{82 )}, which found lettuce harvested during summer had a statistically significant lower nitrate content than lettuce harvested during winter (summer harvest: 1209 mg/kg; winter harvest: 2164 mg/kg). In addition, Ekart et al. ⁽ Reference Ekart, Gorenjal and Madorran ^{82 )} found that washing leafy greens reduced the nitrate content of foods on average by 19 %. Other processing, such as boiling, blanching and sautéing, were found to significantly reduce the nitrate content of spinach by 53, 36 and 30 %, respectively⁽ Reference Ekart, Gorenjal and Madorran ^{82 )}, a finding which could be partly explained due to the water-soluble nature of inorganic nitrate⁽ Reference Omar, Artime and Webb ^{83 )}.

Due to the high variability of nitrate within plant species, accurate and reliable nitrate intake measured from fruit and vegetable consumption is difficult to predict. Despite this, combined vegetable and fruit intake is the major source of exogenous inorganic nitrate exposure and is predicted to constitute 30–90 % of total nitrate intake⁽ Reference Du, Zhang and Lin ^{84 )}. Other sources of nitrate intake include drinking water and meat products; however, their nitrate content is highly regulated to comply with strict government limits^{( 85 –} Reference Shuval and Gruener ^{89 )}.

Nitrate occurs naturally in the water supply; however, in most developed countries water nitrate is generally present in concentrations much lower than allowed in the water guidelines (≤50 mg/l)^{( 85 , 86 ,} Reference Ward, DeKok and Levallois ^{88 )}. Therefore, nitrate from the water supply is unlikely to contribute significantly to total nitrate intake in comparison with food sources.

Nitrate and nitrite salts (for example, potassium nitrite/sodium nitrate) have been used as food additives in cured meats for many years due to their effectiveness in ensuring microbial safety and their ability to enhance the flavour and appearance of the product⁽ Reference Bryan and Loscalzo ^{43 )}. The maximum levels of nitrate and nitrite allowed as food additives have been defined (Table 1)^{( 85 ,} Reference Jukes ^{90 – 92 )}.

Table 1 Permissions for nitrate and nitrite in food productsFootnote *

* Nitrate salt: potassium nitrate and sodium nitrate. Nitrite salt: potassium nitrite and sodium nitrite.

It has been estimated that approximately 60–80 % of dietary nitrates are derived from vegetables (mainly green leafy and root vegetables), indicating that vegetable intake tends to contribute the greatest quantities of dietary nitrate (Table 2)⁽ Reference Hord, Tang and Bryan ^{22 ,} Reference Weitzberg and Lundberg ^{93 )}. This has been further implied by dietary patterns such as the DASH diet, Mediterranean, vegetarian and traditional Japanese diets which tend to include high quantities of vegetables (five or more serves per d) and provide approximately 147–1222 mg nitrate per d⁽ Reference Hord, Tang and Bryan ^{22 –} Reference L’Hirondel and L’Hirondel ^{24 )}. This is a relatively high nitrate intake compared with the typical Western-style diet which tends to be low in vegetables (one to three serves per d) and provides about 60–75 mg nitrate per d⁽ Reference L’Hirondel and L’Hirondel ^{24 )}. In addition, processed and cured meats are frequently cited as the major dietary source of nitrite (Table 3)⁽ Reference Hord, Tang and Bryan ^{22 ,} Reference Lundberg and Weitzberg ^{25 ,} Reference Du, Zhang and Lin ^{84 ,} Reference Machha and Schechter ^{94 )}, followed by various fruits and vegetables (Tables 2, 4 and 5) that have been physically damaged or poorly stored, as enzymes present in the plant tissues and/or contaminating bacteria facilitate the reduction of nitrate to nitrite⁽ Reference Bryan and Loscalzo ^{43 , 85 )}.

Table 2 Vegetable sources of nitrate and nitrite with estimated nitrate and/or nitrite contentsFootnote * (Mean values and ranges)

NA, data not available; ND, not detected.

* Data are combined nitrate and nitrite estimates from various published papers, government documents and reviews.

Table 3 Meat-based sources of nitrate and nitrite with estimated nitrate and/or nitrite contentsFootnote * (Mean values and ranges)

ND, not detected; NA, data not available.

* Data are combined nitrate and nitrite estimates from various published papers, government documents and reviews.

Table 4 Fruit sources of nitrate and nitrite with estimated nitrate and/or nitrite contentsFootnote * (Mean values and ranges)

NA, data not available; ND, not detected.

* Data are combined nitrate and nitrite estimates from various published papers, government documents and reviews.

Table 5 Nitrate- and nitrite-containing herbs with estimated nitrate and/or nitrite contentsFootnote * (Mean values and ranges)

ND, not detected.

* Data are combined nitrate and nitrite estimates from various published papers, government documents and reviews.

Nitric oxide in the cardiovascular system

Within the cardiovascular system, basal endothelial NO has a critical role in maintaining cardiovascular health as it controls vascular tone, smooth muscle cell proliferation and growth, platelet activity and aggregation, leucocyte trafficking, expression of adhesion molecules and inflammation⁽ Reference Li and Förstermann ^{34 ,} Reference Machha and Schechter ^{94 –} Reference De Caterina, Libby and Peng ^{99 )}. However, when the bioavailability of NO is compromised, the beneficial effects of NO are lost and endothelial dysfunction predominates due to the imbalance created between the release of vasoconstrictors and vasodilators (such as NO)⁽ Reference Hobbs, George and Lovegrove ^{53 ,} Reference Versari, Daghini and Virdis ^{100 ,} Reference Vanhoutte ^{101 )}. This idea has been supported in a study conducted by Kleinbongard et al. ⁽ Reference Kleinbongard, Dejam and Lauer ^{102 )} which found that plasma nitrite levels are a reliable indicator of endothelial dysfunction and correlate with cardiovascular risk factors in humans. Additionally, endothelial dysfunction has been strongly linked with atherosclerosis development and a number of cardiovascular disorders such as hypertension, CAD, congestive heart failure and peripheral artery disease in multiple longitudinal studies⁽ Reference Hobbs, George and Lovegrove ^{53 ,} Reference Vanhoutte ^{101 ,} Reference Landmesser and Drexler ^{103 –} Reference Gokce, Keaney and Hunter ^{107 )}.

While in the past most of the evidence suggesting a relationship between endothelial dysfunction and clinical events from atherosclerosis development was considered ‘circumstantial’, more recently conducted cross-sectional studies have indicated that severe endothelial dysfunction of the arteries can trigger events of unstable angina and MI⁽ Reference Vita and Keaney ^{108 ,} Reference Schächinger, Britten and Zeiher ^{109 )}. Al Suwaidi et al. ⁽ Reference Al Suwaidi, Hamasaki and Higano ^{104 )} studied 157 patients with mild CAD for 2·3 years, and found an increased incidence of cardiovascular events in patients with impaired endothelium-dependent vasodilation (NO production of endothelium) of the coronary arteries. In another study by Katz et al. ⁽ Reference Katz, Hryniewicz and Hriljac ^{110 )}, 259 subjects with chronic heart failure were assessed prospectively, to which endothelial dysfunction in chronic heart failure was found to significantly increase risk of mortality, thus supporting the notion that coronary endothelial dysfunction plays a role in the pathogenesis of coronary atherosclerosis, risk of cardiac events and death⁽ Reference Al Suwaidi, Hamasaki and Higano ^{104 ,} Reference Katz, Hryniewicz and Hriljac ^{110 )}.

Many factors are known to predispose to endothelial dysfunction, due to reductions in NO concentrations and bioavailability in humans⁽ Reference Li and Förstermann ^{34 ,} Reference Lidder and Webb ^{111 ,} Reference Lundberg, Gladwin and Weitzberg ^{112 )}. These factors are consistent with the modifiable and non-modifiable risk factors for CVD, including hypertension, hypercholesterolaemia, diabetes, tobacco use, physical inactivity, consumption of unhealthy diets and increased age and sex (NO bioavailability is reduced in postmenopausal women, a period in which CVD risk is drastically increased in women)⁽ Reference Li and Förstermann ^{34 ,} Reference Lundberg, Gladwin and Weitzberg ^{112 –} Reference Celermajer, Sorensen and Spiegelhalter ^{120 )}. Interestingly, improved endothelial function is a common feature of experimental intervention studies, which have shown reductions in cardiovascular risk and improvements in endothelial-dependent vasodilation in the coronary and peripheral circulation⁽ Reference Vita and Keaney ^{108 )}. Such interventions commonly include the use of lipid- and blood pressure-lowering medications, smoking cessation and increased physical activity⁽ Reference Vita and Keaney ^{108 ,} Reference Tsuchiya, Asada and Kasahara ^{117 ,} Reference Celermajer, Sorensen and Georgakopoulos ^{121 –} Reference Hornig, Maier and Drexler ^{124 )}. However, the notion that inorganic nitrate and nitrite either consumed from dietary sources such as green leafy vegetables or supplements is relatively new, and their therapeutic potential as an NO donor via the nitrate-nitrite–NO pathway remains unclear⁽ Reference Lundberg, Gladwin and Weitzberg ^{112 ,} Reference DeVan, Brooks and Evans ^{125 )}.

Cardiovascular protective actions of nitric oxide

NO is non-polar and can diffuse freely across cell plasma membranes and is a key signalling molecule capable of many important functions, acting primarily by stimulating intra-cellular receptors within the target cell⁽ Reference Wilson and Hunt ^{126 )}.

Within the vasculature of the cardiovascular system, the primary role for NO’s action is for the regulation of vascular function and blood pressure, a notion which has been clearly demonstrated in animal models in which synthesis of NO was blocked leading to persistently elevated blood pressure⁽ Reference Lundberg, Gladwin and Weitzberg ^{112 ,} Reference Channon, Qian and George ^{127 )}. In addition, this interaction has been demonstrated in some recently conducted short-term dietary nitrate trials in human subjects, which showed that peak blood pressure-lowering effects were achieved in synchronisation with peak plasma concentrations of NO (NO_x) after a dietary nitrate load⁽ Reference Hobbs, Kaffa and George ^{28 ,} Reference Webb, Patel and Loukogeorgakis ^{33 ,} Reference Hobbs, Goulding and Nguyen ^{128 )}.

The cellular pathway in which NO exerts this vasodilatory action is well established. NO rapidly diffuses across vascular smooth muscle cell membranes. Within the smooth muscle cells, NO binds to and activates guanylyl cyclase to produce cyclic GMP⁽ Reference Wilson and Hunt ^{126 )}. Once produced, cyclic GMP can have a number of effects in the cells, but many of these effects are mediated through the activation of protein kinase G. Activation of protein kinase G via cyclic GMP leads to the activation of myosin phosphatase which in turn leads to smooth muscle cell relaxation and vasodilation⁽ Reference Wilson and Hunt ^{126 ,} Reference Channon, Qian and George ^{127 )}.

In addition to regulating vascular tone, NO can facilitate many other important functions preventing the development of atherosclerosis, which include antiplatelet effects, anti-proliferative effects, anti-inflammatory, and antioxidant effects⁽ Reference Channon, Qian and George ^{127 ,} Reference Simon, Stamler and Jaraki ^{129 ,} Reference Clapp, Hingorani and Kharbanda ^{130 )}. Although the cellular pathways for these actions are yet to be clearly defined, it is clear that NO is capable of binding to or reacting with a variety of chemical modalities within the cellular environment, including metal-containing proteins, membrane receptors, ion channels, enzymes, transcription factors and oxygen species⁽ Reference Channon, Qian and George ^{127 ,} Reference Vallance and Webb ^{131 )}.

Other nitric oxides and possible mechanisms in the cardiovascular system

While NO is the most widely cited bioactive metabolite underpinning the cardiovascular therapeutic benefits of dietary inorganic nitrates and nitrites, it has been suggested that other nitric oxides also play a role⁽ Reference Lundberg and Weitzberg ^{25 ,} Reference Weitzberg and Lundberg ^{93 )}. This may be expected, given that dietary constituents in the stomach may react with each other in order to form a variety of bioactive compounds⁽ Reference Lundberg and Weitzberg ^{25 )}. Examples of such compounds include nitrated fatty acids, nitrosothiols and ethyl nitrite⁽ Reference Lundberg and Weitzberg ^{25 )}.

While the biological significance of these compounds is yet to be made clear, the following actions have been suggested:

1. (1) Ethyl nitrite. Rat models have shown that ethanol from alcoholic drinks can interact with salivary-derived nitrite in the acidic stomach, leading to the production of ethyl nitrite⁽ Reference Lundberg and Weitzberg ^{25 ,} Reference Gago, Nyström and Cavaleiro ^{132 )}. Ethyl nitrate is a potent smooth muscle relaxant and may have a vasodilatory role in the cardiovascular system⁽ Reference Gago, Nyström and Cavaleiro ^{132 )}.

2. (2) Nitrosothiols. In the stomach, nitrite has been shown to induce S-nitrosation within the gastric compartment. S-nitrosothiols are thought to represent a circulating endogenous reservoir of NO acting as an NO donor⁽ Reference Lundberg and Weitzberg ^{25 )}.

3. (3) Nitrated fatty acids (nitroalkenes). Nitric oxides can react with unsaturated fatty acids to produce nitroalkenes. Analysis of synthetic nitroalkenes derivatives of oleic, linoleic and arachidonic acids reveals that these species possess unique chemical reactions which may support multiple cell signalling events such as vasodilation and reduced inflammation⁽ Reference Lundberg and Weitzberg ^{25 )}. Such events may be mediated through their NO donor capabilities.

Currently the systemic capabilities of these bioactive N compounds remain uncertain; however, it highlights a possible whole-diet effect for exerting a beneficial effect on NO and other relevant cardiovascular signalling molecules. This notion is highlighted by Lundberg and Weitzberg⁽ Reference Lundberg and Weitzberg ^{25 ,} Reference Weitzberg and Lundberg ^{93 )}, indicating that various dietary constituents of the Mediterranean diet may interact in the stomach to produce these potentially therapeutic compounds, and may provide an additional explanation for the cardiovascular health benefits/protection seen with this dietary pattern.

Inorganic v. organic nitrate and nitrite

Organic nitrates such as glyceryl trinitrate and isosorbide mononitrate represent the first class of NO donors to reach the clinical setting and have been used extensively in the treatment of various cardiovascular conditions including angina, CAD and heart failure⁽ Reference Omar, Artime and Webb ^{83 )}.

Unlike inorganic nitrates which are relatively simple molecules and naturally occurring in fruits and vegetables, organic nitrates are synthetic compounds produced by a reaction between nitric acid and an alcohol group⁽ Reference Omar, Artime and Webb ^{83 )}. Organic nitrates are complex, non-polar hydrocarbon chains attached to a nitro-oxy-radical (–ONO₂), which is responsible for its biological effects (Fig. 2)⁽ Reference Omar, Artime and Webb ^{83 )}.

Fig. 2 Chemical structure of inorganic nitrate/nitrite compared with organic mono-, di-, tri- and tetra-nitrates/nitrites. 5-ISMN, isosorbide-5-mononitrate; ISDN, isosorbide dinitrate; GTN, glyceryl trinitrate; ETN, erythritol tetranitrate; PETriN, pentaerythrityl trinitrate; PETN, pentaerythritol tetranitrate. Reprinted from Omar et al. ⁽ Reference Omar, Artime and Webb ^{83 )}, with permission from Elsevier.

Once organic nitrates are introduced to the blood system, levels rise quickly leading to the rapid onset of their action⁽ Reference Omar, Artime and Webb ^{83 )}. At low doses (≤1·25 mg/kg body weight) organic nitrate has been demonstrated to dilate large conductance veins and large arteries, while at high doses (2·5–5 mg/kg body weight) organic nitrates can also induce dilation of the arterioles of the microcirculation⁽ Reference Omar, Artime and Webb ^{83 )}. These vasodilatory effects of organic nitrates have been shown to reduce cardiac work and lower myocardial oxygen requirements, which may alleviate or even prevent cases of MI⁽ Reference Klemenska and Beresewicz ^{133 )}. In addition, it has been suggested that organic nitrates have anti-aggregatory properties in patients with stable and unstable angina⁽ Reference Klemenska and Beresewicz ^{133 )}.

Today in clinical practice short-acting organic nitrates, most notably in the form of glyceryl trinitrate, are administered during the symptomatic treatment of MI and angina⁽ Reference Omar, Artime and Webb ^{83 ,} Reference Klemenska and Beresewicz ^{133 )}. Glyceryl tri-nitrates are generally administered in the form of either a mouth spray or intravenous infusion, to which onset of action is rapid (2–3 min)⁽ Reference Klemenska and Beresewicz ^{133 )}. Although short-term treatment with organic nitrates has some positive impact on endothelial function, acute side effects of their use include hypotension, dizziness, nausea and headache⁽ Reference Omar, Artime and Webb ^{83 )}. Also, despite the high potency of organic nitrates and their long history as being used to treat various CVD, nitrate tolerance is a huge limitation and an undesirable side effect of their use⁽ Reference Omar, Artime and Webb ^{83 ,} Reference Klemenska and Beresewicz ^{133 )}.

Nitrate tolerance is a complex phenomenon and is poorly understood; however, it is clearly a result of chronic organic nitrate use to which nitrovasodilator responsiveness is lost⁽ Reference Omar, Artime and Webb ^{83 )}. Nitrate tolerance has been reported to occur within 1–3 d of continuous glyceryl trinitrate treatment in patients with MI, stable angina and chronic congestive heart failure⁽ Reference Klemenska and Beresewicz ^{133 )}. Further, chronic organic nitrate use has also been linked to endothelial dysfunction, increased production of free radicals and the development of vascular tolerance to other endothelium-dependent vasodilators⁽ Reference Omar, Artime and Webb ^{83 )}. Although this phenomenon is poorly understood, recent animal and human studies indicate that increased vascular production of the superoxide anion (O₂ ^–) underlies the mechanism for tolerance⁽ Reference Klemenska and Beresewicz ^{133 )}. This oxidative stress hypothesis of nitrate tolerance is supported by numerous reports demonstrating that the tolerance is prevented by co-administration of antioxidants (for example, vitamin C, vitamin E and folic acid) and interventions which inhibit reactive oxygen species formation (lipid- and blood pressure-lowering medications)⁽ Reference Klemenska and Beresewicz ^{133 –} Reference Fontaine, Otto and Fontaine ^{136 )}.

It is interesting to note that the phenomenon of tolerance is not exhibited with the consumption of inorganic nitrates/nitrites; however, despite showing promise in preventing or treating certain cardiovascular conditions, such as hypertension, they have received little attention by the medical community⁽ Reference Kapil, Weitzberg and Lundberg ^{27 )}.

Inorganic nitrate and nitrite: from dietary contaminant to potential therapeutic nutrient

Throughout history, cases of accidental toxic exposure to nitrate and nitrite have been documented; however, the health risk of excessive inorganic nitrate and nitrite consumption appears specific to population subgroups⁽ Reference Hord, Tang and Bryan ^{22 )}. One of these subgroups includes infants aged less than 6 months, to which excessive nitrite exposure has been linked to cases of methaemoglobinaemia (blue baby syndrome)⁽ Reference Greer and Shannon ^{137 )}. As a result, strict regulatory limits have been established to govern the NO_x content of the drinking water supply and their use as an additive to processed and cured meats in order to limit exposure to the population^{( 85 , 86 )}.

Methaemoglobinaemia can occur when nitrite oxidises ferrous Fe (Fe²⁺) in Hb to the ferric state (Fe³⁺), resulting in methaemoglobin. Methaemoglobin is incapable of binding molecular oxygen, and impairs oxygen delivery to the tissues, causing hypoxia and cyanosis⁽ Reference Greer and Shannon ^{137 )}. While most cases of methomeoglobinaemia have been attributed to the consumption of well water (prone to high nitrate accumulation) used for the preparation of infant formula, there have been reported cases of nitrate poisoning in infants from the ingestion of plant nitrates^{( 86 ,} Reference Greer and Shannon ^{137 )}. While Martinez et al. ⁽ Reference Martinez, Sanchez-Valverde and Gil ^{138 )} found that the use of certain high-nitrate vegetables (herbs and green leafy vegetables) in infant homemade vegetable purée increased methaemoglobinaemia in infants (herbs: OR 5·2, 95% CI 1·1, 24·6; and green leafy vegetables: OR 2·0, 95% CI 0·4, 8·7), the most important factor increasing methaemoglobinaemia was the time lapse between vegetable purée preparation and consumption (OR 17·4, 95% CI 3·5, 86·3 if purée was prepared 24–48 h before; and OR 24·9, 95% CI 3·3, 187·6 if prepared >48 h before)⁽ Reference Martinez, Sanchez-Valverde and Gil ^{138 )}.

To date, human nitrate and nitrite exposure studies have failed to prove a direct link with methaemoglobinaemia, suggesting that NO_x exposure alone may not be responsible for methaemoglobinaemia development⁽ Reference Milkowski, Garg and Coughlin ^{139 ,} Reference Avery ^{140 )}.

Another population subgroup that is thought to be at health risk due to excessive NO_x exposure is high consumers of cured and processed meats⁽ Reference Hord, Tang and Bryan ^{22 ,} Reference Bouvard, Loomis and Guyton ^{141 )}. It has been theorised that nitrates and nitrites from processed meats generate N-nitroso compounds which can be carcinogenic⁽ Reference Bingham ^{142 )}.

In October 2015 the International Agency for Research on Cancer (IARC) summarised more than 800 studies conducted globally, and determined that 50 g of processed meat per d increased the risk of colorectal cancer by 18%, and therefore concluded that processed meats are carcinogenic⁽ Reference Bouvard, Loomis and Guyton ^{141 )}. In animal studies N-nitrosamines and related N-nitrosamides have been shown to be carcinogenic in a variety of molecular structures⁽ Reference Gilchrist, Winyard and Benjamin ^{143 ,} Reference Magee and Barnes ^{144 )}. However, such direct evidence demonstrating nitrate and nitrite as human carcinogens is severely lacking. This has been reflected in the conclusions of the FAO expert committee who found no consistent increased risk of cancer with increasing consumption of nitrate, as available epidemiological studies did not provide evidence that nitrate is carcinogenic to humans^{( 145 )}.

Currently, researchers are interested in understanding whether the health risks associated with inorganic nitrates/nitrites outweigh the recently discovered health benefits; however, there is a growing consensus that any weak and inconclusive data on inorganic NO_x and cancer associations are far outweighed by the potential health benefits of restoring NO homeostasis⁽ Reference Hord, Tang and Bryan ^{22 ,} Reference Du, Zhang and Lin ^{84 ,} Reference Milkowski, Garg and Coughlin ^{139 ,} Reference Gilchrist, Winyard and Benjamin ^{143 )}. In particular this has been demonstrated in various animal and human experimental studies, in which inorganic NO_x has been shown to improve outcomes such as blood pressure, endothelial function, platelet function, ischaemia–reperfusion injury, exercise performance and host defence⁽ Reference Gilchrist, Winyard and Benjamin ^{143 ,} Reference Kapil, Milsom and Okorie ^{146 –} Reference Dykhuizen, Frazer and Duncan ^{151 )}.

Evidence of cardiovascular benefit from animal studies

Intakes of dietary inorganic nitrate have been shown to be strongly cardioprotective in animal studies. Carlström et al. ⁽ Reference Carlström, Persson and Larsson ^{152 )} indicated this in a four-arm dietary intervention trial in rats. The rats were placed on either a normal-salt diet (control), a high-salt diet, a high-salt diet supplemented with a nutritional (low) dose of nitrate, and a high-salt diet supplemented with a pharmacological (high) dose of nitrate for 8–11 weeks⁽ Reference Carlström, Persson and Larsson ^{152 )}. As expected, results demonstrated that chronic consumption of a high-salt diet develops hypertension; however, when combined with a low nitrate dose, blood pressure was non-statistically significantly lower⁽ Reference Carlström, Persson and Larsson ^{152 )}. On the other hand, the higher nitrate dose lowered blood pressure by a significant 24 mmHg compared with the plain high-salt diet, a magnitude of blood pressure reduction considerably magnified compared with blood pressure reductions observed in another study of healthy normotensive rats using the same nitrate dose⁽ Reference Carlström, Persson and Larsson ^{152 ,} Reference Petersson, Carlström and Schreiber ^{153 )}. Similar results were reported by Kanematsu et al. ⁽ Reference Kanematsu, Yamaguchi and Ohnishi ^{154 )}, finding that in hypertensive rats, antihypertensive effects were only apparent with the highest dose of nitrate, yet there was a strong tissue-protective effect seen with lower doses equivalent to modest dietary intakes. Ferguson et al. ⁽ Reference Ferguson, Hirai and Copp ^{155 )} demonstrated clinically significant reductions in mean arterial pressure with beetroot juice supplementation in exercising rats (control: 137 (sem 3); beetroot juice: 127 (sem 4) mmHg; P<0·05), indicating that clinically significant blood pressure reductions may be achievable in doses attained from dietary sources⁽ Reference Ferguson, Hirai and Copp ^{155 )}.

In addition to significant blood pressure control, Carlström et al. ⁽ Reference Carlström, Persson and Larsson ^{152 )} found that dietary nitrate supplementation can partly prevent the development of cardiac hypertrophy and high nitrate doses significantly reduced the fibrotic changes which were observed in the high-salt group, two factors which are major predictors of heart failure⁽ Reference Carlström, Persson and Larsson ^{152 )}. Two other studies found that mice ingesting inorganic nitrate led to a significantly reduced infarct size during myocardial ischaemia, an important finding given that reduced infarct size is associated with lower heart failure risk post-MI and mortality⁽ Reference Bryan, Calvert and Elrod ^{156 –} Reference Minicucci, Azevedo and Polegato ^{158 )}.

When Baker et al. ⁽ Reference Baker, Su and Fu ^{149 )} treated rats with an intravenous bolus of sodium nitrite across various doses (0·04, 0·4, 1·0, 4·0, 7·0 and 10·0 mg/kg), before initialising a blockage of the coronary artery, there was a clear dose-dependent effect of nitrite on infarct size. However, it was intriguing to note that protection was only found in doses up to 4·0 mg/kg, an effect which was absent at higher doses⁽ Reference Baker, Su and Fu ^{149 )}. Rats administered with 4·0 mg/kg nitrite exhibited a significant 32% reduction in infarct size compared with controls⁽ Reference Baker, Su and Fu ^{149 )}. Nitrite was also found most effective when administered before and/or during the ischaemic event, but not at the onset of reperfusion⁽ Reference Baker, Su and Fu ^{149 )}. Further, equivalent doses of sodium nitrate had no effect on infarct size, indicating that administration timing and doses are key considerations for nitrite protection from MI⁽ Reference Baker, Su and Fu ^{149 )}.

Thrombosis is largely a result of platelet adhesion, activation and aggregation, and is a common pathology underlying IHD and ischaemic stroke⁽ Reference Nieswandt, Pleines and Bender ^{159 ,} Reference Raskob, Angchaisuksiri and Blanco ^{160 )}. NO plays a key role in preventing thrombosis development⁽ Reference Park, Piknova and Huang ^{161 )}. Park et al. ⁽ Reference Park, Piknova and Huang ^{161 )} demonstrates this notion upon discovering an inverse correlation between NO_x levels and platelet activity/aggregation in mice. In addition, Apostoli et al. ⁽ Reference Apostoli, Solomon and Smallwood ^{162 )} examined the effect of inorganic nitrite on platelet aggregation in endothelial NOS-deficient mice. This study found that inorganic nitrite exerts an antiplatelet effect during endothelial NOS deficiency and suggested that dietary nitrate may reduce platelet hyperactivity during endothelial dysfunction⁽ Reference Apostoli, Solomon and Smallwood ^{162 )}.

Pulmonary hypertension can lead to the remodelling of the artery wall, causing abnormalities of elastic fibres, intimal fibrosis and medial hypertrophy⁽ Reference Moraes, Colucci and Givertz ^{163 )}. This can result in vascular stiffness and is a condition linked to the development of chronic heart failure⁽ Reference Moraes, Colucci and Givertz ^{163 )}. Sodium nitrite interventions in lamb and mouse models have shown reductions in pulmonary hypertension specifically during hypoxic conditions⁽ Reference Hunter, Dejam and Blood ^{164 ,} Reference Zuckerbraun, Shiva and Ifedigbo ^{165 )}. However, Casey et al. ⁽ Reference Casey, Badejo and Dhaliwal ^{166 )} found that intravenous injections of sodium nitrite during normoxic conditions could lead to reductions in pulmonary and systemic arterial pressure and increased cardiac outputs in adult male rats. This suggests that sodium nitrite may have a role in reducing the workload of the heart during pulmonary hypertension, thus protecting the heart and vascular system from associated damage and dysfunction⁽ Reference Casey, Badejo and Dhaliwal ^{166 )}.

Hendgen-Cotta et al. ⁽ Reference Hendgen-Cotta, Luedike and Totzeck ^{167 )} pre-treated mice with nitrate before inducing chronic limb ischaemia, and nitrate supplementation was found to enhance revascularisation and increased mobilisation of circulating angiogenic cells (CAC), which are important for the recovery and maintenance of healthy endothelial function⁽ Reference Hendgen-Cotta, Luedike and Totzeck ^{167 )}. Heiss et al. ⁽ Reference Heiss, Meyer and Totzeck ^{168 )}, on the other hand, injected inorganic nitrite into healthy mice, and found that nitrite significantly increased CAC at 1 h compared with controls. It is interesting to note, however, that when this test was repeated in endothelial NOS-deficient mice, no CAC mobilisation was observed, indicating that NOS may be required to take part in nitrate-mediated CAC mobilisation⁽ Reference Heiss, Meyer and Totzeck ^{168 )}.

In a study conducted by Sindler et al. ⁽ Reference Sindler, Fleenor and Calvert ^{169 )} the effect of nitrite in aged, but healthy, mice was investigated and high dietary nitrite doses were found to reverse age-related vascular dysfunction, arterial stiffness and reduce levels of oxidative stress. This is in line with Carlström et al. ⁽ Reference Carlström, Persson and Larsson ^{152 )} who found that key plasma and urinary oxidative stress markers (malondialdehyde, type VI isoprostane (iPF_2α-VI) and 8-oxo-2’-deoxyguanosine (8-OHdG)) were significantly reduced (despite co-consumption of a high-salt diet) with both low- (0·1 mmol nitrate/d) and high- (1·0 mmol nitrate/d) dose dietary nitrate supplementation, which may be useful in preventing NO degradation and endothelial dysfunction⁽ Reference Carlström, Persson and Larsson ^{152 ,} Reference Cai and Harrison ^{170 )}. This is an interesting finding, given that oxidative stress is directly linked with an inflammatory response which is thought to have a central role in the development of atherosclerosis⁽ Reference Weitzberg and Lundberg ^{93 )}.

Stokes et al. ⁽ Reference Stokes, Dugas and Tang ^{171 )} found that mice fed cholesterol-enriched diets for 3 weeks tend to develop clear signs of vascular disease pathology, including elevated leucocyte adhesion and endothelial dysfunction, an effect which was prevented with nitrite supplementation in the drinking water. In another study by Carlström et al. ⁽ Reference Carlström, Larsen and Nyström ^{172 )} it was demonstrated that several features of the metabolic syndrome (including visceral fat and circulating TAG, which are strong risk factors for CVD) can be reversed by dietary nitrate supplementation, in amounts which correspond to those derived from endothelial NOS under normal healthy conditions or a vegetable-rich diet⁽ Reference Carlström, Larsen and Nyström ^{172 )}.

Evidence of cardiovascular benefit from human studies

In 2003, Cosby et al. ⁽ Reference Cosby, Partovi and Crawford ^{71 )} conducted one of the first studies demonstrating a relationship between inorganic nitrite supplementation and blood pressure reductions in healthy human subjects. This study chose to use sodium nitrite (NaNO₂ ^–) infusions providing approximately 75 mg NaNO₂ ^– over two 15-min periods, a dose which was found to significantly reduce mean blood pressure by 7 mmHg (P<0·01)⁽ Reference Cosby, Partovi and Crawford ^{71 )}. Similar findings were later established using sodium nitrate (NaNO₃ ^–) in a study conducted by Larsen et al. ⁽ Reference Larsen, Ekblom and Sahlin ^{173 )}. In this study healthy subjects consumed NaNO₃ ^– (8·5 mg/kg per d for 3 d) as a dietary supplement, and although systolic blood pressure was not changed during this time compared with placebo (sodium chloride), diastolic blood pressure was significantly reduced on average by 3·7 mmHg (P<0·02) and mean arterial pressure was lowered by 3·2 mmHg (P<0·03)⁽ Reference Larsen, Ekblom and Sahlin ^{173 )}. Soon after, Webb et al. ⁽ Reference Webb, Patel and Loukogeorgakis ^{33 )} investigated this topic further using beetroot juice (containing approximately 1400 mg inorganic nitrate). Results from Webb et al. ⁽ Reference Webb, Patel and Loukogeorgakis ^{33 )} showed a peak reduction in systolic blood pressure of 10·4 (sem 3) mmHg (P<0·01), a reduction in diastolic blood pressure of 8·1 (sem 2·1) mmHg (P<0·01) and mean arterial pressure reduction of 8·0 (sem 2·1) mmHg (P<0·01), thus indicating that significant blood pressure reductions are possible with the acute consumption of dietary inorganic nitrate in healthy subjects. This is a notion which has been further supported by a recently conducted systematic review and meta-analysis which found that inorganic nitrate and beetroot juice consumption was associated with greater changes in systolic blood pressure (–4·4 (95% CI –5·9, –2·8) mmHg; P<0·001) than diastolic blood pressure (–1·1 (95% CI –2·2, 0·1) mmHg; P=0·06)⁽ Reference Siervo, Lara and Ogbonmwan ^{174 )}. However, it is important to note that these findings have not been consistent across the literature, as a few recently conducted randomised controlled trials have found that inorganic nitrate consumption from either beetroot juice or from a high-nitrate diet (rich in green leafy vegetables) for 1–2 weeks had little/no effect on the blood pressure of study subjects⁽ Reference Bondonno, Liu and Croft ^{57 ,} Reference Bondonno, Liu and Croft ^{175 ,} Reference Gilchrist, Winyard and Aizawa ^{176 )}. The exact cause of this variation across studies remains unclear, yet could be due to methodological differences including the study population (for example, healthy subjects v. hypertensive subjects) or the conditions in which NO_x was consumed (for example, food v. supplement, dosing or altered environmental conditions such as exercise stress). Nevertheless, this question remains unclear and will require further investigation, in order to better understand the usefulness of dietary/inorganic NO_x within the general population.

While the acute effects of dietary inorganic nitrate on blood pressure have been extensively investigated, very few studies have investigated long-term effects. Sobko et al. ⁽ Reference Sobko, Marcus and Govoni ^{23 )} investigated the effects of a traditional Japanese diet on blood pressure which provided approximately 1140 mg of nitrate per d for a 10 d period. The traditional Japanese diet led to a lower diastolic blood pressure than seen in the non-Japanese diet group (71·3 (sd 7·9) v. 75·8 (sd 7·8) mmHg; P=0·0066), indicating that dietary inorganic nitrate consumption for longer-periods of time may have some blood pressure-lowering effects in healthy individuals; however, a 10 d intervention can hardly be classified as a long-term intervention⁽ Reference Sobko, Marcus and Govoni ^{23 )}. In another 4-week intervention, Kapil et al. ⁽ Reference Kapil, Khambata and Robertson ^{29 )} assigned hypertensive patients to receive a daily dose of either 250 ml of beetroot juice or placebo (nitrate-depleted beetroot juice). Notably, Kapil et al. ⁽ Reference Kapil, Khambata and Robertson ^{29 )} found that daily dietary nitrate supplementation significantly reduced mean clinic blood pressure (7·7/2·4 mmHg (range 3·6–11·8/0·0–4·9 mmHg); P<0·001, P=0·05), mean 24 h ambulatory blood pressure (7·7/5·2 mmHg (range 4·1–11·2/2·7–7·7 mmHg); P<0·001 for both) and mean home blood pressure (8·1/3·8 mmHg (range 3·8–12·4/0·7–6·9 mmHg); P<0·001, P<0·01)⁽ Reference Kapil, Khambata and Robertson ^{29 )}.

Currently, the longest intervention study conducted in this area is a 10-week intervention trial from DeVan et al. ⁽ Reference DeVan, Brooks and Evans ^{125 )}. In this study, healthy 50- to 79-year-old subjects were recruited to consume either 0, 80 or 160 mg of sodium nitrite per d for a 10-week period⁽ Reference DeVan, Brooks and Evans ^{125 )}. Results indicated no significant changes in blood pressure at week 10 compared with baseline blood pressure values; however, a significant time × treatment effect for carotid diameter in the nitrite groups was detected, as well as improved endothelial function of the brachial artery, suggesting improved vascular function with chronic inorganic nitrite supplementation despite a lack of an effect seen with blood pressure⁽ Reference DeVan, Brooks and Evans ^{125 )}. However, it is worth noting that the only prospective cohort study on this topic conducted by Golzarand et al. ⁽ Reference Golzarand, Bahadoran and Mirmiran ^{177 )} found that higher dietary intakes of nitrate-containing vegetables (about 427·6 g/d) in normotensive individuals may have a protective effect against the development of hypertension (highest tertile of nitrate-containing vegetables, OR 0·63 (95% CI 0·41–0·98); P=0·05).

Endothelial dysfunction is one of the key early events involved in the development of atherosclerosis⁽ Reference Raitakari and Celermajer ^{178 )}. Flow-mediated dilatation is commonly used as a measure of endothelial function as reduced flow-mediated dilatation is an indicator of endothelial dysfunction (caused by reduced NO bioavailability) and has been associated with increased severity and duration of blood pressure elevations⁽ Reference Hadi, Carr and Suwaidi ^{179 )}. More recently, dietary inorganic nitrate interventions have been shown to significantly improve flow-mediated dilatation in healthy and hypertensive human subjects consuming spinach, beetroot juice or sodium nitrate capsules⁽ Reference Kapil, Khambata and Robertson ^{29 ,} Reference Heiss, Meyer and Totzeck ^{168 ,} Reference Bondonno, Yang and Croft ^{180 ,} Reference Rodriguez-Mateos, Hezel and Aydin ^{181 )}. Joris & Mensink⁽ Reference Joris and Mensink ^{182 )} tested the effects of beetroot juice (containing approximately 500 mg nitrate) with a dietary load of fat (56·6 g fat) in overweight and obese subjects (BMI 30·1 (sd 1·9) kg/m²). While the control drink group saw impaired flow-mediated dilatation with dietary fat intake, the consumption of beetroot juice appeared to attenuate this impairment (beetroot juice: –0·37 (sd 2·92) % v. control: –1·56 (sd 2·9) %; P=0·03)⁽ Reference Joris and Mensink ^{182 )}. Additionally, flow-mediated dilatation has been shown to be reduced by approximately 40% after vascular ischaemia; however, Ingram et al. ⁽ Reference Ingram, Fraser and Bleasdale ^{183 )} demonstrated that sodium nitrite pre-conditioning (providing a nitrite dose before ischaemic event) will prevent ischaemic reperfusion injury by preventing reductions in flow-mediated dilatation and endothelial dysfunction. Similar findings have been reported by Kapil et al. ⁽ Reference Kapil, Khambata and Robertson ^{29 )} and Webb et al. ⁽ Reference Webb, Patel and Loukogeorgakis ^{33 )} with beetroot juice pre-conditioning, indicating that higher plasma NO_x concentrations achieved by inorganic NO_x consumption may have a role for improving cardiovascular outcomes after vascular ischaemic events⁽ Reference Kapil, Khambata and Robertson ^{29 ,} Reference Webb, Patel and Loukogeorgakis ^{33 )}.

In addition to flow-mediated dilatation, CAC have been identified as an important indicator of vascular endothelial function, as they have a critical role in vascular repair⁽ Reference Heiss, Jahn and Taylor ^{184 )}. The number of CAC have also been shown to predict the occurrence of CVD and death⁽ Reference Heiss, Meyer and Totzeck ^{168 )}. Therefore it is of interest to note that Heiss et al. ⁽ Reference Heiss, Meyer and Totzeck ^{168 )} have indicated an important role for dietary nitrate for increasing CAC, showing that a single dose of sodium nitrate (12·7 mg/kg body weight) can double the number of CAC 1–2 h post-nitrate ingestion.

Pulse wave velocity and augmentation index are accepted measurements of arterial stiffness and atherosclerosis, to which higher readings are associated with increased CVD risk^{( 185 ,} Reference Chirinos, Zambrano and Chakko ^{186 )}. The role of dietary inorganic nitrate in preventing arterial stiffness has been established, as Kapil et al. ⁽ Reference Kapil, Khambata and Robertson ^{29 )} found that a 4-week beetroot juice intervention reduced pulse wave velocity and augmentation index in hypertensive subjects. Zamani et al. ⁽ Reference Zamani, Rawat and Shiva-Kumar ^{187 )} also saw a significantly reduced augmentation index with beetroot juice consumption in patients with symptomatic heart failure (beetroot juice: 132·2 (sd 16·7) %; placebo: 141·2 (sd 21·9) %; mean change –9·1 (sd 15·4) %; P=0·03). Rammos et al. ⁽ Reference Rammos, Hendgen-Cotta and Sobierajski ^{188 )} investigated the effect of a 4-week sodium nitrate supplementation trial in elderly volunteers with mild hypertension, and found that vascular stiffness was significantly improved in the nitrate-supplemented volunteers. This is a very significant finding given that vascular stiffness tends to naturally increase with age⁽ Reference Liu, Bondonno and Croft ^{189 )}.

In an randomized controlled trial conducted by Jones et al. ⁽ Reference Jones, Pellaton and Velmurugan ^{190 )}, participants prone to MI and undergoing primary percutaneous coronary intervention (non-surgical intervention to treat stenosis) were administered with either a high-dose bolus injection of NaNO₂ ^– (1·8 µmol) or NaCl placebo. The nitrite group experienced a significantly (P=0·05) improved myocardial salvage index (established indicator of cardioprotective benefit) relative to placebo⁽ Reference Jones, Pellaton and Velmurugan ^{190 )}. In addition, a subset of participants who exhibited a blocked blood vessel experienced a 19% reduction in infarct size with nitrite treatment compared with placebo⁽ Reference Jones, Pellaton and Velmurugan ^{190 )}. A 1-year follow-up of study participants also found that the nitrite group experienced a significant reduction in major adverse cardiac events (NaNO₂ ^–: 2·6% v. NaCl: 15·8%; P=0·04)⁽ Reference Jones, Pellaton and Velmurugan ^{190 )}.