SEARCH

SEARCH BY CITATION

Keywords:

  • dietary nitrate;
  • inorganic nitrite;
  • blood pressure;
  • platelets;
  • endothelial dysfunction;
  • exercise performance;
  • peripheral arterial disease;
  • Nitrate ‘Veg-Table’ ‘Nitrate Units’

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Effects of vegetables on blood pressure and cardiovascular disease events
  5. Dietary sources of nitrate
  6. Acceptable daily intake (ADI)?
  7. Vascular effects of dietary nitrate
  8. Conclusion
  9. Competing Interests
  10. References

The discovery that dietary (inorganic) nitrate has important vascular effects came from the relatively recent realization of the ‘nitrate-nitrite-nitric oxide (NO) pathway’. Dietary nitrate has been demonstrated to have a range of beneficial vascular effects, including reducing blood pressure, inhibiting platelet aggregation, preserving or improving endothelial dysfunction, enhancing exercise performance in healthy individuals and patients with peripheral arterial disease. Pre-clinical studies with nitrate or nitrite also show the potential to protect against ischaemia-reperfusion injury and reduce arterial stiffness, inflammation and intimal thickness. However, there is a need for good evidence for hard endpoints beyond epidemiological studies. Whilst these suggest reduction in cardiovascular risk with diets high in nitrate-rich vegetables (such as a Mediterranean diet), others have suggested possible small positive and negative associations with dietary nitrate and cancer, but these remain unproven. Interactions with other nutrients, such as vitamin C, polyphenols and fatty acids may enhance or inhibit these effects. In order to provide simple guidance on nitrate intake from different vegetables, we have developed the Nitrate ‘Veg-Table’ with ‘Nitrate Units’ [each unit being 1 mmol of nitrate (62 mg)] to achieve a nitrate intake that is likely to be sufficient to derive benefit, but also to minimize the risk of potential side effects from excessive ingestion, given the current available evidence. The lack of data concerning the long term effects of dietary nitrate is a limitation, and this will need to be addressed in future trials.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Effects of vegetables on blood pressure and cardiovascular disease events
  5. Dietary sources of nitrate
  6. Acceptable daily intake (ADI)?
  7. Vascular effects of dietary nitrate
  8. Conclusion
  9. Competing Interests
  10. References

Until recently dietary nitrate (NO3) and nitrite (NO2) were considered to lack any useful physiological activity in the circulation [1], despite these inorganic anions also being derived from nitric oxide (NO, produced endogenously by the action of the nitric oxide synthases (NOS), on the substrate amino acid, L-arginine [2]), a reactive free radical and potent vasodilator [3]. Rather, there have been longstanding major concerns regarding their toxicity, particularly potential carcinogenicity. However, a definite link still remains to be established. From 2001, nitrite was discovered to provide an important alternative source of nitric oxide, particularly when oxygen levels are reduced [4, 5], such as in the microcirculation, causing vasodilatation [6] and in 2004 in ischaemic hearts, with protective effects [7]. Even then, dietary nitrate was still thought to lack any effect in the circulation as it was not thought to increase circulating nitrite concentrations [8]. However, over the last 5 years or so there has been increasing evidence of physiological effects of nitrate, particularly on the cardiovascular system and the realization of the ‘nitrate-nitrite-NO pathway’. This review will describe some of the key background to the field, provide an update on the latest cardiovascular studies, review the current situation with some of the potential carcinogenic effects and introduce a Nitrate ‘Veg-Table’ and nitrate units to guide patients and health professionals on what may be effective and safe amounts of nitrate to ingest.

Effects of vegetables on blood pressure and cardiovascular disease events

  1. Top of page
  2. Abstract
  3. Introduction
  4. Effects of vegetables on blood pressure and cardiovascular disease events
  5. Dietary sources of nitrate
  6. Acceptable daily intake (ADI)?
  7. Vascular effects of dietary nitrate
  8. Conclusion
  9. Competing Interests
  10. References

Mediterranean and Japanese traditional diets are generally regarded as healthy. Both are associated with a low incidence of cardiovascular disease and longevity [9-13]. These diets share several similarities in that both are high in fruit and vegetables, fish (and olive oil – Mediterranean), and low in red meat. A key beneficial common component may be the high dietary nitrate content, particularly of green leafy vegetables such as lettuce, spinach and rocket in the Mediterranean diet and ta cai, garland chrisantemum, laver, spinach and chin gin c. in the Japanese traditional diet [14]. Indeed, vegetarian diets are associated with lower blood pressure [15-18] and green leafy vegetables have been associated with a reduced incidence of non-fatal myocardial infarction or fatal coronary heart disease and stroke [19, 20]. However, given the observational nature of these studies, they should be interpreted with caution [21]. More definitive evidence of an effect (albeit short term) on the surrogate outcome of blood pressure reduction is available from prospective controlled intervention trials, such as the DASH study [22-24].

Dietary sources of nitrate

  1. Top of page
  2. Abstract
  3. Introduction
  4. Effects of vegetables on blood pressure and cardiovascular disease events
  5. Dietary sources of nitrate
  6. Acceptable daily intake (ADI)?
  7. Vascular effects of dietary nitrate
  8. Conclusion
  9. Competing Interests
  10. References

Typically, around 85% of dietary nitrate (the inorganic nitrate anion, NO3) is derived from vegetables [25-27], with most of the remainder from drinking water, though the concentration of this may vary considerably [28]. Dietary nitrite (NO2) is mainly derived from cured meats, where it is added to prevent the development of botulinum toxin [29] and is reviewed elsewhere [30-33]. Inorganic nitrate has also been used for meat curing for centuries, and as the major component of gun powder since medieval times [1], and has several similarities, but also marked differences from the organic nitrates, such as nitroglycerin which also has an important history as an explosive and medicinal product. These differences are reviewed elsewhere [34]. Vegetables can be categorized according to their nitrate content (see Table 1, Nitrate ‘Veg-Table’): high nitrate content vegetables (>1000 mg kg−1) belong to the following families: Brassicaceae (rocket, the highest nitrate accumulating vegetable), Chenopodiaceae (beetroot, spinach), Asteraceae (lettuce) and Apiaceae (celery) [35-37]. Nitrate content also varies across the plant: leaf > stem > root [37-39]. Most common vegetables are in the medium range for nitrate content (100–1000 mg kg−1), including peppers, garlic, potatoes and carrots at the lower end, and green beans, cabbage and turnip at the upper end. Vegetables notable for their low nitrate content (<100 mg kg−1) are onions and tomatoes.

Table 1. The Nitrate ‘Veg-Table’: vegetables, ranked from highest to lowest according to mean nitrate content [range] expressed in mg kg−1, mmol per UK portion (80 g) and as a guide as the approximate number of nitrate units per portion (1 nitrate unit = 1 mmol) to facilitate estimation of nitrate intake or to modify intake as desired. Also included is tap water and bottled water for comparison. Nitrate content sourced from the following references [35, 37, 40, 54-62]:Thumbnail image of

Nitrate content is influenced by environmental, agricultural and genetic factors [28, 36]. The main environmental factors are humidity, temperature, water content and exposure to sunlight. For example, summer grown lettuce (longer sunshine hours and less rainfall) has lower mean nitrate concentration than winter grown [40]. Agricultural factors such as nitrogen fertilization and concomitant use of herbicides also play their part in varying degrees to influence the nitrate content of vegetables [38, 41, 42]. Agricultural factors include nitrogen fertilization, degree of nitrogen fixation of atmospheric nitrogen by symbiotic bacteria in non-leguminous plants in addition to leguminous plants [43] and the nitrate reductase activity in the root, genetically deficient in the lettuce, but very active in peas which contain low nitrate concentrations [44-46].

Juicing vegetables is a popular and convenient way of increasing vegetable intake and a range of commercially produced juices are available. While the nitrite content of freshly prepared homemade juice is negligible, after 2 days at room temperature the nitrite concentration of beetroot juice increases dramatically to almost 600 mg l−1, though remains low if kept refrigerated below 4°C [47]. The conversion is due to bacterial nitrate reductases which are less of a problem with industrially-produced raw juices which are lightly pasteurized.

Acceptable daily intake (ADI)?

  1. Top of page
  2. Abstract
  3. Introduction
  4. Effects of vegetables on blood pressure and cardiovascular disease events
  5. Dietary sources of nitrate
  6. Acceptable daily intake (ADI)?
  7. Vascular effects of dietary nitrate
  8. Conclusion
  9. Competing Interests
  10. References

The Acceptable Daily Intake (ADI) set by the European Food Safety Authority for nitrate is 3.7 mg kg−1 (0.06 mmol kg−1). This equates to ∼260 mg day–1 for a 70 kg adult (∼4.2 mmol). The World Health Organization (WHO) first set an upper limit for nitrate in food in 1962 derived from studies showing that daily intakes of ∼500 mg of sodium nitrate kg−1 body weight had been found to be harmless to rats and dogs. This figure was divided by 100 to yield an ADI for humans of 5 mg sodium nitrate (=3.7 mg nitrate kg−1), a figure that has stood ever since [48].

The vegetarian diet has been demonstrated to contain ∼4.3 mmol nitrate day−1, close to the ADI, almost four times greater than a ‘normal’ diet, which contains ∼1.2 mmol nitrate [49]. A study of 3 days' supplementation with ∼120 g of round green lettuce demonstrated that ∼70% of the nitrate load was excreted and the total amount of urinary nitrate excretion in a 24 h period increased from 53 mg (∼0.9 mmol) on the normal diet to 223, 241 and 243 mg on days 2, 3 and 4 respectively; equivalent to ∼4 mmol nitrate [8]. It should be remembered that nitrate is also produced endogenously. This was discovered in 1916 by Mitchell et al. [50] and confirmed by Green et al. [51] and Leaf et al. [52], who found that [15N]-L-arginine was the source of [15N]-nitrate. Indeed, the main source of nitrite in plasma was thought to be from the L-arginine-NO pathway [53] with no contribution from dietary nitrate [8].

The Nitrate ‘Veg-Table’ (Table 1) provides a guide to the nitrate content of high, medium and low nitrate-containing vegetables. Nitrate content was obtained from a range of sources [35, 37, 40, 54-62]. The average nitrate content is also expressed per UK portion of 80 g. Also, for ease of use for patients and health professionals, we propose that ‘nitrate units’ are used, 1 nitrate unit being equivalent to 1 mmol nitrate. This is a new concept that we put forward, analagous to the use of existing potassium points [63], primarily used as a guide to avoid excessive potassium intake in patients at risk of hyperkalaemia. Thus ‘nitrate units’ may be used as a guide to avoid excessive nitrate ingestion, or to avoid exceeding the ADI, if this is desired, or indeed to increase nitrate intake for functional benefit, such as blood pressure lowering or enhancement of exercise performance. The ADIs of nitrate for a range of given body weights is presented in Table 2. A UK portion (80 g) of a high nitrate-containing vegetable, such as beetroot or a green leafy vegetable, will therefore contain ∼2 mmol (or 2 units) of nitrate, about half the current ADI in a 70 kg person, which is 4.2 mmol. A portion of a medium nitrate-containing vegetable will provide ∼0.5 mmol nitrate, whereas low nitrate containing vegetables (onions and tomatoes) would provide just 0.1 mmol nitrate. A non-vegetarian ‘normal’ diet, containing ∼1 mmol nitrate day−1 [8, 49], therefore has the equivalent of ∼2 UK portions of medium nitrate containing vegetables/ day. It is easy to underestimate how much should be ingested for a UK portion, particularly for green leafy salad leaves: 80 g would fill a fairly large serving bowl.

Table 2. Weight-based guide to the ‘acceptable daily intake’ (ADI) of dietary nitrate in mg, mmol and ‘nitrate units’ (1 Nitrate unit = 1 mmol). ADI = 3.7 mg kg−1, and 62 mg nitrate = 1 mmol
Weight (kg)Acceptable Daily Intake, ADI (mg)Acceptable Daily Intake, ADI in mmol or ‘Nitrate units’
501853.0
602223.6
702594.2
802964.8
903335.4
1003706.0

The limits for drinking water stem from the association between infantile methaemoglobinaemia, also known as ‘blue baby syndrome’ and high nitrate concentrations in well water, which was established in the 1940s [64]. Since nitrate concentrations below 44 mg l−1 were rarely associated with methaemoglobinaemia [65], this resulted in the imposition of the limits for drinking water of 45 and 50 mg l−1 in the USA and Europe respectively, even though methaemoglobinaemia is highly unlikely in the absence of the contaminating bacteria found in wells, which are required to generate high concentrations of nitrite, via reduction of nitrate [66].

Vascular effects of dietary nitrate

  1. Top of page
  2. Abstract
  3. Introduction
  4. Effects of vegetables on blood pressure and cardiovascular disease events
  5. Dietary sources of nitrate
  6. Acceptable daily intake (ADI)?
  7. Vascular effects of dietary nitrate
  8. Conclusion
  9. Competing Interests
  10. References

Blood pressure

Potassium nitrate had been used as a diuretic for oedema since the 16th century [67] and different nitrate salts were in use until the mid-1930s when alternative diuretics became available [68, 69]. While small intravenous doses of potassium nitrite in cats (e.g. 0.08 g) and dogs (0.2 g) had been found to reduce arterial blood pressure by Reichert & Mitchell in 1880 [70], nitrite concentrations were not thought to increase after nitrate ingestion [8] and accounts of syncope and hypotension due to over-indulgence in sausages [71], are more likely to have been a result of nitrite than the suggested nitrate. While high concentrations of acidified nitrite had been shown to relax rabbit aorta strips in 1953 [72], nitrite was thought to lack any physiological effect until the demonstration of arterial-venous plasma nitrite gradients in 2000 [73], dilatation of arterial rings [4, 5], and in 2003 the dilatation of forearm resistance vessels with a concomitant drop in MABP of ∼7 mmHg [6]. The effect of nitrite appeared to be due to its conversion to NO by deoxyhaemoglobin [6, 74]. The dilatory effect of nitrite has been shown to be greatly enhanced during hypoxia in humans in both arteries and veins [75] and in tissues, oxygen is a potent regulator of the rate and products of tissue nitrite metabolism. At low oxygen concentrations nitrite reduction to NO predominates, whereas at normal to high oxygen levels oxidation of nitrite to nitrate predominates [76]. Other ‘nitrite reductases’ have been demonstrated and are reviewed elsewhere [34] and are summarized in Table 3.

Table 3. Kinetic processes in the handling of dietary nitrate and inorganic nitrite derived from nitrate. XOR (xanthine oxidoreductase), AO (aldehyde oxidase), ALDH2 (aldehyde dehydrogenase type 2)
KineticsDietary nitrateNitrite (derived from nitrate)
Absorption

Readily absorbed across upper gastrointestinal tract [110].

Do not undergo first pass metabolism [112].

Only a small fraction reaches the large bowel: <1% ingested nitrate excreted in faeces [113]; <2% is present in ileostomy fluid in patients with a total colectomy [113, 114], with negligible nitrite concentrations.

Bioavailability of nitrate from cooked spinach, raw lettuce and cooked beetroot ∼100% [111].

Plasma tmax of 1.5–1.8 h [111].

Bioavailability of nitrite ∼95–98% [112].

Plasma tmax ∼3 h, when derived from oral nitrate [82], and 15–45 min following oral nitrite administration [112].

Distribution

Volume of distribution moderate ∼0.3 l kg−1 [111], compared with that of water (∼0.6 l kg−1).

Plasma half-life of 5–8 h [77, 79, 111, 141, 142].

Concentrated in the salivary glands: 20–28% of a nitrate load is secreted in saliva [107, 113, 115, 116]. Entero-salivary circulation [1].

Volume of distribution of nitrite at steady-state is similar to that of nitrate, ∼0.35 l kg−1 [86].

Rapid uptake into most tissues [108].

Half-life of 1–5 min ex vivo [77, 142] to ∼20–45 min in vivo [86, 112].

Metabolism

Symbiotic bacteria on the posterior third of the tongue which contain nitrate reductases, predominantly Veillonella species, as well as Actinomyces, Rothia and Staphylococcus epidermidis convert nitrate into nitrite which is then swallowed [117, 118].

May be inhibited by mouthwash [80].

Minimal or no conversion of nitrate to nitrite in the circulation in humans (though demonstrated in germ-free mice).

In the stomach, nitrite reacts with the acidic gastric environment producing nitrous acid which decomposes to form nitric oxide as well as other reactive nitrogen oxides [95].

Vitamin C and polyphenols have been shown to reduce nitrite to NO [188].

In the circulation and tissues, nitrite is metabolized to nitric oxide by: deoxyhaemoglobin [6] [119-121], deoxymyoglobin [122], cytoglobin and neuroglobin [123, 124], XOR [7, 125], aldehyde oxidase [126], ALDH2 [127], carbonic anhydrase [137], eNOS [125, 128-130], cytochrome P450 [131-134].

Additional pathways: formation of nitro-fatty acids [139], nitrosothiols [138].

Excretion∼65–75% of absorbed nitrate is renally excreted [110].Renally excreted, with renal carbonic anhydrases involved in nitrite reabsorption [140].

Therefore, while nitrite now had an obvious potential to reduce BP, dietary nitrate was not thought to result in an increase in circulating nitrosothiols or nitrite [8], and therefore was thought to lack any effect on the vasculature. However, in 2004, Lundberg & Govoni detected an increase in plasma [nitrite] after ingestion of sodium nitrate [77] and in 2006 Larsen et al. demonstrated that diastolic BP was reduced by 3.7 mmHg following 3 days' dietary supplementation with sodium nitrate (0.1 mmol kg−1 day−1) compared with placebo sodium chloride in 17 healthy volunteers (randomized, double-blind, crossover design) [78]. Using a single ingestion of 500 ml beetroot juice [22.5 mmol (∼0.35 mmol kg−1)], Webb et al. demonstrated a more acute and more profound fall in both systolic BP (∼10.4 mmHg, baseline of ∼108 mmHg) and diastolic BP (∼8 mmHg) compared with placebo (500 ml water, crossover study) [79]. Importantly, the beetroot juice did not contain any detectable nitrite, and whilst plasma nitrate levels were already increasing by 30 min, the BP did not start to fall until the plasma nitrite concentration started to rise, with maximum changes in both occurring at ∼2.5–3 h, reflecting the time to produce nitrite from nitrate and for it to accumulate via the enterosalivary circulation (described below). Indeed, interruption of this process by asking volunteers to spit out all their saliva for 3 h immediately following beetroot juice ingestion completely blocked the rise in plasma nitrite and the reduction BP. This increase in plasma nitrite is also inhibited by the use of an antibacterial mouthwash just prior to nitrate ingestion in humans [80] and also blocks the 5 mmHg blood pressure reduction consequent to nitrate supplemented drinking water in Sprague-Dawley rats [81], in addition to attenuating the gastric mucus thickness with loss of gastroprotective effects against ulcerogenic insults.

These studies therefore provided evidence for a ‘nitrate-nitrite-NO’ pathway. In order to provide further evidence that this effect was due to nitrite, Kapil et al., used potassium nitrate capsules, and demonstrated a dose dependent reduction in BP (with 4, 12 and 24 mmol nitrate) equivalent to beetroot juice [nitrate] with no effect seen with potassium chloride as control [82], suggesting a BP lowering effect of nitrate rich vegetables independent of any potential effect due to their potassium content [83, 84]. This study also demonstrated that the peak increase in plasma nitrite at ∼3 h was associated with a significant increase in cGMP, the most sensitive indicator of NO bioactivity [85], thus providing evidence of bioactive NO generation from nitrite.

The Kapil et al. study also provided a clue to the heterogeneity of blood pressure responses to dietary nitrate. Post hoc analysis revealed that nitrate reduced blood pressure in males – from a higher baseline (associated with a lower baseline plasma nitrite concentration) compared with BP in females, who had no response to nitrate (possibly a result of their higher baseline plasma nitrite concentrations). Whether there is a sex difference per se, or whether the BP response to dietary nitrate is dependent on baseline plasma nitrite/BP remains to be determined. However, strong inverse correlations were demonstrated between the peak decrease in BP and the baseline BP (both systolic and diastolic). This therefore holds promise that dietary nitrate will be more effective in reducing blood pressure when it is needed, i.e. in people with high normal BP or hypertension, and results of dietary nitrate in hypertensives are awaited. Similarly, dietary nitrate would appear not to induce unwanted hypotension in people with low normal BP. The enterosalivary circulation also provides an inherent limiting mechanism to prevent excessive conversion of nitrate to nitrite, avoiding the risk of nitrite toxicity.

An important potential advantage of inorganic nitrate/nitrite is the apparent lack of tolerance induction [86], which commonly limits the therapeutic use of organic nitrates [87]. This was supported by the dietary intervention study of Sobko et al. which showed a sustained reduction in diastolic BP of ∼4.5 mmHg over 10 days, when subjects took a Japanese traditional diet, rich in dietary nitrate, compared with a low nitrate control diet [9], and by the study of Vanhatalo et al., which showed a reduction in BP of ∼7/5 mmHg following 15 days' supplementation with 500 ml beetroot juice [88]. Studies showing effects of dietary nitrate, including other forms such as spinach [89], and very recently with red and white beetroot-enriched bread [90] on BP are summarized in Table 4.

Table 4. Effect of dietary nitrate on BP in human studies, showing duration, amount of nitrate given in mmol (*0.1 mmol kg−1 day−1 for 3 days = 7 mmol day−1 in 70 kg person) peak systolic and diastolic BP effect (SBP and DBP respectively) and timing of peak effect following ingestion
StudySourceNitrate (mmol)Peak ΔSBP (mmHg) (time)Peak ΔDBP (mmHg) (time)
Larsen et al. (2006) [78]Sodium nitrate 3 days∼7*−3.7
Webb et al. (2008) [79]Beetroot juice 500 ml × 122.5−10.4 (2.5–3 h)−8
Kapil et al. (2010) [82]Potassium nitrate × 124−9.4 (6 h)−6.0 (2.75 h)
12−6 (2.25 h)−4.5 (3 h)
4−2.5 (1.75 h)−4.5 (2.25 h)
Kapil et al. (2010) [82]Beetroot juice 250 ml × 15.5−5.4
Sobko et al. (2010) [9]Japanese Traditional Diet 10 days∼18−4.5
Vanhatalo et al. (2010) [88]Beetroot juice (500 ml) 15 days5.2−7−5
Bahra et al. (2012) [148]Potassium nitrate × 18−5
Bondonno et al. (2012) [89]Spinach (200 g) × 13−2.7
Hobbs et al. (2012) [90]Beetroot juice (100, 250, 500 g)2.3−13.1 (2–3 h)−16.6 (2–3 h)
5.7−20.5 (2–3 h)−14.6 (2–3 h)
11.4−22.2 (2–3 h)−18.3 (2–3 h)
Hobbs et al. (2012) [90]

Beetroot-enriched bread (100 g)

with white beetroot

1.8−16.5 (2–3 h)−23.2 (2–3 h)
1.6−19.3 (2–3 h)−23.6 (2–3 h)

Platelet function

Platelets influence occlusive vascular disease through their interaction with the vessel wall [91], and play a significant part in the pathogenesis of acute coronary syndromes [92]. Nitric oxide inhibits platelet adhesion to the endothelium [93], and platelet aggregation [94]. Following the discovery of stomach NO synthesis from dietary nitrate, with potentially important protective effects against pathogens [95], and to maintain the integrity of the gastric mucosa [96], studies in healthy volunteers showed that oral potassium nitrate (2 mmol: equivalent to half a British flat lettuce [28]) inhibited platelet aggregation [97]. This was thought to be through the formation of nitrosothiols, which were detected in gastric juice, but not in the systemic or portal circulation [97, 98]. Nitrite was not a likely candidate as it was thought to be inert [99, 100], or possibly damaging [101], requiring hypoxic/ischaemic conditions, and millimolar nitrite concentrations [102-105] to generate NO, and not to increase in plasma following oral nitrate intake anyway [8, 106, 107]. Even if it did, Bryan et al. demonstrated that nitrite had no effect on platelet aggregation in rats [108]. While the inhibition of platelet aggregation by beetroot juice (500 ml, 22.5 mmol nitrate) [79], could have been anticipated from the previous studies [97, 109], the demonstration that this was due to nitrite, by asking the volunteers to spit out all their nitrite-containing saliva for 3 h immediately following beetroot juice ingestion, in order to interrupt the enterosalivary circulation and thus prevent absorption of nitrite, was surprising.

Kinetics of the nitrate-nitrite-NO pathway

With the realization of the nitrate-nitrite-NO pathway, the kinetics of dietary nitrate and nitrite (i.e. that derived from nitrate) are summarized in Table 3. Some of the key reactions of nitrate and nitrite in the upper gastrointestinal tract and the circulation are shown in the Figure 1.

figure

Figure 1. Kinetic processes in the handling of dietary nitrate (NO3-) and inorganic nitrite (NO2-) derived from nitrate, from the gut to the circulation to the urinary tract. HNO2 (nitrous acid), N2O3 (dinitrogen trioxide), NO+ (nitrosonium ion), RSNO (nitrosothiol), RR'NNO (N-nitrosamine), eNO (exhaled NO), Vit C (vitamin C), deoxyHb (deoxyhaemoglobin), deoxyMb (deoxymyoglobin), XOR (xanthine oxidoreductase), AO (aldehyde oxidase), ETC (electron transport chain in mitochondria), CA (carbonic anhydrase), ALDH2 (aldehyde dehydrogenase type 2), NOS/eNOS (endothelial nitric oxide synthase), L-arg* (L-arginine), *non-essential amino acid, but also derived from the diet. Nitrate reductase-possessing bacteria on the posterior third of the tongue and in some urinary tract infections convert nitrate to nitrite. Red and blue arrows represent pathways that are favoured under oxygenated and deoxygenated conditions, respectively

Download figure to PowerPoint

Absorption

Dietary nitrate is exceptionally well absorbed in the upper gastrointestinal tract [110], with the bioavailability of nitrate from cooked spinach, raw lettuce and cooked beetroot being ∼100%, with plasma concentrations of nitrate peaking after 1 h [79], (tmax of 1.5–1.8 h) [111], and the bioavailability of nitrite, following ingestion of large amounts, being ∼95–98% [112]. This is despite numerous reactions in the acidic environment of the stomach (see below). Indeed, nitrite is rapidly absorbed, with a plasma tmax of 15–45 min following oral nitrite administration [112], but this increases to ∼3 h, when nitrite is derived from oral nitrate ingestion via the enterosalivary circulation [82]. Only a small fraction of nitrate reaches the large bowel, as <1% of ingested nitrate is excreted in the faeces [113] and <2% is present in ileostomy fluid in patients with a total colectomy [113, 114], with negligible nitrite concentrations. Nitrate and nitrite do not undergo significant first pass metabolism [112].

Distribution

The volumes of distribution of nitrate (∼0.3 l kg−1) [111] and nitrite (∼0.35 l kg−1) [86], are similar and about half that of water (∼0.6 l kg−1). Nitrite is taken up rapidly into most tissues [108]. One of the curiosities of nitrate handling is that such a large proportion is concentrated in the salivary glands. Most estimates suggest that 20–28% of a nitrate load is secreted in saliva [107, 113, 115, 116].

Metabolism

The site of nitrate reduction to nitrite in humans is almost exclusively on the posterior third of the tongue, by nitrate reductase-containing symbiotic bacteria, predominantly Veillonella species, as well as Actinomyces, Rothia and Staphylococcus epidermidis [117, 118]. Whilst some conversion of nitrate to nitrite occurs on the ‘first pass’ of nitrate-containing food over the surface of the tongue before it is initially swallowed, the majority of nitrate reduction to nitrite occurs over the subsequent few hours, via the entero-salivary circulation [1], following concentration of nitrate in the salivary glands, and secretion in saliva, as described above [107, 113, 115, 116]. When nitrite reaches the stomach, some of it reacts with the acidic gastric environment producing nitrous acid which decomposes to form nitric oxide as well as other reactive nitrogen oxides [95] (see Figure 1 and Equations (1)-(3)):

  • display math(1)
  • display math(2)
  • display math(3)

Additional reactions between nitrite and vitamin C or polyphenols in the stomach also generate NO. Despite these numerous reactions in the stomach, the bioavailability of nitrite, when administered in large doses, is normally high (95–98%) [112]. Nitrite appears to mediate the majority of its effects through bioactivation to NO. This occurs mainly via nitrite reductases, which have selective activity under oxygen/hypoxic/ischaemic conditions; these include the deoxygenated globins (haemoglobin [6, 119-121], myoglobin [122], cytoglobin and neuroglobin [123, 124]), the molybdoflavoproteins, which have similar structures to some bacterial nitrite reductases (xanthine oxidoreductase [7, 125], aldehyde oxidase [126]), aldehyde dehydrogenase type 2, ALDH2 [127], eNOS [125, 128-130], cytochrome P450 [131-134] and the mitochondrial electron transport chain [135, 136]. In addition, carbonic anhydrase appears to possess a nitrite anhydrase function [137]. Alternative pathways of nitrite bioactivation include the formation of nitrosothiols [138] and the formation of nitro-fatty acids, such as nitro-oleic acid [139]. It is likely that a varying spectrum of bioactivation pathways operate, dependent on the tissue involved and the prevailing conditions, with different enzymes becoming more active under different conditions.

Excretion

The majority of an ingested load of nitrate, ∼65–75% is renally excreted [110]. Small amounts of nitrite are excreted in the urine, with renal carbonic anhydrases being involved in nitrite reabsorption [140]. Certain bacteria that possess nitrate reductases and are associated with urinary tract infections increase urinary nitrite concentrations, the basis of the nitrite test on the urine dip stick. The half-life of nitrate is 5–8 h [77, 79, 111, 141, 142], which is much longer than that of nitrite, which has been reported as being 1–5 min ex vivo [77, 142] and ∼20–45 min in vivo [86, 112]. This is a reflection of nitrate's stability in the circulation, and nitrite's propensity to be rapidly metabolized via oxidation to nitrate in oxygenated conditions and reduction to NO, as described above.

Arterial ageing and atherosclerosis

The risk factors for atherosclerosis also contribute to several important surrogate markers. The potential impact of dietary nitrate on each of these is considered below.

Endothelial dysfunction

Hypertension, diabetes, dyslipidemia, smoking and ageing are strong risk factors contributing towards atherogenesis and are associated with impaired endothelial function [143], a key step in the pathogenesis of atherosclerosis and a surrogate marker. Such endothelial dysfunction usually results, at least in part, from decreased NO production from endothelial NOS and/or reduced NO bioavailability, which both result in, and are further diminished by inflammation. Plasma nitrite concentration has been shown to reflect constitutive NOS activity in mammals [144] and is inversely correlated with the number of cardiovascular risk factors, and positively correlated with endothelial function as assessed by flow mediated dilatation (FMD) in humans [145]. Also, changes in nitrite (nitrite reserve) with reactive hyperaemia during FMD also reflected endothelial function [146]. Hence, it may be anticipated that provision of dietary nitrate/nitrite may enhance endothelial function and suppress microvascular inflammation. Indeed, this was demonstrated in a hypercholesterolaemia model by Stokes et al. who fed mice a cholesterol-enriched diet for 3 weeks, resulting in impaired endothelium-dependent arteriolar vasodilator responses to acetylcholine which were restored to normal by administration of nitrite in the drinking water [147]. Similarly, nitrite inhibited the elevated leucocyte adhesion to, and emigration through, the venular endothelium resulting from the high cholesterol diet and also reversed the concomitantly elevated CRP to normal concentrations. Whilst inorganic nitrate/nitrite has the potential to improve endothelial dysfunction in patients, Bahra et al. found no effect in healthy volunteers with normal endothelial function. FMD was not altered at 3 h following the ingestion of dietary nitrate (8 mmol). However, a reduction in pulse wave velocity of ∼0.3 m s−1 accompanied the reduction in SBP of ∼5 mmHg [148]. Bondonno et al., (2012) found a small increase in mean FMD over 4 min of ∼0.5% in 30 healthy volunteers following ingestion of 200 mg spinach, but this was not as great as flavanoid-rich apple (∼1.1%) or apple and spinach (∼0.9%) suggesting a potentially greater effect of flavanoids on eNOS function than nitrate in the context of normal endothelial function in healthy volunteers [89]. Whilst many nutraceuticals and drugs have been tested for their capacity to enhance endothelium dependent dilatation via NO production, some have also been demonstrated to enhance endothelium-independent dilatation with nitrovasodilators, suggesting improved NO bioavailability [149]. It is likely that provision of nitrate/nitrite enhances NO bioavailability predominantly by inhibiting inflammation and inactivating reactive oxygen species (ROS), in addition to providing a source of NO per se, rather than by enhancing eNOS.

Arterial stiffness

In addition to finding decreased plasma nitrite concentrations in old (26–28 months) male C57BL6 control mice, Sindler et al. also detected depleted concentrations in the arteries and heart, which were restored to youthful levels (4–6 month controls) by 3 weeks' supplementation with sodium nitrite (50 mg l−1 in the drinking water) [150]. Nitrite also restored the impaired acetylcholine-dependent carotid dilatation and reduced markers of oxidative stress, including nitrotyrosine. Ageing [151-153], hypertension [154], endothelial dysfunction [155], inflammation [156] (and possibly ROS [157]) are also associated with stiffening of large arteries, which when assessed by pulse wave velocity is a strong independent predictor of cardiovascular events [158]. Thus, Sindler et al. found that nitrite reduced the elevated pulse wave velocity and levels of inflammatory cytokines IL-1b, IL6, INFγ and TNFα [150]. However, to what extent the reduction in PWV may have been due to a reduction in BP is not clear as the BP was not reported in this study.

Intimal hyperplasia

Intimal hyperplasia is also a pathological process in the development of atherosclerosis, associated with reduced NO bioavailability [159]. In a compelling series of experiments, Alef et al. demonstrated that, following balloon injury of the rat carotid artery, sodium nitrite (whether through 24 h oral supplementation, or a single intraperitoneal injection, or via inhalation) markedly limited the development of intimal hyperplasia and inhibited smooth muscle cell proliferation, whereas a low nitrate/nitrite diet increased intimal hyperplasia. Even the late introduction of nitrite was able to reverse intimal hyperplasia [160]. Furthermore, the process was demonstrated to be dependent on xanthine oxidoreductase (XOR), which has previously been shown to reduce nitrite to NO in human vessels [125] in addition to animal vessels where it was upregulated following vascular injury. Here, XOR was protective in the presence of nitrite, through the generation of NO species as evidenced by S-nitrosothiols in the vessel wall. Chronic inhibition of XOR with allopurinol or 3 weeks of tungsten-rich diet increased intimal hyperplasia.

Ischaemia-reperfusion injury

Ischaemia-reperfusion injury is damage caused to tissues when blood supply returns or is restored after a period of ischaemia, which deprives the tissue of oxygen and nutrients. The restoring blood causes oxidative damage to the affected organ. Webb et al. demonstrated that infusions of sodium nitrite (10 and 100 μm) before and during an ischaemic insult in the isolated perfused rat heart (Langendorff preparation) resulted in the generation of nitric oxide with involvement of XOR and preserved left ventricular function and reduced infarct size compared with control saline [7]. This protective effect of nitrite in ischaemia-reperfusion injury has now been replicated by many other in vivo and in vitro studies showing protection in most organs tested such as the liver [161, 162], brain [163], heart [164] and kidneys [165] and reviewed by Dezfulian C et al. [166] and Webb & Ahluwalia [167]. Webb et al. translated this into a forearm model of ischaemia-reperfusion and demonstrated that nitrate supplementation in the form of beetroot juice attenuated endothelial function impairment following ischaemia-reperfusion injury during FMD of the brachial artery in healthy human subjects [79]. A similar effect was achieved with potassium nitrate [82].

Improving blood flow in hypoxic and ischaemic tissues

Given the capacity of dietary nitrate to result in vasodilatation, particularly of hypoxic or ischaemic vascular beds, Presley et al. studied the effect of a nitrate-rich diet on cerebral perfusion in older adults, aged ∼75 years using arterial spin labelling magnetic resonance imaging [168]. While there was no global effect, regional cerebral perfusion to the frontal white matter was enhanced, suggesting dietary nitrate may have the potential to enhance executive function and combat cognitive decline.

Plasma nitrite concentration has been shown to increase following exercise in young healthy individuals, but not in older individuals with impaired endothelial function due to impaired production and bioactivity of NO [169]. In patients with peripheral arterial disease (PAD), plasma nitrite falls during exercise. Therefore provision of dietary nitrate may be useful to supplement such deficiency in nitrite flux. Indeed, Kenjale et al. demonstrated that a single dose of 500 ml beetroot juice (9 mmol nitrate) increased both the time before onset of claudication by 18% and the total walking time by 17% in patients with PAD whilst maintaining elevated plasma nitrite concentrations, associated with a reduction in fractional O2 extraction by the gastrocnemius [170].

Exercise performance

A fundamental principle of exercise physiology was that during exercise, the oxygen cost is predictable for a given submaximal work rate [171-173]. However, when Larsen et al. tested the effects of 3 days of supplementation of sodium nitrate (0.1 mmol kg−1), as a potential source of NO, on exercise performance they surprisingly found that it resulted in reduction in oxygen consumption (of ∼0.16 l min−1) during submaximal exercise (between 45–80% of peak exercise) [174] with no effect on plasma lactate concentration, suggesting that exercise had become more efficient and the oxygen cost had been reduced. In a subsequent study, Larsen et al. found that nitrate supplementation (0.033 mmol kg−1 three times daily for 2 days) also reduced maximal oxygen consumption (VO2 max) by ∼0.10 l min−1 [175]. Bailey et al. found similar results with beetroot juice (∼11 mmol nitrate for 6 days), which reduced the increase in pulmonary oxygen uptake during moderate exercise by ∼19%, reduced the slow component of O2 uptake during severe exercise, and increased time to exhaustion [176].

Using 31P-MRS, Bailey et al. subsequently demonstrated that the ∼20% reduction in oxygen cost due to beetroot juice ingestion (5.1 mmol day−1 for 6 days) was due to a reduction in ATP cost of muscle force production (during knee-extension exercises) at both low and high intensity exercise [177]. There was also a 16% improvement in the time to task failure during severe exercise. Vanhatalo et al. extended the duration of observed beneficial effects of beetroot juice (nitrate 5.2 mmol day−1) on exercise performance and blood pressure lowering from acute effects at 2.5 h, to sustained effects over 15 days [88]. Using a nitrate-depleted beetroot juice as placebo, Lansley et al. demonstrated that it was the nitrate content of the active juice, rather than other components such as betaine or polyphenols, that was responsible for the improvements in the O2 cost of walking and moderate and severe intensity running and a 15% increase in time-to-exhaustion [178]. Other studies have demonstrated acute improvements in power output with nitrate during cycling (4 and 16.1 km) [179]. Also during exercise in hypoxia (with subjects breathing 14.5% O2), ingestion of 750 ml of beetroot juice over the preceding 24 h (9.3 mmol nitrate), resulted in reduced muscle metabolic perturbation and remarkably restored exercise tolerance and oxidative function to values observed in normoxia [180]. Most recently, supplementation with beetroot juice (∼8 mmol day−1) for 6 days has been found to reduce the time for trained cyclists to complete a 10 km time trial by ∼12 s, in a double-blind study compared with placebo (nitrate-depleted) beetroot juice [181]. The effects of dietary nitrate on exercise performance in human studies are summarized in Table 5.

Table 5. Effects of dietary nitrate on exercise performance in human studies, showing duration, amount of nitrate given in mmol (*0.1 mmol kg−1 day−1 or *0.033 mmol kg−1 3 times day−1 = ∼7 mmol day−1 in 70 kg person) and summary of effect
StudySourceNitrate (mmol d−1)DurationEffect on exercise
Larsen et al. (2007) [174]

Sodium nitrate

*0.1 mmol kg−1 day−1

∼7*3 daysReduction in O2 consumption (of ∼0.16 l min−1) during submaximal exercise (between 45–80% of peak exercise) with no effect on plasma lactate concentration, suggesting that exercise had become more efficient and the O2 cost had been reduced
Bailey et al. (2009) [176]Beetroot juice 500 ml day−1∼116 daysReduced the increase in pulmonary O2 uptake during moderate exercise by ∼19%, as well as reducing the slow component of O2 uptake during severe exercise, and increased time to exhaustion by ∼16%
Bailey et al. (2010) [177]Beetroot juice 500 ml day−15.16 days∼20% reduction in O2 cost due to a reduction in ATP cost of muscle force production (during knee-extension exercises) at both low and high intensity exercise, as well as ∼16% improvement in time to task failure during severe exercise
Larsen et al. (2010) [175]

Sodium nitrate

*0.033 mmol kg−1 three times day−1

∼7*2 daysReduced maximal O2 consumption (VO2 max) by ∼0.10 l min−1
Vanhatalo et al. (2010) [88]Beetroot juice 500 ml day−15.215 daysAcute effects at 2.5 h, and sustained effects over 15 days observed with continued supplementation, demonstrating a reduction in steady state VO2 by ∼4% with elevations in peak power and work rate
Lansley et al. (2011) [178]

Beetroot juice

500 ml day−1

∼6.26 daysImprovements in the O2 cost of walking and moderate and severe intensity running and a 15% increase in time-to-exhaustion
Lansley et al. (2011) [179]Beetroot juice 500 ml∼6.2Single doseAcute improvements in power output have been demonstrated by ∼2.8% during cycling (4 and 16.1 km distances) for the same VO2
Vanhatalo et al. (2011) [180]Beetroot juice 750 ml 24 h−19.3250 ml: 24 h, 12 h & 2.5 h pre-Exercise in hypoxia (subjects breathing 14.5% O2) restored exercise tolerance and oxidative function to values observed in normoxia
Kenjale et al. (2011) [170]Beetroot juice 500 ml x1∼9Single doseIncreased time before onset of claudication by 18% and the total walking time by 17% in patients with peripheral arterial disease
Cermak et al. (2012) [181]Beetroot juice shots (140 ml day−1)∼86 daysReduced time for trained cyclists to complete 10 km time trial by ∼12 s

A major advance in the mechanistic understanding of the reduced oxygen and ATP cost, came when Larsen et al. harvested skeletal muscle mitochondria and found that prior dietary nitrate supplementation improved oxidative phosphorylation efficiency (increased P : O ratio) indicating diversion of membrane potential away from uncoupling actions such as proton leak towards ATP synthesis [182]. Nitrate achieved this by reducing the expression of adenine nucleotide translocase (ANT), a site of proton leak. Additional effects include the nitrosation (-SNO) of complex I by nitrite and competition between NO and O2 with complex IV. Preservation of mitochondrial complex I activity, oxidative phosphorylation and attenuation of hydrogen peroxide generation and tissue lipid peroxidation may also be mechanisms by which dietary nitrate supplementation prevents doxorubicin-induced impairment of ventricular contractility and cell death, as recently demonstrated by Zhu et al. in a mouse model of doxorubicin-induced cardiomyopathy [183].

Pulmonary circulation

Nitrite also leads to dilatation in pulmonary vascular beds reducing pulmonary arterial pressure in humans and animals [184-186], and therefore has the potential to ameliorate pulmonary hypertension. Indeed, this is supported by a very recent study demonstrating a reduction in right ventricular pressure and hypertrophy, and pulmonary vascular remodelling with dietary nitrate treatment in mice exposed to 3 weeks of hypoxia, conditions associated with preferential reduction (of nitrite) to NO [187].

Interaction of nitrate-nitrite with other nutrients

The effects of dietary nitrate may be considerably enhanced or altered through interactions with other nutrients. For example, in addition to polyphenols in fruit and vegetables, Gago et al. found that red wine polyphenols [anthocyanin fraction and catechol (caffeic acid)] are very effective at converting nitrite to NO in vitro and in the human stomach [188]. Indeed, nitrite reductase activity has been associated with a broad range of dietary phenols (greatest to least activity): epicatechin-3-O-gallate, quercetin, procyanidin B8 dimer, oleuropein, procyanidin B2 dimer, chlorogenic acid, epicatechin, catechin, procyanidin B5 dimer [189]. Hawthorn berry extract has been found to have extremely potent nitrite reductase activity, in addition to containing polyphenols (∼5%) and Zand et al. recently performed a placebo-controlled study of 30 days twice daily supplementation with a formulation containing sodium nitrite, hawthorn berry extract, vitamin C, beetroot powder, vitamin B12 and L-citrulline in patients with three or more cardiovascular risk factors and found that it reduced triglycerides in patients with elevated triglycerides (>150 mg dl–1) at baseline. No significant reduction in blood pressure was seen however [190]. In addition, in wine, ethanol is nitrosated resulting in the formation of ethylnitrite, an organic nitrite, which has been shown to be a potent vasodilator [191]. Indeed, Moya et al. have previously demonstrated that inhaled ethyl nitrite results in sustained improvements in arterial oxygenation and haemodynamics in persistent pulmonary hypertension of the newborn [192].

Unsaturated fatty acids in the diet, such as linoleic and oleic acid are nitrated by nitrous acid, derived from nitrite in the acid environment of the stomach, forming nitro-fatty acids (NO2-FAs) [193]. NO2-FAs have several anti-inflammatory actions, inhibiting neutrophils, platelets and macrophages. Signalling pathways are mediated through S-alkylation of, for example, nuclear factor κ B (resulting in inhibition of macrophage cytokine and iNOS expression) [194] and peroxisome proliferator-activated receptor-γ (PPAR-γ) [195]. Rudolph et al. demonstrated that subcutaneous injection of nitro-oleic acid markedly reduced atherosclerotic lesion formation in apoliproprotein E-deficient mice associated with a variety of anti-inflammatory effects including reduction in foam cell formation through attenuation of oxidized LDL-induced phosphorylation of signal transducer and activator of transcription-1 (STAT-1) [139]. The oral administration of nitro-oleic acid has also been shown to be effective in suppressing inflammation in experimental inflammatory bowel disease [196]. Also, Kelley et al., have found that nitro-oleic acid inhibits XOR, and is surprisingly more potent than allopurinol in terms of inhibition of superoxide production [197] and may be part of the mechanism by which nitro-oleic acid confers protection in a mouse model of renal ischaemia-reperfusion [198].

The study by Bondonno et al. examined the interaction of apple skin rich in flavonoids, quercetin and (−)-epicatechin and spinach as a source of dietary nitrate. Whilst apple and spinach reduced SBP (by ∼−3.3 mmHg and ∼−2.7 mmHg after 2 h respectively) compared with control, the combination of apple and spinach had no effect and resulted in an intermediate plasma nitrite concentration suggesting possible extravascular (e.g. stomach) reduction of nitrite by the apple flavonoids and ascorbic acid, as intravascular reduction might be expected to enhance BP reduction [89].

Cancer risk

Nitrate and nitrite have been used for curing meat for centuries, and remain the most effective method to reduce bacterial growth and kill botulinum spores. Major concern emerged in the 1960s [199], with the demonstration of carcinogenic dimethylnitrosamine formation (known to disrupt nucleic acids in the rat and cause liver tumours [200]) from sodium nitrite [201, 202]. However, chronic feeding of nitrite to rats, even when diethylamine was given at the same time, did not induce tumours [203]. Concern for dietary nitrate arose in 1976, when Spiegelhalder et al. [116] and Tannenbaum et al. [115], both independently suggested that conversion to nitrite in the enterosalivary circulation, could result in the formation of N-nitrosoamines. Whilst N-nitroso compounds have repeatedly been shown to be carcinogenic in animals [200, 204], reviews such as the 2003 Joint FAO/WHO Expert Committee on Food Additives (JEFCA) of the epidemiological and toxicological studies in humans have failed to establish a definite link between nitrate intake and risk of developing cancer [205-207]. However, as with most substances, some groups of individuals are likely to be more susceptible than others. For example, work from McColl's group has elegantly demonstrated that patients with Barrett's oesophagus have increased nitrosation at the gastro-oesophageal junction, the major site of adenocarcinoma of the human upper gastrointestinal tract [208].

The World Cancer Research Fund/American Institute of Cancer Research, Second Report 2007, did not show any increased risk of cancers, such as stomach or ovarian, with green leafy vegetables, and even showed trends towards beneficial effects [209]. Indeed, the recommendation from the report was to eat at least five portions/servings (at least 400 g) of a variety of non-starchy vegetables and fruits every day, specifically including green, leafy vegetables. Some epidemiological studies have shown that fruit and vegetables have protective effects against certain cancers [210]. Indeed, there may be other components in fruit and vegetables which protect against damaging effects of nitrite such as ascorbate [211-213], vitamin E, phenolic compounds and fruit and vegetable juices [204, 214-217], although some of these interactions appear complex [212, 218, 219].

The National Institutes of Health-American Association of Retired Persons (NIH-AARP) Diet and Health Study prospectively studied large cohorts and have recently identified some potential associations between high nitrate intake, assessed using a 124-item food frequency questionnaire, and epithelial ovarian cancer [220] and thyroid cancer (nitrate competitively inhibits iodide uptake) [221]. However, no associations have been demonstrated between total nitrate or nitrite intake (processed meat) and pancreatic cancer [222], adenocarcinoma of the stomach and oesophagus [223], non-Hodgkin lymphoma risk overall [224], renal cell carcinoma [225] or bladder cancer. However, most of the associations are small, do not prove causality and should be interpreted with extreme caution. The limitations of extrapolating from epidemiological studies has been recently reviewed by Milkowski et al. [32]. Thus, the lack of data concerning the long term effects of dietary nitrate is a limitation, and this will need to be addressed in future trials.

Potential anti-cancer effects of beetroot

Quite independently of the nitrate/nitrite field, several in vitro and in vivo studies have demonstrated that red beet/beetroot extract has protective effects in various cancer cell lines, such as prostate and breast, liver, lung, oesophagus and skin [226-229]. These effects of beetroot (juice) have generally been ascribed to betanin, the major betacyanin constituent, which has strong antioxidant activity, and is particularly high in betalain extracts obtained from hairy root cultures of the red beetroot B. vulgaris [230]. Beetroot may represent a particularly safe source of dietary nitrate, with the potential to reduce, rather than increase cancer risk. Indeed, beetroot juice has even achieved a considerable degree of acceptance as an alternative medicine for cancer patients [231, 232]. However, the mechanisms require further clarification.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Effects of vegetables on blood pressure and cardiovascular disease events
  5. Dietary sources of nitrate
  6. Acceptable daily intake (ADI)?
  7. Vascular effects of dietary nitrate
  8. Conclusion
  9. Competing Interests
  10. References

Dietary nitrate has important vascular effects mediated via the nitrate-nitrite-NO pathway. Whilst these effects are promising and suggestive that dietary nitrate underlies the apparent beneficial effects of vegetable rich diets, such as the Mediterranean and Japanese diets, they represent surrogate endpoints and further long term studies with hard endpoints will eventually be required to substantiate the beneficial effects and demonstrate that they outweigh any risk of cancer. Until such data are available, the quantity of nitrate consumed is largely the decision of the individual, depending on their requirement (which may be different in patients with hypertension, healthy individuals or athletes). Since health professionals may be called upon to give advice, and to guide individuals, we provide practical advice in the form of summary tables of nitrate content, ADIs for given weights, comparisons of the body's handling of nitrate and nitrite, and studies showing efficacy in exercise, and on blood pressure in healthy individuals. The results of studies in patients with hypertension are awaited.

Competing Interests

  1. Top of page
  2. Abstract
  3. Introduction
  4. Effects of vegetables on blood pressure and cardiovascular disease events
  5. Dietary sources of nitrate
  6. Acceptable daily intake (ADI)?
  7. Vascular effects of dietary nitrate
  8. Conclusion
  9. Competing Interests
  10. References

All authors have completed the Unified Competing Interest form at http://www.icmje.org/coi_disclosure.pdf Vascular effects of dietary nitrate (available on request from the corresponding author) and declare no support from any organization for the submitted work and no other relationships or activities that could appear to have influenced the submitted work.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Effects of vegetables on blood pressure and cardiovascular disease events
  5. Dietary sources of nitrate
  6. Acceptable daily intake (ADI)?
  7. Vascular effects of dietary nitrate
  8. Conclusion
  9. Competing Interests
  10. References
  • 1
    Lundberg JO, Weitzberg E, Gladwin MT. The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov 2008; 7: 156167.
  • 2
    Palmer RM, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 1988; 333: 664666.
  • 3
    Moncada S, Palmer RM, Higgs EA. The discovery of nitric oxide as the endogenous nitrovasodilator. Hypertension 1988; 12: 365372.
  • 4
    Modin A, Bjorne H, Herulf M, Alving K, Weitzberg E, Lundberg JO. Nitrite-derived nitric oxide: a possible mediator of ‘acidic-metabolic’ vasodilation. Acta Physiol Scand 2001; 171: 916.
  • 5
    Demoncheaux EA, Higenbottam TW, Foster PJ, Borland CD, Smith AP, Marriott HM, Bee D, Akamine S, Davies MB. Circulating nitrite anions are a directly acting vasodilator and are donors for nitric oxide. Clin Sci 2002; 102: 7783.
  • 6
    Cosby K, Partovi KS, Crawford JH, Patel RP, Reiter CD, Martyr S, Yang BK, Waclawiw MA, Zalos G, Xu X, Huang KT, Shields H, Kim-Shapiro DB, Schechter AN, Cannon RO 3rd, Gladwin MT. Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nat Med 2003; 9: 14981505.
  • 7
    Webb A, Bond R, McLean P, Uppal R, Benjamin N, Ahluwalia A. Reduction of nitrite to nitric oxide during ischemia protects against myocardial ischemia-reperfusion damage. Proc Natl Acad Sci U S A 2004; 101: 1368313688.
  • 8
    Pannala AS, Mani AR, Spencer JP, Skinner V, Bruckdorfer KR, Moore KP, Rice-Evans CA. The effect of dietary nitrate on salivary, plasma, and urinary nitrate metabolism in humans. Free Radic Biol Med 2003; 34: 576584.
  • 9
    Sobko T, Marcus C, Govoni M, Kamiya S. Dietary nitrate in Japanese traditional foods lowers diastolic blood pressure in healthy volunteers. Nitric Oxide 2010; 22: 136140.
  • 10
    Yamori Y, Miura A, Taira K. Implications from and for food cultures for cardiovascular diseases: Japanese food, particularly Okinawan diets. Asia Pac J Clin Nutr 2001; 10: 144145.
  • 11
    FB H. The Mediterranean diet and mortality – olive oil and beyond. N Engl J Med 2003; 348: 25952596.
  • 12
    Keys A. Mediterranean diet and public health: personal reflections. Am J Clin Nutr 1995; 61: 1321S1323.
  • 13
    Mente A, de Koning L, Shannon HS, Anand SS. A systematic review of the evidence supporting a causal link between dietary factors and coronary heart disease. Arch Intern Med 2009; 169: 659669.
  • 14
    Tsuji KMS, Morita Y, Shibata T, Kaneta N, Wakabayashi K, Uchibori-Hase S, Ide S, Fujiwara K, Suzuki H, Ito Y. Naturally occurring of nitrite and nitrate existing in various raw and processed foods. J Food Hyg Soc Jpn 1993; 34: 294302.
  • 15
    Donaldson AN. The Relation of Protein Foods to Hypertension. Cal West Med 1926; 24: 328331.
  • 16
    Sacks FM, Rosner B, Kass EH. Blood pressure in vegetarians. Am J Epidemiol 1974; 100: 390398.
  • 17
    Armstrong B, van Merwyk AJ, Coates H. Blood pressure in Seventh-day Adventist vegetarians. Am J Epidemiol 1977; 105: 444449.
  • 18
    Armstrong B, Clarke H, Martin C, Ward W, Norman N, Masarei J. Urinary sodium and blood pressure in vegetarians. Am J Clin Nutr 1979; 32: 24722476.
  • 19
    Joshipura KJ, Ascherio A, Manson JE, Stampfer MJ, Rimm EB, Speizer FE, Hennekens CH, Spiegelman D, Willett WC. Fruit and vegetable intake in relation to risk of ischemic stroke. JAMA 1999; 282: 12331239.
  • 20
    Joshipura KJ, Hu FB, Manson JE, Stampfer MJ, Rimm EB, Speizer FE, Colditz G, Ascherio A, Rosner B, Spiegelman D, Willett WC. The effect of fruit and vegetable intake on risk for coronary heart disease. Ann Intern Med 2001; 134: 11061114.
  • 21
    Kapil V, Webb AJ, Ahluwalia A. Inorganic nitrate and the cardiovascular system. Heart 2010; 96: 17031709.
  • 22
    Appel LJ, Moore TJ, Obarzanek E, Vollmer WM, Svetkey LP, Sacks FM, Bray GA, Vogt TM, Cutler JA, Windhauser MM, Lin PH, Karanja N. A clinical trial of the effects of dietary patterns on blood pressure. DASH Collaborative Research Group. N Engl J Med 1997; 336: 11171124.
  • 23
    Rouse IL, Beilin LJ, Armstrong BK, Vandongen R. Blood-pressure-lowering effect of a vegetarian diet: controlled trial in normotensive subjects. Lancet 1983; 1: 510.
  • 24
    Lindahl O, Lindwall L, Spangberg A, Stenram A, Ockerman PA. A vegan regimen with reduced medication in the treatment of hypertension. Br J Nutr 1984; 52: 1120.
  • 25
    Gangolli SD, van den Brandt PA, Feron VJ, Janzowsky C, Koeman JH, Speijers GJ, Spiegelhalder B, Walker R, Wisnok JS. Nitrate, nitrite and N-nitroso compounds. Eur J Pharmacol 1994; 292: 138.
  • 26
    White JW Jr. Relative significance of dietary sources of nitrate and nitrite. J Agric Food Chem 1975; 23: 886891.
  • 27
    van Loon AIM, van Klaveren JD. Nitraatinname van de Nederiandse bevolking. Voeding 1991; 52: 96100.
  • 28
    Knight TM, Forman D, Al-Dabbagh SA, Doll R. Estimation of dietary intake of nitrate and nitrite in Great Britain. Food and Chemical Toxicology : An International Journal Published for the British Industrial Biological Research Association 1987; 25: 277285.
  • 29
    Binkerd EF, Kolari OE. The history and use of nitrate and nitrite in the curing of meat. Food Cosmet Toxicol 1975; 13: 655661.
  • 30
    Machha A, Schechter A. Dietary nitrite and nitrate: a review of potential mechanisms of cardiovascular benefits. Eur J Nutr 2011; 50: 293303.
  • 31
    Rocha BS, Gago B, Pereira C, Barbosa RM, Bartesaghi S, Lundberg JO, Radi R, Laranjinha J. Dietary nitrite in nitric oxide biology: a redox interplay with implications for pathophysiology and therapeutics. Curr Drug Targets 2011; 12: 13511363.
  • 32
    Milkowski A, Garg HK, Coughlin JR, Bryan NS. Nutritional epidemiology in the context of nitric oxide biology: a risk-benefit evaluation for dietary nitrite and nitrate. Nitric Oxide 2010; 22: 110119.
  • 33
    Kevil CG, Kolluru GK, Pattillo CB, Giordano T. Inorganic nitrite therapy: historical perspective and future directions. Free Radic Biol Med 2011; 51: 576593.
  • 34
    Omar SA, Artime E, Webb AJ. A comparison of organic and inorganic nitrates/nitrites. Nitric Oxide 2012; 26: 229240.
  • 35
    Tamme T, Reinik M, Roasto M, Juhkam K, Tenno T, Kiis A. Nitrates and nitrites in vegetables and vegetable-based products and their intakes by the Estonian population. Food Addit Contam 2006; 23: 355361.
  • 36
    Santamaria P. Nitrate in vegetables: toxicity, content, intake and EC regulation. J Sci Food Agric 2006; 86: 1017.
  • 37
    Santamaria P, Elia A, Serio F, Todaro E. A survey of nitrate and oxalate content in fresh vegetables. J Sci Food Agric 1999; 79: 18821888.
  • 38
    Santamaria P, Gonnella M, Elia A, Parente A, Serio F. Ways of reducing rocket salad nitrate content. Acta Hortic 2001; 529536.
  • 39
    Meah MN, Harrison N, Davies A. Nitrate and nitrite in foods and the diet. Food Addit Contam 1994; 11: 519532.
  • 40
    Ysart G, Clifford R, Harrison N. Monitoring for nitrate in UK-grown lettuce and spinach. Food Addit Contam 1999; 16: 301306.
  • 41
    Maynard DN, Barker AV, Minotti PL, Peck NH. Nitrate accumulation in vegetables. Adv Agron 1976; 28: 71118.
  • 42
    Corr WJ, Breimer T. Nitrate and Nitrite in Vegetables. Wageningen: Centre for Agricultural Publishing and Documentation (PUDOC), 1979.
  • 43
    Bhattacharjee R, Singh A, Mukhopadhyay S. Use of nitrogen-fixing bacteria as biofertiliser for non-legumes: prospects and challenges. Appl Microbiol Biotechnol 2008; 80: 199209.
  • 44
    Pate JS. Uptake, assimilation and transport of nitrogen compounds by plants. Soil Biol Biochem 1973; 5: 109119.
  • 45
    Andrews M. The partitioning of nitrate assimilation between root and shoot of higher plants. Plant Cell Environ 1986; 9: 511519.
  • 46
    Wallace W. Distribution of nitrate assimilation between the root and shoot of legumes and a comparison with wheat. Physiol Plant 1986; 66: 630636.
  • 47
    Tamme T, Reinik M, Pussa T, Roasto M, Meremae K, Kiis A. Dynamics of nitrate and nitrite content during storage of home-made and small-scale industrially produced raw vegetable juices and their dietary intake. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 2010; 27: 487495.
  • 48
    Katan MB. Nitrate in foods: harmful or healthy? Am J Clin Nutr 2009; 90: 1112.
  • 49
    Taylor S. Relative Exposure to Nitrite, Nitrate, and N-Nitroso Compounds from Endogenous and Exogenous Sources. Food Toxicology: A Perspective on the Relative Risks Marcel Dekker Inc 1989; 6364, ISBN 0-8247-8141-4.
  • 50
    Mitchell HH, Shonle HA, Grindley HS. The origin of the nitrates in the urine. J Biol Chem 1916; 24: 461490.
  • 51
    Green LC, Ruiz de Luzuriaga K, Wagner DA, Rand W, Istfan N, Young VR, Tannenbaum SR. Nitrate biosynthesis in man. Proc Natl Acad Sci U S A 1981; 78: 77647768.
  • 52
    Leaf CD, Wishnok JS, Tannenbaum SR. L-arginine is a precursor for nitrate biosynthesis in humans. Biochem Biophys Res Commun 1989; 163: 10321037.
  • 53
    Rhodes P, Leone AM, Francis PL, Struthers AD, Moncada S, Rhodes PM. The L-arginine: nitric oxide pathway is the major source of plasma nitrite in fasted humans. Biochem Biophys Res Commun 1995; 209: 590596.
  • 54
    Penttila PL. Estimation of Food Additive and Pesticide Intakes by Means of A Stepwise Method [Dissertation]. Turku: University of Turku, 1995.
  • 55
    Dejonckheere W, Streubaut W, Drieghe S, Verstraeten R, Braeckman H. Nitrate in food commodities of vegetable origin and the total diet in Belgium, 1992–1993. Microbiologie – Aliments – Nutrition 1994; 12: 359370.
  • 56
    Petersen A, Stoltze S. Nitrate and nitrite in vegetables on the Danish market: content and intake. Food Addit Contam 1999; 16: 291299.
  • 57
    Belitz HD, Grosch W. Food Chemistry, 992. Berlin: Springer-Verlag, 1999.
  • 58
    Chung SY, Kim JS, Kim M, Hong MK, Lee JO, Kim CM, Song IS. Survey of nitrate and nitrite contents of vegetables grown in Korea. Food Addit Contam 2003; 20: 621628.
  • 59
    Pardo-Marín O, Yusà-Pelechà V, Villalba-Martín P, Perez-Dasí JA. Monitoring programme on nitrates in vegetables and vegetable-based baby foods marketed in the Region of Valencia, Spain: levels and estimated daily intake. Food Additives & Contaminants: Part A 2010; 27: 478486.
  • 60
    Menard C, Heraud F, Volatier JL, Leblanc JC. Assessment of Dietary Exposure of Nitrate and Nitrite in France. Food Additives & Contaminants: Part A 2008; 25: 971988.
  • 61
    Thames Water. Water quality report – 2011 data [online]. Available at http://secure.thameswater.co.uk/water-quality-reports/2011%20WQ%20Report_Z0329_north%20lambeth.pdf (last accessed 20 February 2012).
  • 62
    The Open University, Water for life, Section 8. Water and its impurities [online]. Available at http://openlearn.open.ac.uk/mod/oucontent/view.php?id=398688&section=8 (last accessed 20 February 2012).
  • 63
    Queensland Health Dietitians. Foods that contain potassium (K+), April 2012 [online]. Available at http://www.health.qld.gov.au/nutrition/resources/renal_klong.pdf (last accessed 14 February 2012).
  • 64
    Comly HH. Landmark article Sept 8, 1945: cyanosis in infants caused by nitrates in well-water. By Hunter H. Comly. JAMA 1987; 257: 27882792.
  • 65
    Walton G. Survey of literature relating to infant methemoglobinemia due to nitrate-contaminated water. Am J Public Health Nations Health 1951; 41: 986996.
  • 66
    Avery AA. Infantile methemoglobinemia: reexamining the role of drinking water nitrates. Environ Health Perspect 1999; 107: 583586.
  • 67
    Willis T. Pharmaceutice Rationalis: Or An Exercitation of the Medicines in Humane Bodies. London: T. Dring, C. Harper, and J. Leigh, 1697.
  • 68
    Hiatt EP. Comparison of the diuretic effects of nitrate salts with other diuretic agents. Am J Physiol 1957; 189: 173176.
  • 69
    Butler AR, Feelisch M. Therapeutic uses of inorganic nitrite and nitrate: from the past to the future. Circulation 2008; 117: 21512159.
  • 70
    Reichert ET, Mitchell SW. On the physiological action of potassium nitrite. Am J Med Sci 1880; 156: 158180.
  • 71
    Stebbing J. Post-prandial syncope due to nitrates in food. Postgrad Med J 1997; 73: 606.
  • 72
    Furchgott RF, Bhadrakom S. Reactions of strips of rabbit aorta to epinephrine, isopropylarterenol, sodium nitrite and other drugs. J Pharmacol Exp Ther 1953; 108: 129143.
  • 73
    Gladwin MT, Shelhamer JH, Schechter AN, Pease-Fye ME, Waclawiw MA, Panza JA, Ognibene FP, Cannon RO 3rd. Role of circulating nitrite and S-nitrosohemoglobin in the regulation of regional blood flow in humans. Proc Natl Acad Sci U S A 2000; 97: 1148211487.
  • 74
    Tsuchiya K, Takiguchi Y, Okamoto M, Izawa Y, Kanematsu Y, Yoshizumi M, Tamaki T. Malfunction of vascular control in lifestyle-related diseases: formation of systemic hemoglobin-nitric oxide complex (HbNO) from dietary nitrite. J Pharmacol Sci 2004; 96: 395400.
  • 75
    Maher AR, Milsom AB, Gunaruwan P, Abozguia K, Ahmed I, Weaver RA, Thomas P, Ashrafian H, Born GV, James PE, Frenneaux MP. Hypoxic modulation of exogenous nitrite-induced vasodilation in humans. Circulation 2008; 117: 670677.
  • 76
    Curtis E, Hsu LL, Noguchi AC, Geary L, Shiva S. Oxygen regulates tissue nitrite metabolism. Antioxid Redox Signal 2012; 17: 951961.
  • 77
    Lundberg JO, Govoni M. Inorganic nitrate is a possible source for systemic generation of nitric oxide. Free Radic Biol Med 2004; 37: 395400.
  • 78
    Larsen FJ, Ekblom B, Sahlin K, Lundberg JO, Weitzberg E. Effects of dietary nitrate on blood pressure in healthy volunteers. N Engl J Med 2006; 355: 27922793.
  • 79
    Webb AJ, Patel N, Loukogeorgakis S, Okorie M, Aboud Z, Misra S, Rashid R, Miall P, Deanfield J, Benjamin N, MacAllister R, Hobbs AJ, Ahluwalia A. Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite. Hypertension 2008; 51: 784790.
  • 80
    Govoni M, Jansson EA, Weitzberg E, Lundberg JO. The increase in plasma nitrite after a dietary nitrate load is markedly attenuated by an antibacterial mouthwash. Nitric Oxide 2008; 19: 333337.
  • 81
    Petersson J, Carlstrom M, Schreiber O, Phillipson M, Christoffersson G, Jagare A, Roos S, Jansson EA, Persson AE, Lundberg JO, Holm L. Gastroprotective and blood pressure lowering effects of dietary nitrate are abolished by an antiseptic mouthwash. Free Radic Biol Med 2009; 46: 10681075.
  • 82
    Kapil V, Milsom AB, Okorie M, Maleki-Toyserkani S, Akram F, Rehman F, Arghandawi S, Pearl V, Benjamin N, Loukogeorgakis S, Macallister R, Hobbs AJ, Webb AJ, Ahluwalia A. Inorganic nitrate supplementation lowers blood pressure in humans: role for nitrite-derived NO. Hypertension 2010; 56: 274281.
  • 83
    Berry SE, Mulla UZ, Chowienczyk PJ, Sanders TA. Increased potassium intake from fruit and vegetables or supplements does not lower blood pressure or improve vascular function in UK men and women with early hypertension: a randomised controlled trial. Br J Nutr 2010; 104: 18391847.
  • 84
    He FJ, Markandu ND, Coltart R, Barron J, MacGregor GA. Effect of short-term supplementation of potassium chloride and potassium citrate on blood pressure in hypertensives. Hypertension 2005; 45: 571574.
  • 85
    Hobbs AJ. Soluble guanylate cyclase: the forgotten sibling. Trends Pharmacol Sci 1997; 18: 484491.
  • 86
    Dejam A, Hunter CJ, Tremonti C, Pluta RM, Hon YY, Grimes G, Partovi K, Pelletier MM, Oldfield EH, Cannon RO 3rd, Schechter AN, Gladwin MT. Nitrite infusion in humans and nonhuman primates: endocrine effects, pharmacokinetics, and tolerance formation. Circulation 2007; 116: 18211831.
  • 87
    Parker JD, Gori T. Tolerance to the organic nitrates: new ideas, new mechanisms, continued mystery. Circulation 2001; 104: 22632265.
  • 88
    Vanhatalo A, Bailey SJ, Blackwell JR, DiMenna FJ, Pavey TG, Wilkerson DP, Benjamin N, Winyard PG, Jones AM. Acute and chronic effects of dietary nitrate supplementation on blood pressure and the physiological responses to moderate-intensity and incremental exercise. Am J Physiol Regul Integr Comp Physiol 2010; 299: R11211131.
  • 89
    Bondonno CP, Yang X, Croft KD, Considine MJ, Ward NC, Rich L, Puddey IB, Swinny E, Mubarak A, Hodgson JM. Flavonoid-rich apples and nitrate-rich spinach augment nitric oxide status and improve endothelial function in healthy men and women: a randomized controlled trial. Free Radic Biol Med 2012; 52: 95102.
  • 90
    Hobbs DA, Kaffa N, George TW, Methven L, Lovegrove JA. Blood pressure-lowering effects of beetroot juice and novel beetroot-enriched bread products in normotensive male subjects. Br J Nutr 2012; 14: 19. [Epub ahead of print].
  • 91
    Mustard JF, Moore S, Packham MA, Kinlough-Rathbone RL. Platelets, thrombosis and atherosclerosis. Prog Biochem Pharmacol 1977; 13: 312325.
  • 92
    Fitzgerald DJ, Roy L, Catella F, FitzGerald GA. Platelet activation in unstable coronary disease. N Engl J Med 1986; 315: 983989.
  • 93
    Radomski MW, Palmer RM, Moncada S. Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet 1987; 2: 10571058.
  • 94
    Radomski MW, Palmer RM, Moncada S. An L-arginine/nitric oxide pathway present in human platelets regulates aggregation. Proc Natl Acad Sci U S A 1990; 87: 51935197.
  • 95
    Benjamin N, O'Driscoll F, Dougall H, Duncan C, Smith L, Golden M, McKenzie H. Stomach NO synthesis. Nature 1994; 368: 502.
  • 96
    Lundberg JO, Weitzberg E, Lundberg JM, Alving K. Intragastric nitric oxide production in humans: measurements in expelled air. Gut 1994; 35: 15431546.
  • 97
    Richardson G, Hicks SL, O'Byrne S, Frost MT, Moore K, Benjamin N, McKnight GM. The ingestion of inorganic nitrate increases gastric S-nitrosothiol levels and inhibits platelet function in humans. Nitric Oxide 2002; 7: 2429.
  • 98
    Rocks SA, Davies CA, Hicks SL, Webb AJ, Klocke R, Timmins GS, Johnston A, Jawad AS, Blake DR, Benjamin N, Winyard PG. Measurement of S-nitrosothiols in extracellular fluids from healthy human volunteers and rheumatoid arthritis patients, using electron paramagnetic resonance spectrometry. Free Radic Biol Med 2005; 39: 937948.
  • 99
    Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med 1993; 329: 20022012.
  • 100
    Lauer T, Preik M, Rassaf T, Strauer BE, Deussen A, Feelisch M, Kelm M. Plasma nitrite rather than nitrate reflects regional endothelial nitric oxide synthase activity but lacks intrinsic vasodilator action. Proc Natl Acad Sci U S A 2001; 98: 1281412819.
  • 101
    Zweier JL, Wang P, Samouilov A, Kuppusamy P. Enzyme-independent formation of nitric oxide in biological tissues. Nat Med 1995; 1: 804809.
  • 102
    Godber BL, Doel JJ, Sapkota GP, Blake DR, Stevens CR, Eisenthal R, Harrison R. Reduction of nitrite to nitric oxide catalyzed by xanthine oxidoreductase. J Biol Chem 2000; 275: 77577763.
  • 103
    Li H, Samouilov A, Liu X, Zweier JL. Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrite reduction. Evaluation of its role in nitric oxide generation in anoxic tissues. J Biol Chem 2001; 276: 2448224489.
  • 104
    Millar TM, Stevens CR, Benjamin N, Eisenthal R, Harrison R, Blake DR. Xanthine oxidoreductase catalyses the reduction of nitrates and nitrite to nitric oxide under hypoxic conditions. FEBS Lett 1998; 427: 225228.
  • 105
    Zhang Z, Naughton D, Winyard PG, Benjamin N, Blake DR, Symons MC. Generation of nitric oxide by a nitrite reductase activity of xanthine oxidase: a potential pathway for nitric oxide formation in the absence of nitric oxide synthase activity. Biochem Biophys Res Commun 1998; 249: 767772.
  • 106
    Cortas NK, Wakid NW. Pharmacokinetic aspects of inorganic nitrate ingestion in man. Pharmacol Toxicol 1991; 68: 192195.
  • 107
    Kortboyer JM, Colbers EPH, Vaessen HAMG, Groen K, Zeilmaker MJ, Slob W, Speijers GJA, Meulenbelt J. A Pilot-Study to Investigate Nitrate and Nitrite Kinetics in Healthy Volunteers with Normal and Artificially Increased Gastric Ph after Sodium Nitrate Ingestion Proceeds of the International Workshop on Health Aspects of Nitrate and Its Metabolites (Partcularly Nitrite), November 8–10, 1994. Bilthoven: Council of Europe Press, 1995; 269284.
  • 108
    Bryan NS, Fernandez BO, Bauer SM, Garcia-Saura MF, Milsom AB, Rassaf T, Maloney RE, Bharti A, Rodriguez J, Feelisch M. Nitrite is a signaling molecule and regulator of gene expression in mammalian tissues. Nat Chem Biol 2005; 1: 290297.
  • 109
    McKnight GM, Duncan CW, Leifert C, Golden MH. Dietary nitrate in man: friend or foe? Br J Nutr 1999; 81: 349358.
  • 110
    Wagner DA, Schultz DS, Deen WM, Young VR, Tannenbaum SR. Metabolic fate of an oral dose of N-15-labeled nitrate in humans – effect of diet supplementation with ascorbic-acid. Cancer Res 1983; 43: 19211925.
  • 111
    van Velzen AG, Sips AJ, Schothorst RC, Lambers AC, Meulenbelt J. The oral bioavailability of nitrate from nitrate-rich vegetables in humans. Toxicol Lett 2008; 181: 177181.
  • 112
    Hunault CC, van Velzen AG, Sips AJ, Schothorst RC, Meulenbelt J. Bioavailability of sodium nitrite from an aqueous solution in healthy adults. Toxicol Lett 2009; 190: 4853.
  • 113
    Bartholomew B, Hill MJ. The pharmacology of dietary nitrate and the origin of urinary nitrate. Food Chem Toxicol 1984; 22: 789795.
  • 114
    Florin TH, Neale G, Cummings JH. The effect of dietary nitrate on nitrate and nitrite excretion in man. Br J Nutr 1990; 64: 387397.
  • 115
    Tannenbaum SR, Weisman M, Fett D. The effect of nitrate intake on nitrite formation in human saliva. Food Cosmet Toxicol 1976; 14: 549552.
  • 116
    Spiegelhalder B, Eisenbrand G, Preussmann R. Influence of dietary nitrate on nitrite content of human saliva: possible relevance to in vivo formation of N-nitroso compounds. Food Cosmet Toxicol 1976; 14: 545548.
  • 117
    Duncan C, Dougall H, Johnston P, Green S, Brogan R, Leifert C, Smith L, Golden M, Benjamin N. Chemical generation of nitric oxide in the mouth from the enterosalivary circulation of dietary nitrate. Nat Med 1995; 1: 546551.
  • 118
    Doel JJ, Benjamin N, Hector MP, Rogers M, Allaker RP. Evaluation of bacterial nitrate reduction in the human oral cavity. Eur J Oral Sci 2005; 113: 1419.
  • 119
    Gladwin MT, Schechter AN, Kim-Shapiro DB, Patel RP, Hogg N, Shiva S, Cannon RO 3rd, Kelm M, Wink DA, Espey MG, Oldfield EH, Pluta RM, Freeman BA, Lancaster JR Jr, Feelisch M, Lundberg JO. The emerging biology of the nitrite anion. Nat Chem Biol 2005; 1: 308314.
  • 120
    Huang Z, Shiva S, Kim-Shapiro DB, Patel RP, Ringwood LA, Irby CE, Huang KT, Ho C, Hogg N, Schechter AN, Gladwin MT. Enzymatic function of hemoglobin as a nitrite reductase that produces NO under allosteric control. J Clin Invest 2005; 115: 20992107.
  • 121
    Gladwin MT, Kim-Shapiro DB. The functional nitrite reductase activity of the heme-globins. Blood 2008; 112: 26362647.
  • 122
    Shiva S, Huang Z, Grubina R, Sun J, Ringwood LA, MacArthur PH, Xu X, Murphy E, Darley-Usmar VM, Gladwin MT. Deoxymyoglobin is a nitrite reductase that generates nitric oxide and regulates mitochondrial respiration. Circ Res 2007; 100: 654661.
  • 123
    Tiso M, Tejero J, Basu S, Azarov I, Wang X, Simplaceanu V, Frizzell S, Jayaraman T, Geary L, Shapiro C, Ho C, Shiva S, Kim-Shapiro DB, Gladwin MT. Human neuroglobin functions as a redox-regulated nitrite reductase. J Biol Chem 2011; 286: 1827718289.
  • 124
    Petersen MG, Dewilde S, Fago A. Reactions of ferrous neuroglobin and cytoglobin with nitrite under anaerobic conditions. J Inorg Biochem 2008; 102: 17771782.
  • 125
    Webb AJ, Milsom AB, Rathod KS, Chu WL, Qureshi S, Lovell MJ, Lecomte FM, Perrett D, Raimondo C, Khoshbin E, Ahmed Z, Uppal R, Benjamin N, Hobbs AJ, Ahluwalia A. Mechanisms underlying erythrocyte and endothelial nitrite reduction to nitric oxide in hypoxia: role for xanthine oxidoreductase and endothelial nitric oxide synthase. Circ Res 2008; 103: 957964.
  • 126
    Li H, Cui H, Kundu TK, Alzawahra W, Zweier JL. Nitric oxide production from nitrite occurs primarily in tissues not in the blood: critical role of xanthine oxidase and aldehyde oxidase. J Biol Chem 2008; 283: 1785517863.
  • 127
    Badejo AM, Hodnette C, Dhaliwal JS, Casey DB, Pankey E, Murthy SN, Nossaman BD, Hyman AL, Kadowitz PJ. Mitochondrial aldehyde dehydrogenase mediates vasodilator responses of glyceryl trinitrate and sodium nitrite in the pulmonary vascular bed of the rat. Am J of Physiol – Heart and Circulatory Physiology 2010; 299: H819826.
  • 128
    Gautier C, van Faassen E, Mikula I, Martasek P, Slama-Schwok A. Endothelial nitric oxide synthase reduces nitrite anions to NO under anoxia. Biochem Biophys Res Commun 2006; 341: 816821.
  • 129
    Vanin AF, Bevers LM, Slama-Schwok A, van Faassen EE. Nitric oxide synthase reduces nitrite to NO under anoxia. Cell Mol Life Sci 2007; 64: 96103.
  • 130
    Milsom AB, Patel NS, Mazzon E, Tripatara P, Storey A, Mota-Filipe H, Sepodes B, Webb AJ, Cuzzocrea S, Hobbs AJ, Thiemermann C, Ahluwalia A. Role for endothelial nitric oxide synthase in nitrite-induced protection against renal ischemia-reperfusion injury in mice. Nitric Oxide 2010; 22: 141148.
  • 131
    Li H, Liu X, Cui H, Chen YR, Cardounel AJ, Zweier JL. Characterization of the mechanism of cytochrome P450 reductase-cytochrome P450-mediated nitric oxide and nitrosothiol generation from organic nitrates. J Biol Chem 2006; 281: 1254612554.
  • 132
    Raikhman LM, Annaev BB. [Nature of the free-radical states in microsomes]. Biofizika 1971; 16: 11351137.
  • 133
    Duthu GS, Shertzer HG. Effect of nitrite on rabbit liver mixed-function oxidase activity. Drug Metab Dispos 1979; 7: 263269.
  • 134
    Reutov VP, Sorokina EG. NO-synthase and nitrite-reductase components of nitric oxide cycle. Biochemistry (Mosc) 1998; 63: 874884.
  • 135
    Nohl H, Staniek K, Sobhian B, Bahrami S, Redl H, Kozlov AV. Mitochondria recycle nitrite back to the bioregulator nitric monoxide. Acta Biochim Pol 2000; 47: 913921.
  • 136
    Kozlov AV, Staniek K, Nohl H. Nitrite reductase activity is a novel function of mammalian mitochondria. FEBS Lett 1999; 454: 127130.
  • 137
    Aamand R, Dalsgaard T, Jensen FB, Simonsen U, Roepstorff A, Fago A. Generation of nitric oxide from nitrite by carbonic anhydrase: a possible link between metabolic activity and vasodilation. Am J Physiol Heart Circ Physiol 2009; 297: H20682074.
  • 138
    Dalsgaard T, Simonsen U, Fago A. Nitrite-dependent vasodilation is facilitated by hypoxia and is independent of known NO-generating nitrite reductase activities. Am J Physiol Heart Circ Physiol 2007; 292: H30723078.
  • 139
    Rudolph TK, Rudolph V, Edreira MM, Cole MP, Bonacci G, Schopfer FJ, Woodcock SR, Franek A, Pekarova M, Khoo NK, Hasty AH, Baldus S, Freeman BA. Nitro-fatty acids reduce atherosclerosis in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 2010; 30: 938945.
  • 140
    Chobanyan-Jürgens K, Schwarz A, Böhmer A, Beckmann B, Gutzki F-M, Michaelsen JT, Stichtenoth DO, Tsikas D. Renal carbonic anhydrases are involved in the reabsorption of endogenous nitrite. Nitric Oxide 2012; 26: 126131.
  • 141
    McKnight GM, Smith LM, Drummond RS, Duncan CW, Golden M, Benjamin N. Chemical synthesis of nitric oxide in the stomach from dietary nitrate in humans. Gut 1997; 40: 211214.
  • 142
    Lundberg JO, Weitzberg E. NO generation from nitrite and its role in vascular control. Arterioscler Thromb Vasc Biol 2005; 25: 915922.
  • 143
    Widlansky ME, Gokce N, Keaney JF Jr, Vita JA. The clinical implications of endothelial dysfunction. J Am Coll Cardiol 2003; 42: 11491160.
  • 144
    Kleinbongard P, Dejam A, Lauer T, Rassaf T, Schindler A, Picker O, Scheeren T, Godecke A, Schrader J, Schulz R, Heusch G, Schaub GA, Bryan NS, Feelisch M, Kelm M. Plasma nitrite reflects constitutive nitric oxide synthase activity in mammals. Free Radic Biol Med 2003; 35: 790796.
  • 145
    Kleinbongard P, Dejam A, Lauer T, Jax T, Kerber S, Gharini P, Balzer J, Zotz RB, Scharf RE, Willers R, Schechter AN, Feelisch M, Kelm M. Plasma nitrite concentrations reflect the degree of endothelial dysfunction in humans. Free Radic Biol Med 2006; 40: 295302.
  • 146
    Rassaf T, Heiss C, Hendgen-Cotta U, Balzer J, Matern S, Kleinbongard P, Lee A, Lauer T, Kelm M. Plasma nitrite reserve and endothelial function in the human forearm circulation. Free Radic Biol Med 2006; 41: 295301.
  • 147
    Stokes KY, Dugas TR, Tang Y, Garg H, Guidry E, Bryan NS. Dietary nitrite prevents hypercholesterolemic microvascular inflammation and reverses endothelial dysfunction. Am J Physiol Heart Circ Physiol 2009; 296: H12811288.
  • 148
    Bahra M, Kapil V, Pearl V, Ghosh S, Ahluwalia A. Inorganic nitrate ingestion improves vascular compliance but does not alter flow-mediated dilatation in healthy volunteers. Nitric Oxide 2012; 26: 197202.
  • 149
    Cheriyan J, Webb AJ, Sarov-Blat L, Elkhawad M, Wallace SM, Maki-Petaja KM, Collier DJ, Morgan J, Fang Z, Willette RN, Lepore JJ, Cockcroft JR, Sprecher DL, Wilkinson IB. Inhibition of p38 mitogen-activated protein kinase improves nitric oxide-mediated vasodilatation and reduces inflammation in hypercholesterolemia. Circulation 2011; 123: 515523.
  • 150
    Sindler AL, Fleenor BS, Calvert JW, Marshall KD, Zigler ML, Lefer DJ, Seals DR. Nitrite supplementation reverses vascular endothelial dysfunction and large elastic artery stiffness with aging. Aging Cell 2011; 10: 429437.
  • 151
    Lakatta EG, Levy D. Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises: part I: aging arteries: a ‘set up’ for vascular disease. Circulation 2003; 107: 139146.
  • 152
    Lakatta EG, Levy D. Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises: part II: the aging heart in health: links to heart disease. Circulation 2003; 107: 346354.
  • 153
    Lakatta EG. Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises: part III: cellular and molecular clues to heart and arterial aging. Circulation 2003; 107: 490497.
  • 154
    Cecelja M, Chowienczyk P. Dissociation of aortic pulse wave velocity with risk factors for cardiovascular disease other than hypertension: a systematic review. Hypertension 2009; 54: 13281336.
  • 155
    Wilkinson IB, Franklin SS, Cockcroft JR. Nitric oxide and the regulation of large artery stiffness: from physiology to pharmacology. Hypertension 2004; 44: 112116.
  • 156
    McEniery CM, Wilkinson IB. Large artery stiffness and inflammation. J Hum Hypertens 2005; 19: 507509.
  • 157
    Lalaoui MZ, El Midaoui A, de Champlain J, Moreau P. Is there a role for reactive oxygen species in arterial medial elastocalcinosis? Vascul Pharmacol 2007; 46: 201206.
  • 158
    Vlachopoulos C, Aznaouridis K, Stefanadis C. Prediction of cardiovascular events and all-cause mortality with arterial stiffness: a systematic review and meta-analysis. J Am Coll Cardiol 2010; 55: 13181327.
  • 159
    Ahanchi SS, Tsihlis ND, Kibbe MR. The role of nitric oxide in the pathophysiology of intimal hyperplasia. J Vasc Surg 2007; 45: A64A73.
  • 160
    Alef MJ, Vallabhaneni R, Carchman E, Morris SM, Shiva S, Wang Y, Kelley EE, Tarpey MM, Gladwin MT, Tzeng E, Zuckerbraun BS. Nitrite-generated NO circumvents dysregulated arginine/NOS signaling to protect against intimal hyperplasia in Sprague-Dawley rats. J Clin Invest 2011; 121: 16461656.
  • 161
    Duranski MR, Greer JJ, Dejam A, Jaganmohan S, Hogg N, Langston W, Patel RP, Yet SF, Wang X, Kevil CG, Gladwin MT, Lefer DJ. Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver. J Clin Invest 2005; 115: 12321240.
  • 162
    Lu P, Liu F, Yao Z, Wang CY, Chen DD, Tian Y, Zhang JH, Wu YH. Nitrite-derived nitric oxide by xanthine oxidoreductase protects the liver against ischemia-reperfusion injury. Hepatobiliary Pancreat Dis Int 2005; 4: 350355.
  • 163
    Jung KH, Chu K, Ko SY, Lee ST, Sinn DI, Park DK, Kim JM, Song EC, Kim M, Roh JK. Early intravenous infusion of sodium nitrite protects brain against in vivo ischemia-reperfusion injury. Stroke 2006; 37: 27442750.
  • 164
    Bryan NS, Calvert JW, Elrod JW, Gundewar S, Ji SY, Lefer DJ. Dietary nitrite supplementation protects against myocardial ischemia-reperfusion injury. Proc Natl Acad Sci U S A 2007; 104: 1914419149.
  • 165
    Tripatara P, Patel NS, Webb A, Rathod K, Lecomte FM, Mazzon E, Cuzzocrea S, Yaqoob MM, Ahluwalia A, Thiemermann C. Nitrite-derived nitric oxide protects the rat kidney against ischemia/reperfusion injury in vivo: role for xanthine oxidoreductase. J Am Soc Nephrol 2007; 18: 570580.
  • 166
    Dezfulian C, Raat N, Shiva S, Gladwin MT. Role of the anion nitrite in ischemia-reperfusion cytoprotection and therapeutics. Cardiovasc Res 2007; 75: 327338.
  • 167
    Webb A, Ahluwalia A. Mechanisms of nitrite reduction in ischemia in the cardiovascular system: therapeutic potential. In: Nitric Oxide (2nd Edition) Biology and Pathobiology, ed. Ignarro L . London: Academic Press, 2010; 555586.
  • 168
    Presley TD, Morgan AR, Bechtold E, Clodfelter W, Dove RW, Jennings JM, Kraft RA, Bruce King S, Laurienti PJ, Jack Rejeski W, Burdette JH, Kim-Shapiro DB, Miller GD. Acute effect of a high nitrate diet on brain perfusion in older adults. Nitric Oxide 2011; 24: 3442.
  • 169
    Lauer T, Heiss C, Balzer J, Kehmeier E, Mangold S, Leyendecker T, Rottler J, Meyer C, Merx M, Kelm M, Rassaf T. Age-dependent endothelial dysfunction is associated with failure to increase plasma nitrite in response to exercise. Basic Res Cardiol 2008; 103: 291297.
  • 170
    Kenjale AA, Ham KL, Stabler T, Robbins JL, Johnson JL, Vanbruggen M, Privette G, Yim E, Kraus WE, Allen JD. Dietary nitrate supplementation enhances exercise performance in peripheral arterial disease. J Appl Physiol 2011; 110: 15821591.
  • 171
    Barstow TJ, Lamarra N, Whipp BJ. Modulation of muscle and pulmonary O2 uptakes by circulatory dynamics during exercise. J Appl Physiol 1990; 68: 979989.
  • 172
    Grassi B, Poole DC, Richardson RS, Knight DR, Erickson BK, Wagner PD. Muscle O2 uptake kinetics in humans: implications for metabolic control. J Appl Physiol 1996; 80: 988998.
  • 173
    Jones AM, Wilkerson DP, Koppo K, Wilmshurst S, Campbell IT. Inhibition of nitric oxide synthase by L-NAME speeds phase II pulmonary.VO2 kinetics in the transition to moderate-intensity exercise in man. J Physiol 2003; 552: 265272.
  • 174
    Larsen FJ, Weitzberg E, Lundberg JO, Ekblom B. Effects of dietary nitrate on oxygen cost during exercise. Acta Physiol 2007; 191: 5966.
  • 175
    Larsen FJ, Weitzberg E, Lundberg JO, Ekblom B. Dietary nitrate reduces maximal oxygen consumption while maintaining work performance in maximal exercise. Free Radic Biol Med 2010; 48: 342347.
  • 176
    Bailey SJ, Winyard P, Vanhatalo A, Blackwell JR, Dimenna FJ, Wilkerson DP, Tarr J, Benjamin N, Jones AM. Dietary nitrate supplementation reduces the O2 cost of low-intensity exercise and enhances tolerance to high-intensity exercise in humans. J Appl Physiol 2009; 107: 11441155.
  • 177
    Bailey SJ, Fulford J, Vanhatalo A, Winyard PG, Blackwell JR, DiMenna FJ, Wilkerson DP, Benjamin N, Jones AM. Dietary nitrate supplementation enhances muscle contractile efficiency during knee-extensor exercise in humans. J Appl Physiol 2010; 109: 135148.
  • 178
    Lansley KE, Winyard PG, Fulford J, Vanhatalo A, Bailey SJ, Blackwell JR, DiMenna FJ, Gilchrist M, Benjamin N, Jones AM. Dietary nitrate supplementation reduces the O2 cost of walking and running: a placebo-controlled study. J Appl Physiol 2011; 110: 591600.
  • 179
    Lansley KE, Winyard PG, Bailey SJ, Vanhatalo A, Wilkerson DP, Blackwell JR, Gilchrist M, Benjamin N, Jones AM. Acute dietary nitrate supplementation improves cycling time trial performance. Med Sci Sports Exerc 2011; 43: 11251131.
  • 180
    Vanhatalo A, Fulford J, Bailey SJ, Blackwell JR, Winyard PG, Jones AM. Dietary nitrate reduces muscle metabolic perturbation and improves exercise tolerance in hypoxia. J Physiol 2011; 589: 55175528.
  • 181
    Cermak NM, Gibala MJ, van Loon LJ. Nitrate supplementation's improvement of 10-km time-trial performance in trained cyclists. Int J Sport Nutr Exerc Metab 2012; 22: 6471.
  • 182
    Larsen FJ, Schiffer TA, Borniquel S, Sahlin K, Ekblom B, Lundberg JO, Weitzberg E. Dietary inorganic nitrate improves mitochondrial efficiency in humans. Cell Metab 2011; 13: 149159.
  • 183
    Zhu S-G, Kukreja RC, Das A, Chen Q, Lesnefsky EJ, Xi L. Dietary Nitrate Supplementation Protects Against Doxorubicin-Induced Cardiomyopathy by Improving Mitochondrial Function. J Am Coll Cardiol 2011; 57: 21812189.
  • 184
    Gladwin MT, Hunter CJ, Dejam A, Blood AB, Shields H, Kim-Shapiro D, Machado RF, Tarekegn S, Mulla N, Hopper AO, Schechter AN, Power GG. Inhaled nebulized nitrite is a hypoxia-sensitive NO-dependent selective pulmonary vasodilator. Nat Med 2004; 10: 11221127.
  • 185
    Ingram TE, Pinder AG, Bailey DM, Fraser AG, James PE. Low-dose sodium nitrite vasodilates hypoxic human pulmonary vasculature by a means that is not dependent on a simultaneous elevation in plasma nitrite. Am J Physiol Heart Circ Physiol 2010; 298: H331339.
  • 186
    Dias-Junior CA, Gladwin MT, Tanus-Santos JE. Low-dose intravenous nitrite improves hemodynamics in a canine model of acute pulmonary thromboembolism. Free Radic Biol Med 2006; 41: 17641770.
  • 187
    Baliga RS, Milsom AB, Ghosh SM, Trinder SL, Macallister RJ, Ahluwalia A, Hobbs AJ. Dietary nitrate ameliorates pulmonary hypertension: cytoprotective role for endothelial nitric oxide synthase and xanthine oxidoreductase. Circulation 2012; 125: 29222932.
  • 188
    Gago B, Lundberg JO, Barbosa RM, Laranjinha J. Red wine-dependent reduction of nitrite to nitric oxide in the stomach. Free Radic Biol Med 2007; 43: 12331242.
  • 189
    Rocha BS, Gago B, Barbosa RM, Laranjinha J. Dietary polyphenols generate nitric oxide from nitrite in the stomach and induce smooth muscle relaxation. Toxicology 2009; 265: 4148.
  • 190
    Zand J, Lanza F, Garg HK, Bryan NS. All-natural nitrite and nitrate containing dietary supplement promotes nitric oxide production and reduces triglycerides in humans. Nutr Res 2011; 31: 262269.
  • 191
    Gago B, Nyström T, Cavaleiro C, Rocha BS, Barbosa RM, Laranjinha J, Lundberg JO. The potent vasodilator ethyl nitrite is formed upon reaction of nitrite and ethanol under gastric conditions. Free Radic Biol Med 2008; 45: 404412.
  • 192
    Moya MP, Gow AJ, Califf RM, Goldberg RN, Stamler JS. Inhaled ethyl nitrite gas for persistent pulmonary hypertension of the newborn. Lancet 2002; 360: 141143.
  • 193
    O'Donnell VB, Eiserich JP, Chumley PH, Jablonsky MJ, Krishna NR, Kirk M, Barnes S, Darley-Usmar VM, Freeman BA. Nitration of unsaturated fatty acids by nitric oxide-derived reactive nitrogen species peroxynitrite, nitrous acid, nitrogen dioxide, and nitronium ion. Chem Res Toxicol 1998; 12: 8392.
  • 194
    Cui T, Schopfer FJ, Zhang J, Chen K, Ichikawa T, Baker PR, Batthyany C, Chacko BK, Feng X, Patel RP, Agarwal A, Freeman BA, Chen YE. Nitrated fatty acids: endogenous anti-inflammatory signaling mediators. J Biol Chem 2006; 281: 3568635698.
  • 195
    Li Y, Zhang J, Schopfer FJ, Martynowski D, Garcia-Barrio MT, Kovach A, Suino-Powell K, Baker PRS, Freeman BA, Chen YE, Xu HE. Molecular recognition of nitrated fatty acids by PPAR[gamma]. Nat Struct Mol Biol 2008; 15: 865867.
  • 196
    Borniquel S, Jansson EÅ, Cole MP, Freeman BA, Lundberg JO. Nitrated oleic acid up-regulates PPARγ and attenuates experimental inflammatory bowel disease. Free Radic Biol Med 2010; 48: 499505.
  • 197
    Kelley EE, Batthyany CI, Hundley NJ, Woodcock SR, Bonacci G, Del Rio JM, Schopfer FJ, Lancaster JR Jr, Freeman BA, Tarpey MM. Nitro-oleic acid, a novel and irreversible inhibitor of xanthine oxidoreductase. J Biol Chem 2008; 283: 3617636184.
  • 198
    Liu H, Jia Z, Soodvilai S, Guan G, Wang MH, Dong Z, Symons JD, Yang T. Nitro-oleic acid protects the mouse kidney from ischemia and reperfusion injury. Am J Physiol Renal Physiol 2008; 295: F942949.
  • 199
    Nitrites, nitrosamines, and cancer. Lancet 1968; 1: 10711072.
  • 200
    Magee PN, Barnes JM. The production of malignant primary hepatic tumours in the rat by feeding dimethylnitrosamine. Br J Cancer 1956; 10: 114122.
  • 201
    Sakshaug J, Sognen E, Hansen MA, Koppang N. Dimethylnitrosamine; its hepatotoxic effect in sheep and its occurrence in toxic batches of herring meal. Nature 1965; 206: 12611262.
  • 202
    Sander J. [Can nitrites in the human diet be the cause of cancerogenesis through formation of nitrosamines?]. Arch Hyg Bakteriol 1967; 151: 2228.
  • 203
    Druckrey H, Schildbach A, Schmaehl D, Preussmann R, Ivankovic S. [Quantitative Analysis of the Carcinogenic Effect of Diethylnitrosamine]. Arzneimittelforschung 1963; 13: 841851.
  • 204
    Bartsch H, Ohshima H, Pignatelli B. Inhibitors of endogenous nitrosation. Mechanisms and implications in human cancer prevention. Mutat Res 1988; 202: 307324.
  • 205
    World Health Organisation. Fifty-ninth Report of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Geneva: Evaluation of Certain Food Additives, 2002.
  • 206
    Mensinga TT, Speijers GJ, Meulenbelt J. Health implications of exposure to environmental nitrogenous compounds. Toxicol Rev 2003; 22: 4151.
  • 207
    Alexander J, Benford D, Cockburn A, Cravedi JP, Dogliotti E, Domenico AD, Fernández-Cruz ML, Fink-Gremmels J, Fürst P, Galli C, Grandjean P, Gzyl J, Heinemeyer G, Johansson N, Mutti A, Schlatter J, van Leeuwen R, van Peteghem C, Verger P. Opinion of the Scientific Panel on Contaminants in the Food chain on a request from the European Commission to perform a scientific risk assessment on nitrate in vegetables. The European Food Safety Authority Journal 2008; 689: 179.
  • 208
    Winter JW, Paterson S, Scobie G, Wirz A, Preston T, McColl KE. N-nitrosamine generation from ingested nitrate via nitric oxide in subjects with and without gastroesophageal reflux. Gastroenterology 2007; 133: 164174.
  • 209
    World Cancer Research Fund / American Institute for Cancer Research. Food, Nutrition, Physical Activity, and the Prevention of Cancer: A Global Perspective. Washington DC: AICR, 2007.
  • 210
    Steinmetz KA, Potter JD. Vegetables, fruit, and cancer prevention: a review. J Am Diet Assoc 1996; 96: 10271039.
  • 211
    Mirvish SS, Wallcave L, Eagen M, Shubik P. Ascorbate-nitrite reaction: possible means of blocking the formation of carcinogenic N-nitroso compounds. Science 1972; 177: 6568.
  • 212
    Combet E, Paterson S, Iijima K, Winter J, Mullen W, Crozier A, Preston T, McColl KEL. Fat transforms ascorbic acid from inhibiting to promoting acid-catalysed N-nitrosation. Gut 2007; 56: 16781684.
  • 213
    Kuroiwa Y, Yamada M, Matsui K, Okamura T, Ishii Y, Masumura K, Tasaki M, Umemura T, Mitsumori K, Nohmi T, Hirose M, Nishikawa A. Combined ascorbic acid and sodium nitrite treatment induces oxidative DNA damage-associated mutagenicity in vitro, but lacks initiation activity in rat forestomach epithelium. Toxicol Sci 2008; 104: 274282.
  • 214
    Hirose M, Tanaka H, Takahashi S, Futakuchi M, Fukushima S, Ito N. Effects of sodium nitrite and catechol, 3-methoxycatechol, or butylated hydroxyanisole in combination in a rat multiorgan carcinogenesis model. Cancer Res 1993; 53: 3237.
  • 215
    Kawabe M, Takaba K, Yoshida Y, Hirose M. Effects of combined treatment with phenolic compounds and sodium nitrite on two-stage carcinogenesis and cell proliferation in the rat stomach. Jpn J Cancer Res 1994; 85: 1725.
  • 216
    Yoshida Y, Hirose M, Takaba K, Kimura J, Ito N. Induction and promotion of forestomach tumors by sodium nitrite in combination with ascorbic acid or sodium ascorbate in rats with or without N-methyl-N'-nitro-N-nitrosoguanidine pre-treatment. Int J Cancer 1994; 56: 124128.
  • 217
    Miyauchi M, Nakamura H, Furukawa F, Son HY, Nishikawa A, Hirose M. Promoting effects of combined antioxidant and sodium nitrite treatment on forestomach carcinogenesis in rats after initiation with N-methyl-N'-nitro-N-nitrosoguanidine. Cancer Lett 2002; 178: 1924.
  • 218
    Combet E, El Mesmari A, Preston T, Crozier A, McColl KE. Dietary phenolic acids and ascorbic acid: influence on acid-catalyzed nitrosative chemistry in the presence and absence of lipids. Free Radic Biol Med 2010; 48: 763771.
  • 219
    Kuroiwa Y, Ishii Y, Umemura T, Kanki K, Mitsumori K, Nishikawa A, Nakazawa H, Hirose M. Combined treatment with green tea catechins and sodium nitrite selectively promotes rat forestomach carcinogenesis after initiation with N-methyl-N'- nitro-N-nitrosoguanidine. Cancer Sci 2007; 98: 949957.
  • 220
    Aschebrook-Kilfoy B, Ward MH, Gierach GL, Schatzkin A, Hollenbeck AR, Sinha R, Cross AJ. Epithelial ovarian cancer and exposure to dietary nitrate and nitrite in the NIH-AARP Diet and Health Study. Eur J Cancer Prev 2012; 21: 6572. 10.1097/CEJ.0b013e328347622f.
  • 221
    Ward MH, Kilfoy BA, Weyer PJ, Anderson KE, Folsom AR, Cerhan JR. Nitrate intake and the risk of thyroid cancer and thyroid disease. Epidemiology 2010; 21: 389395.
  • 222
    Aschebrook-Kilfoy B, Cross AJ, Stolzenberg-Solomon RZ, Schatzkin A, Hollenbeck AR, Sinha R, Ward MH. Pancreatic cancer and exposure to dietary nitrate and nitrite in the NIH-AARP Diet and Health Study. Am J Epidemiol 2011; 174: 305315.
  • 223
    Ward MH, Heineman EF, Markin RS, Weisenburger DD. Adenocarcinoma of the stomach and esophagus and drinking water and dietary sources of nitrate and nitrite. Int J Occup Environ Health 2008; 14: 193197.
  • 224
    Ward MH, Cerhan JR, Colt JS, Hartge P. Risk of non-Hodgkin lymphoma and nitrate and nitrite from drinking water and diet. Epidemiology 2006; 17: 375382.
  • 225
    Ward M, Rusiecki J, Lynch C, Cantor K. Nitrate in public water supplies and the risk of renal cell carcinoma. Cancer Causes Control 2007; 18: 11411151.
  • 226
    Kapadia GJ, Azuine MA, Rao GS, Arai T, Iida A, Tokuda H. Cytotoxic effect of the red beetroot (Beta vulgaris L.) extract compared to doxorubicin (Adriamycin) in the human prostate (PC-3) and breast (MCF-7) cancer cell lines. Anticancer Agents Med Chem 2011; 11: 280284.
  • 227
    Kapadia GJ, Tokuda H, Konoshima T, Nishino H. Chemoprevention of lung and skin cancer by Beta vulgaris (beet) root extract. Cancer Lett 1996; 100: 211214.
  • 228
    Kapadia GJ, Azuine MA, Sridhar R, Okuda Y, Tsuruta A, Ichiishi E, Mukainake T, Takasaki M, Konoshima T, Nishino H, Tokuda H. Chemoprevention of DMBA-induced UV-B promoted, NOR-1-induced TPA promoted skin carcinogenesis, and DEN-induced phenobarbital promoted liver tumors in mice by extract of beetroot. Pharmacol Res 2003; 47: 141148.
  • 229
    Lechner JF, Wang LS, Rocha CM, Larue B, Henry C, McIntyre CM, Riedl KM, Schwartz SJ, Stoner GD. Drinking water with red beetroot food color antagonizes esophageal carcinogenesis in N-nitrosomethylbenzylamine-treated rats. J Med Food 2010; 13: 733739.
  • 230
    Georgiev V, Weber J, Kneschke E-M, Denev P, Bley T, Pavlov A. Antioxidant activity and phenolic content of betalain extracts from intact plants and hairy root cultures of the red beetroot Beta vulgaris cv. Detroit dark red. Plant Foods Hum Nutr 2010; 65: 105111.
  • 231
    Morant R, Jungi WF, Koehli C, Senn HJ. [Why do cancer patients use alternative medicine?]. Schweiz Med Wochenschr 1991; 121: 10291034.
  • 232
    Obrist R, von Meiss M, Obrecht JP. [The use of paramedical treatment methods by cancer patients. A inquiry on 101 ambulatory patients]. Dtsch Med Wochenschr 1986; 111: 283287.