1. Dietary NO₃⁻ and Endothelial Dysfunction Therapy

Until a decade ago, NO₃⁻ was considered an unfavorable dietary-derived toxic compound, as it was wrongly associated with the development of some malignancies, such as gastric cancer. Strict standards regarding the levels of this anion were regulated in food ^[1]. The World Health Organization (WHO) defined an acceptable daily intake (ADI) of 3.7 mg of NO₃⁻·kg⁻¹ body weight, the same content adopted by the European Food Safety Authority. For a normal adult weighing 70 kg, this content is equivalent to ~260 mg of NO₃⁻·day⁻¹. However, vegetarian diets commonly contain >300 mg of NO₃⁻·day⁻¹ for 70 kg adults, higher than the ADI ^[2].

Recently, researchers have become interested in the biological NO₃⁻ role. Findings regarding the improvement of cardiovascular function have raised a biologically plausible and widely recognized hypothesis that the NO₃⁻ present in vegetables may serve as a physiological substrate for NO generation which, in turn, promotes vasodilation and, consequently, improves cardiovascular function ^[3][4].

NO₃⁻ is a nitric acid salt, while NO₂⁻ is a nitrous acid salt compound, formed by a single nitrogen bonded to three or two oxygen atoms, respectively. Both compounds can be obtained from endogenous and/or exogenous sources. The endogenous formation of NO₃⁻ and NO₂⁻ occurs through the NO metabolism via the L-arginine/NO pathway, as mentioned previously. On the other hand, the main potential exogenous source for the acquisition of NO₃⁻ and NO₂⁻ is through the dietary route. Through this pathway, NO is then generated by a non-enzymatic pathway from NO₂⁻. Dietary NO₃⁻ is reduced to NO₂⁻ in the oral cavity by bacteria that produce the NO₃⁻-reductase enzyme ^[5]. The metabolic activities of commensal bacteria species, such as Granulicatella, Actinomyces, Veillonella, Prevotella, Neisseria, Haemophilus, and Rothia genera that inhabit the oral cavity have a significant influence on NO₃⁻ to NO pathway. Previous studies have shown that individuals with a higher abundance of NO₃⁻-reducing bacteria are able to generate more salivary NO₂⁻ and, consequently, NO, at a faster rate following dietary NO₃⁻ ingestion ^[6]. However, enzymatic activity in the mouth and, consequently, the conversion of NO₃⁻ to NO₂⁻ may be disrupted by antibiotic use or mouthwash rinsing, since both substances inactivate bacteria cells ^[7]. Subsequently, NO₂⁻ reaches the stomach and, in this acidic environment, is protonated, forming nitrous acid (HNO₂), which decomposes non-enzymatically to NO and other bioactive nitrogen oxides such as nitrogen dioxide (NO₂), dinitrogen trioxide (N₂O₃) and the nitrosonium ion (NO⁺) ^[5][4]. The remaining NO₃⁻ and NO₂⁻ in the jejunum are rapidly absorbed into the bloodstream or tissues, where their accumulation occurs in tandem with molecules endogenously synthesized by the L-arginine/NO pathway. Most NO₃⁻ is excreted in urine, whereas a small portion is extracted by the salivary glands, concentrating this compound in the saliva, continuing the entero-salivary cycle ^[3][5]. A small part of plasma NO₃⁻ and NO₂⁻ concentrations may suffer the action of xanthine oxidoreductase (XOR), which displays similar activity to NO₃⁻-reductase. NO₂⁻ can also be reduced to bioactive NO by deoxyhemoglobin (deoxyHb) and deoxymyoglobin (deoxyMb), especially when O₂ levels are low. Other enzymes and compounds exhibiting redox potential, such as aldehyde oxidase (AO), aldehyde dehydrogenase (ALDH), carbonic anhydrase (CA), vitamin C (Vit C.) and polyphenols, display the ability to synthesize NO from NO₂⁻ reduction ^[3].

Several studies report beneficial effects of dietary NO₃⁻ sources as a new physiological, therapeutic and nutritional approach to attain the intended cardioprotective effects by NO production stimulation ^[3][8][9]. However, dosage, supplementation regimen and individual health status must be considered to obtain the maximum cardioprotective effect following NO₃⁻ intake. Furthermore, environmental factors such as temperature, exposure to sunlight, atmospheric humidity, water content and irradiation, as well as agricultural factors like plant genotype, fertilization, herbicide use, amount of available nitrogen, type of crop, soil conditions, nutrient availability and transport and, finally, storage conditions also influence NO₃⁻ contents in plants, and, consequently dietary NO₃⁻ supplementation ^[10].

2. Dietary NO₃⁻ Vegetable Sources

Vegetables are the main source of dietary NO₃⁻, corresponding to 85% of the daily intake, although NO₃⁻ content can vary widely within members of distinct botanical families ^[11]. The NO₃⁻ contents in plant organs can be classified from highest to lowest, as petiole ˃ leaf ˃ stem ˃ root ˃ tuber ˃ bulb ˃ fruit ˃ seed ^[12]. Table 1 presents a list of vegetables commonly included in Western diets considered NO₃⁻ sources, classified according to NO₃⁻ contents, from the highest to the lowest.

The NO₃⁻-rich vegetables within the Amaranthaceae family comprise beetroot (1300 mg·kg⁻¹), beet greens (1852 mg·kg⁻¹), Swiss chard (1690 mg·kg⁻¹), and green spinach (≈2500 mg·kg⁻¹), while a Lamiaceae family representative consists of basil (2292 mg·kg⁻¹). Concerning the Brassicaceae family, the most representative members are bok choy (1933 mg·kg⁻¹), black radish (1271 mg·kg⁻¹), turnip (1018 mg·kg⁻¹), mustard greens (1160 mg·kg⁻¹), rocket or arugula (4677 mg·kg⁻¹), kohlrabi (1769 mg·kg⁻¹), and radish (≈2000 mg·kg⁻¹). Apiaceae family members include coriander (2445 mg·kg⁻¹), celery (1100 mg·kg⁻¹) and parsley (2134 mg·kg⁻¹), whereas Asteraceae family members include lettuce (≈1800 mg·kg⁻¹), leaf chicory (1452 mg·kg⁻¹), and butter leaf lettuce (2000 mg·kg⁻¹). All these vegetables are included in the high NO₃⁻-containing vegetable category of > 1000 mg·kg⁻¹. Vegetables such as cabbage (513 mg·kg⁻¹), curly kale (987 mg·kg⁻¹), broccoli (≈300 mg·kg⁻¹), broccoli raab (905 mg·kg⁻¹), cauliflower (202 mg·kg⁻¹) and Savoy cabbage (324 mg·kg⁻¹), which belong to the Brassicaceae family; carrot (≈300 mg·kg⁻¹) and fennel (363 mg·kg⁻¹), both members of the Apiaceae family; artichokes (174 mg·kg⁻¹), asparagus chicory (355 mg·kg⁻¹), and endive (663 mg·kg⁻¹), belonging to the Asteraceae family, garlic (183 mg·kg⁻¹) and green onion (≈450 mg·kg⁻¹) from the Liliaceae family; aubergine (314 mg·kg⁻¹), capsicum (108 mg·kg⁻¹) and potato (220 mg·kg⁻¹), belonging Solanaceae family; courgette (416 mg·kg⁻¹) and cucumber (240 mg·kg⁻¹), pumpkin (894 mg·kg⁻¹), from the Cucurbitaceae family member all contain intermediate NO₃⁻ concentrations ranging from 100 to 1000 mg·kg^{−1 [12][13][14][15]}.

Among the vegetables considered the richest dietary NO₃⁻ sources, as listed in Table 1, beetroot, rocket and spinach have been the most tested concerning dietary interventions, and all resulted in effective improvements in cardiovascular performance estimated through blood pressure reduction and vascular function amelioration (Figure 1).

Figure 1. The richest sources of dietary NO₃⁻ tested in clinical interventions are beetroot, rocket and spinach. Beetroot formulation choice to supplement dietary NO₃⁻ relies on the design of beetroot-derived formulations containing pharmacological NO₃⁻ doses in a small serving portion.

NO₃⁻ vascular-effects depend on digestibility and bioavailability (bioacessibility), and better performances are obtained when NO₃⁻ intake originates from food matrices compared to NaNO₃⁻ salt administration ^[16]. The beneficial effects of different NO₃⁻-rich vegetables and NO₃⁻ doses in NO stimulation production and biochemical, hemodynamic, and vascular parameters in healthy or cardiovascular-compromised patients are summarized in Table 2. It is important to note that, to the best of our knowledge, NO₃⁻ supplementation from green leaves has only been performed in healthy individuals, and it is unknown whether their effects can be extended to individuals presenting cardiovascular risk factors. In addition, although the cardiovascular protective effects of NO₃⁻-enriched vegetables have been clearly demonstrated in clinical trials with healthy individuals, the large volume of juice vegetables used to achieve effective dietary NO₃⁻ concentrations can be a limiting factor in ensuring adherence to long-term nutritional interventions. However, this NO₃⁻ limitation does not impact supplementation by beetroot juice. Beet juice and other beetroot formulations can be ingested in comfortable serving portions to achieve threshold NO₃⁻ concentrations in order to promote beneficial cardiovascular function effects.

A large volume of spinach comprising a serving portion of 250 g leaves containing 220 mg of NO₃⁻ were administrated to twenty-six healthy individuals, resulting in an increase in NO synthesis evidenced by an eight-fold increase in salivary NO₂⁻ and a seven-fold increase in salivary NO₃⁻ at 120 min post-meal. Large artery elasticity indices were increased alongside lower pulse pressure and reduced systolic blood pressure (SBP) ^[25].

An amount of 800 mg NO₃⁻ intake was supplied through four different vegetable drinks, namely beetroot juice (116 g), rocket salad (196 g), spinach (365 g) or NaNO₃⁻ (1.1 g) prepared in water, which triggered an increase in NO₃⁻ and NO₂⁻ plasma concentrations. SBP declined after 150 min of beetroot juice ingestion (from 118 ± 2 to 113 ± 2 mm Hg) and a rocket salad beverage (from 122 ± 3 to 116 ± 2 mm Hg), which was sustained for at least 300 min after ingestion of the spinach beverage (from 118 ± 2 to 111 ± 3 mm Hg). Diastolic blood pressure (DBP) also declined after 150 min ingestion of all beverages and was sustained at lower levels for 300 min after rocket salad or spinach ingestion ^[27].

All NO₃⁻ rich-vegetable drinks were more efficient than NaNO₃⁻ in reducing both SBP and DBP, and beetroot was the most effective considering the food weight/NO₃⁻ content ratio ^[27]. However, to the best of our knowledge, the lowest effective volume of beetroot able to promote beneficial vascular effects was 70 mL of beetroot juice containing 6.45 mmol NO₃⁻ (403 mg), which was administered to 24 older and overweight volunteers for three weeks. This supplementation regimen and the offered dose promoted 2.3-fold and 7.3-fold increases in urinary and salivary NO₃⁻, respectively, and resulted in a 7.3 mm Hg decrease in SBP ^[29].

Beetroot consumption is noteworthy as a convenient and attractive alternative to obtain cardioprotective NO₃⁻ effects in both healthy individuals and those presenting risk factors for CVD diseases, due to the distinct but smart formulations (traditional or novel) that can be prepared to fulfill effective pharmacological dietary NO₃⁻ concentrations. An attractive and compact NO₃⁻-enriched-beetroot gel has been formulated in an attempt to provide an enriched NO₃⁻ food product able to promote the claimed cardioprotective effects while still being easy to administer and facilitate adherence to nutritional therapy ^[23]. Acute supplementation with 100 g of beetroot gel containing 390 mg of NO₃⁻ promoted a decrease in SBP (−6.2 mm Hg), DBP (−5.2 mm Hg), and heart rate (−7 bpm) in a pilot study conducted with healthy individuals. However, NO₃⁻ supplementation had to be adjusted to treat hypertensive individuals, since similar doses in compromised vascular individuals do not alter hemodynamic parameters. The non-susceptibility of 27 treated hypertensive patients was clearly demonstrated by the intake of 7.0 mmol (434 mg) of NO₃⁻ in 140 mL of beetroot juice for 7 days, resulting in increased NO synthesis, assessed by plasmatic, urinary and salivary NO₃⁻ and NO₂⁻, but with no differences in home and 24 h ambulatory, SBP and DBP ^[26]. These results indicate that, in order to ameliorate primary hemodynamic parameters, high doses of dietary NO₃⁻ combined with a long-term intervention can be applied to treat individuals presenting impaired endothelial function. Furthermore, in an unprecedented clinical trial, patients displaying at least three risk factors for the development of CVD, including hypertension, were chronically supplemented for three weeks with an enriched NO₃⁻ beetroot-cereal bar providing 589 mg of NO₃⁻ in 60 g of the intervention product, resulting in 14.0 mm Hg and 6.5 mm Hg decreases in SBP and DBP, respectively, in response to ~15-fold or ~7-fold increased plasma NO₃⁻ and NO₂⁻ concentrations, respectively. Endothelial function in the treated volunteers was improved and arterial stiffness was reduced by 14% ^[24][30].

3. Plasma NO₃⁻/NO₂⁻ Increments on Cardiovascular Health and Impaired Cardiovascular Functions

It is known that plasma NO₃⁻ and NO₂⁻ concentrations are dependent on ingested NO₃^{− [31]}, but the minimum increase in NO₃⁻/NO₂⁻ plasma levels necessary to promote hemodynamic responses may differ between healthy individuals and those with compromised cardiovascular function. In a clinical trial where healthy men received dietary supplementation, 3.5-fold and 1.6-fold increases of plasma NO₃⁻ and NO₂⁻, respectively, resulted in significant DBP reductions and increases in endothelium-independent vasodilatation. This small but effective plasma increase was generated after the acute intake of beetroot bread (NO₃⁻ 1.1 mmol) ^[18]. On the other hand, Haun et al. ^[28] reported plasma NO_x (~3-fold) and NO₂⁻ (less than 1.5-fold) increases, albeit without any changes in hemodynamic parameters such as heart rate, DBP, SBP, FMD, radial artery pulse waves (PWA), central mean arterial pressure (CMAP) and central pulse pressure (CPP), after the acute intake of red spinach extract (NO₃⁻ 1.45 mmol) by 15 healthy subjects. Although the dose used by Haun et al. ^[28] was slightly higher than by Hobbs et al. ^[18] trial, plasma NO₃⁻ (>3.5-fold) and NO₂⁻ (>1.6-fold) increases should be a determinant factor in choosing the dose required to benefit healthy populations.

In individuals with impaired cardiovascular function, the administered NO₃⁻ dose should be able to meet two requirements: (i) promote an increase in systemic NO₃⁻ and NO₂⁻ higher than observed in healthy individuals; (ii) be administered in a chronic and uninterrupted manner.

Hypertensive pregnant women exhibited ~10- and ~1.5-fold increases in plasma NO₃⁻ and NO₂⁻, respectively, after 7 days of daily supplementation with NO₃⁻ (6.45 mmol in beetroot juice). No significant differences were observed in plasma NO₃⁻ and NO₂⁻ levels measured 24 h after the initial dose, and even in the following 7-days of daily supplementation ^[32]. Similarly, a 1-week intake of beetroot juice (NO₃⁻ ~6.45 mmol) in 27 treated hypertensive individuals resulted in a three-fold increase in plasma NO₃⁻ and NO₂⁻, with no differences in home and 24-h ambulatory blood pressures ^[33]. Finally, 24 overweight older subjects supplemented for 3 weeks with concentrated beet juice (~4.8–6.45 mmol) exhibited urinary NO₃⁻ values ~3-fold higher greater than the baseline and beneficial SBP effects after juice intake. However, both urinary NO₃⁻ and SBP returned to baseline levels 24 h after ingestion and in the first week following the end of supplementation ^[19]. These findings demonstrate that acute treatments able to promote systemic increases in NO₃⁻ and NO₂⁻ at levels similar to those observed in healthy individuals do not benefit individuals presenting cardiovascular risks.

On the other hand, clinical trials lasting more than 3 weeks or comprising higher NO₃⁻ doses than usually applied (6–7 mmol) resulted in better hemodynamic outcomes. Hypertensive subjects treated for 4 weeks with beetroot juice (NO₃⁻ 6.4 mmol) exhibited substantial increases in NO₃⁻ and NO₂⁻ plasma levels (~5.5 and ~2.7, respectively). In addition, this intervention provided sustained BP lowering of 7.7/5.2 mm Hg 24 h after the treatment, with clinical BP reduced by 7.7/2.4 mm Hg and home BP, by 8.1/3.8 mm Hg ^[20]. In this trial, SBP and DBP reduction peaks occurred only in the last week, highlighting the importance of a prolonged intervention.

In another trial, supplementation for 3 weeks with a high dose of dietary NO₃⁻ concentrate in a 60 g beetroot cereal bar (9.5 mmol) resulted in ~15- and ~7-fold increases in plasma NO₃⁻ and NO₂⁻, respectively. This was accompanied by a considerable reduction in BP (−14.0/−6.5 mm Hg) and improvement in central hemodynamic and endothelial function parameters such as arterial stiffness, augmentation and index pressures, aortic systolic and pulse pressures and cutaneous microvascular conductance ^[24].

Based on these reports, individuals presenting physiopathological conditions that affect the cardiovascular system require a dietary therapy that associates high NO₃⁻ doses capable of promoting systemic increases in NO₃⁻ and NO₂⁻ to levels higher than found in healthy individuals and in addition, is administered continuously (Figure 2).

Figure 2. Food formulations and supplementation regimen of dietary NO₃⁻ in healthy or cardiovascular-compromised patients. For individuals presenting risk factors for the development of cardiovascular disease, the dietary NO₃⁻ dose should be higher to promote the systemic elevation of plasma NO₃⁻ and NO₂⁻ levels compared to healthy individuals, increasing NO generation by the NO₃⁻/NO₂⁻ pathway, where increased levels must be administered through chronic and uninterrupted supplementation.

In short, the aforementioned studies discussed herein suggest that frequent daily dietary NO₃⁻ doses for long periods of time would be necessary to promote beneficial effects on blood pressure and endothelial function in populations presenting compromised vascular responsiveness. A systematic review and meta-analysis study of randomized controlled trials demonstrated that supplementation of inorganic NO₃⁻ from beetroot juice over 14 days provoked decreases in SBP (−3.55 mm Hg; 95% CI: −4.55, −2.54 mm Hg) and DBP (−1.32 mm Hg; 95% CI: −1.97, −0.68 mm Hg). Furthermore, beneficial dietary NO₃⁻ effects on endothelial function were associated with dose, age, and body mass index (BMI), where chronic beetroot juice supplementation improved flow-mediated dilation (FMD) and endothelium functional effects according to the administered NO₃⁻ contents (β = 0.04, SE = 0.01, p < 0.001), age (β = −0.01, SE = 0.004, p = 0.02) and BMI (β = −0.04, SE = 0.02, p = 0.05) ^[34]. A critical review of experimental data shows that chronic dietary NO₃⁻ ingestion is a positive vascular endothelium effector promoting vasodilatation and reducing blood pressure in compromised vascular responsiveness individuals.

However, only beetroot supplementation has been tested in acute and chronic assays in individuals with impaired cardiovascular function. Although the NO₃⁻ content of green leaves is able to fulfill the effective NO₃⁻ concentration in such patients, beetroot formulations may be the best non-drug strategy, since beetroot-derived formulations can concentrate the pharmacological NO₃⁻ dosage in a small serving portion of an attractive food product, favoring continuous intake and better adherence to this nutritional intervention. This may explain the well-documented and consistent cardioprotective effects of beet products in both healthy individuals and those presenting risk factors for the development of CVD when compared with other rich-NO₃⁻ vegetables, such as green leaves, assayed in clinical trials.

Furthermore, it is important to note that, in addition to NO₃, vegetables are also a source of numerous phytochemicals able to increase eNOS activity in endothelial cells and contribute to NO synthesis ^[35][36]. Due to the great variety of polyphenols and other bioactive compounds in vegetables, it is difficult to point out individual or synergistic effects on NO generation. However, only NO₃⁻ has been directly associated to the cardioprotective effect, since it provides the physiological substrate for NO generation via the NO₃⁻-NO₂⁻/NO enterosalivary pathway ^[3]. The administration of the same food matrix, depleted in NO₃⁻, used as a placebo in the clinical trials had no effect on NO synthesis and hemodynamic parameters, proving that NO₃⁻ is probably the active principle. The remaining phytochemicals after NO₃⁻ removal, including polyphenols, which are preserved in the placebo, may promote a discrete increase in NO production but it seems they are not effective in promoting hemodynamic improvements, similar to the effect observed when NO₃⁻ at concentrations under the pharmacological threshold is administered.