BEETROOT GAINS

 

The Potential Benefits of Red Beetroot Supplementation in Health and Disease

In recent years there has been a growing interest in the biological activity of red beetroot (Beta vulgaris rubra) and its potential utility as a health promoting and disease preventing functional food. As a source of nitrate, beetroot ingestion provides a natural means of increasing in vivo nitric oxide (NO) availability and has emerged as a potential strategy to prevent and manage pathologies associated with diminished NO bioavailability, notably hypertension and endothelial function. Beetroot is also being considered as a promising therapeutic treatment in a range of clinical pathologies associated with oxidative stress and inflammation. Its constituents, most notably the betalain pigments, display potent antioxidant, anti-inflammatory and chemo-preventive activity in vitro and in vivo. The purpose of this review is to discuss beetroot’s biological activity and to evaluate evidence from studies that specifically investigated the effect of beetroot supplementation on inflammation, oxidative stress, cognition and endothelial function.

Keywords: beetroot, betalains, nitrate, antioxidants, inflammation, oxidative stress

 

1. Introduction

The well-documented health benefits of a diet high in fruit and vegetables has led to a growing interest in so-called “functional foods” and their application in health and disease. In recent years, the root vegetable Beta vulgaris rubra, otherwise known as red beetroot (herein referred to as beetroot) has attracted much attention as a health promoting functional food. While scientific interest in beetroot has only gained momentum in the past few decades, reports of its use as a natural medicine date back to Roman times [1]. Today, beetroot is grown in many countries worldwide, is regularly consumed as part of the normal diet, and commonly used in manufacturing as a food colouring agent known as E162 [2,3].

The recent interest in beetroot has been primarily driven by the discovery that sources of dietary nitrate may have important implications for managing cardiovascular health [4]. However, beetroot is rich in several other bioactive compounds that may provide health benefits, particularly for disorders characterised by chronic inflammation. Consequently, the potential role for beetroot as an adjunct treatment in several clinical conditions will be presented; Specifically, the aims of this review are twofold: (1) to highlight evidence from recent studies showing the physiological and biological actions of beetroot; and (2) to evaluate its use as a nutritional intervention in health and disease, with a special emphasis on experimental studies relating to oxidative stress, inflammation, endothelial function and cognition.

Recent studies have provided compelling evidence that beetroot ingestion offers beneficial physiological effects that may translate to improved clinical outcomes for several pathologies, such as; hypertension, atherosclerosis, type 2 diabetes and dementia [1,5,6,7,8]. Hypertension in particular has been the target of many therapeutic interventions and there are numerous studies that show beetroot, delivered acutely as a juice supplement [9,10,11] or in bread [12,13] significantly reduce systolic and diastolic blood pressure. Further discussion of beetroot’s anti-hypertensive potential is summarised in several reviews: [14,15,16].

Beetroot’s effect on the vasculature is largely attributed to its high inorganic nitrate content (250 mg∙kg−1 of fresh weight; [17]). Nitrate itself is not considered to mediate any specific physiological function; rather, nitrates beneficial effects are attributed to its in vivo reduction to nitric oxide (NO), a multifarious messenger molecule with important vascular and metabolic functions [14,18]. The generation of NO via nitrate involves a series of sequential steps that have been well described in the literature [4,19]. Briefly, ingested nitrate is first absorbed through the upper part of the small intestine into the systemic circulation [4,15]. It is then estimated that 25% of the circulating nitrate enters the entero-salivary cycle where bacterial species located at the posterior aspect of the tongue bioactivate or reduce salivary nitrate to nitrite [16,19]. Because salivary bacteria facilitate the reduction reaction that converts nitrate to nitrite, spitting out saliva or taking oral anti-bacterial treatments, like dental mouthwash for example, has been shown to diminish nitrate-nitrite conversion [10,18]. Under normal circumstances, however, salivary nitrite is re-absorbed into the circulation via the stomach where it is metabolised to NO and other nitrogen oxides by a variety of reductase enzymes [4,10,13].

However, as previously mentioned, nitrate is not the only constituent of beetroot proposed to have beneficial effects in health and disease. Beetroot is a rich source of phytochemical compounds (Figure 1), that includes ascorbic acid, carotenoids, phenolic acids and flavonoids [2,20,21]. Beetroot is also one of the few vegetables that contain a group of highly bioactive pigments known as betalains [22,23]. Members of the betalain family are categorised as either betacyanin pigments that are red-violet in colour or betaxanthin pigments that are yellow-orange in colour [1]. A number of investigations have reported betalains to have high antioxidant and anti-inflammatory capabilities in vitro and a variety of in vivo animal models [3,23,24,25,26]. This has sparked interest in a possible role for beetroot in clinical pathologies characterised by oxidative stress and chronic inflammation such as liver disease [1,23], arthritis [27] and even cancer [28,29,30,31].

 

Figure 1

Overview of potentially bioactive compounds in beetroot (based on data from [1,2,20]).

 

2. Bioavailability

For a food component to be considered beneficial for health it must be bioavailable in vivo, that is, following ingestion, the active compounds are absorbed through the gastro-intestinal tract and made available in the circulation, in sufficient quantities, to be utilized by cells [21,32]. However, in order to reach the systemic circulation and exert any salubrious functions, a food component must maintain its molecular structure through several phases of digestion that each present a significant metabolic challenge for the molecule and affect its eventual rate and extent of absorption [33,34]. It is therefore critically important that any alleged health benefit of a food source be firstly verified with well-designed bioavailability studies that characterise the extent of its in vivo absorption [34]. In this respect, the bioavailability of both inorganic nitrate and the betalains, the major bioactive components of beetroot, have been considered in the literature. The high bioavailability of inorganic dietary nitrate is well established and there are reports of close to 100% absorption following digestion [35]. The extent to which betalains are absorbed is, however, less clear.

Two studies have directly investigated betalain bioavailability by measuring their appearance in human urine after ingesting a single bolus of beetroot juice [36,37]. Kanner et al. [37] identified 0.5%–0.9% of the ingested betacyanins (betanin and isobetanin) in volunteer’s urine in the 12 h after consuming 300 mL of beetroot juice. This indicates that although in small amounts, betacyanins can be successfully absorbed in humans. They also showed that the peak urinary elimination rate of betacyanains (indicative of absorption), occurred 2–4 h after ingestion; however, there was a high level of inter-individual variability within this time period. Frank et al. [36] reported similar findings while investigating betacyanin bioavailability. After providing six healthy participants with 500 mL of beetroot juice, they identified betacyanins in urine at concentrations equivalent to ~0.3% of the ingested dose over a 24 h period. These studies might be interpreted to suggest only small level of bioavailability; however, it is important to realise that betacyanins are unlikely to be exclusively eliminated via the renal pathway [36] Indeed, the use of urinary excretion as a sole indicator of bioavailability has received criticism because it does not account for the biliary and circulatory clearance of compounds, thus underestimating true bioavailability [33]. In addition, the extent to which betalains are metabolised and structurally transformed to secondary metabolites is yet to be characterized, but should be taken into consideration when examining their bioavailability [36].

Given these limitations, Tesoriere et al. [38] employed a different approach to investigate the bioavailability of betalains. Tesoriere and colleagues developed a simulated in vitro model of the human intestinal epithelium using Caco-2 cell monolayers to mimic a functional barrier. This model allowed them to examine whether betalains can be absorbed through a functioning intestinal barrier and hence give an indication of their bioavailability. They demonstrated that two betalains; betanin and to a greater extent indicaxanthin were well absorbed through the simulated model of the intestinal lining (Caco-2 cell monolayer) and mostly in their unmetabolised form via paracellular transport. The latter finding is particularly important, because it reveals that betalains can be absorbed into the systemic circulation in their unchanged form, allowing them to retain their molecular structure and high biological activity [34]. There was some evidence that betanin may be absorbed through transcellular transport as well. Nevertheless, it is important to note that results from in vitro experiments, even when designed to mimic the biological milieu of the human GI tract, do not necessarily translate in vivo, given that several other factors (i.e., first pass metabolism, interactions with gut microflora and protease enzyme degradation) have a significant influence on the concentration of the nutrient that eventually reaches the circulation [33,34].

In addition to the betalain family, other aforementioned plant derived antioxidants have been identified in beetroot, including epicatechin, rutin, and caffeic acid [2], which to varying degrees appear to be well absorbed and bioavailable in humans [39]. Although, the bioavailability of these compounds and other phenolics from beetroot have not been individually determined, there are data describing the bioavailability of the total phenolic compounds present in beetroot. Netzel et al. [40] measured the urinary excretion of total phenolic substances following a single 500 mL bolus of beetroot juice. They identified ~685 mg of phenolic compounds in participant’s urine ≤24 h following beetroot juice ingestion; 97% more than the ~347 mg identified after consuming water (i.e., basal concentrations). While the relative bioavailability from the individual compounds could not be determined, these findings clearly show that beetroots phenolic constituents are extremely well absorbed and likely increase beetroot’s in vivo antioxidant power.

Taken together, the results of the aforementioned studies provide a good base of evidence that beetroot is a bioavailable source of bioactive compounds in humans. With that said, further work is still required to firstly; elucidate the bioavailability of beetroot’s individual bioactive components and secondly; to establish the extent that plasma, biliary and other metabolic pathways contribute to the excretion of these components. Together, these data would give a better understanding of beetroots phytochemical bioavailability and thus elucidate the potential as a health-promoting intervention for humans.

3. Oxidative Stress

Beetroot supplementation might serve as a useful strategy to strengthen endogenous antioxidant defences, helping to protect cellular components from oxidative damage. Under normal metabolic conditions, the biological environment of a cell is considered to be in a state of redox balance, or in other words, an equilibrium exists between reducing (antioxidants) and oxidising (pro-oxidants) agents [41,42]. Molecules capable of oxidation are commonly known as reactive oxygen and nitrogen species (RONS) and are continuously generated in cellular metabolism [42]. At these low concentrations, RONS play an important role in a diverse multitude of cellular and biochemical processes, including gene expression, cell proliferation, apoptosis and muscular contraction [41,42,43,44]. However, excess exposure of a cell to exogenously generated RONS (UV radiation, xenobiotics) or endogenously synthesised RONS (aberrant cell metabolism, inflammation), can overwhelm the cells antioxidant defences, causing an imbalance in redox homeostasis, which gives rise to the condition typically referred to as oxidative stress [42]. This imbalance may overwhelm the endogenous antioxidant defence network leaving DNA, carbohydrate, protein and lipid structures susceptible to oxidation and functional impairments [45,46,47].

In some instances, cells can suffer from acute spells of oxidative stress that temporarily weaken antioxidant defences [48]. This can occur through excessive heat exposure, infectious pathogens and strenuous physical exercise, which are capable of generating RONS that leave cells vulnerable to transient oxidation [44,48]. However, in many human disease states, such as cancer, oxidative stress is a chronic disorder perpetuated by continual and excess production of RONS that induce long-term cellular disruption [48,49]. A previous estimate [42] suggested that oxidative stress plays a role in the pathophysiology of over 200 clinical conditions. It is therefore unsurprising that many antioxidant food sources have been evaluated for their ability to scavenge RONS and avert oxidative stress.

Beetroot is as an exceptionally rich source of antioxidant compounds. The betalain pigments in particular, has been shown by several in vitro studies to protect cellular components from oxidative injury [37,50,51]. For example, in the study by Kanner et al. [37] two betalain metabolites (betanin and betanidin) were shown to reduce linoleate damage induced by cytochrome C oxidase and lipid membrane oxidation induced by H2O2-activated metmyoglobin and free iron (AA-Fe). The authors also reported that betanin, the most abundant betalain found in beetroot (300–600 mg∙kg−1), was the most effective inhibitor of lipid peroxidation. Betanin’s high antioxidant activity appeared to stem from its exceptional electron donating capacity and ability to defuse highly reactive radicals targeting cell membranes [37]. However, as alluded to earlier, betalains are not the only antioxidant compounds present in beetroot. Beetroot contains several highly bioactive phenolics, such as rutin, epicatechin and caffeic acid which are also known to be excellent antioxidants [2,36,39]. Furthermore, nitrite and other NO donors akin to beetroot have been shown to suppress radical formation and directly scavenge potentially damaging RONS such as superoxide and hydrogen peroxide, suggesting nitrate may also exhibit antioxidant effects [19,52,53].

A number of studies report that beetroot, in the form of a juice supplement, protects against oxidative damage to DNA, lipid and protein structures in vitro [27,54,55]. A study by Wootton-Beard and Colleagues suggests that a key mechanism by which beetroot juice exerts its antioxidant effects is by scavenging radical species [56]. They found that two commercially available beetroot juices inhibited in vitro radical formation in the 2,2-diphenyl-1-picrylhydrazyl (DPPH•) and (3-ethylbenzothiazoline-6-sulfonicacid) ABTS• assays by 100% and 92%, respectively. Importantly, when these assays were repeated, but in conditions designed to simulate the human digestive process, the juices still retained ≥55% of their pre-digestion radical scavenging capacity. Furthermore, the antioxidant capacity of both drinks, as measured by ferric reducing antioxidant power (FRAP), was higher than the other 22 vegetable juice drinks under investigation. In another study [21], they showed that the FRAP of beetroot juice actually increases following simulated digestion. This is probably a consequence of several compounds being structurally altered to secondary metabolites that possess antioxidant functions [21,56]. Further work from this group has shown that the antioxidant capacity of beetroot juice is comparable to or higher than a variety of fruit and vegetable juices (See Figure 2 and Figure 3) [56,57]. Interestingly, the antioxidant capacity of beetroot juice in both the (DPPH•) and FRAP assays was far greater than more well-known vegetable juices, such as tomato and carrot, and fruit juices, such as orange and pineapple, with only pomegranate juice displaying a higher antioxidant capacity in the FRAP assay.

 

Figure 2

A comparison of the 2,2-diphenyl-1-picrylhydrazyl (DPPH•) inhibiting capacity (%) exhibited by 10 popular fruit and vegetable beverages available in the UK (values based on data from [56,57]).

 

Figure 3

The free radical antioxidant power (FRAP) of 10 commercially available fruit and vegetable beverages post a simulated in vitro model of human digestion (values based on data from [56,57]).

In addition to being a source of antioxidants in vitro, a growing body of evidence using animal models illustrates that beetroot exhibits radical scavenging ability in vivo (see Table 1). In a recent study [23], rats were provided with 1–3 mL∙kg∙bm−1 of a beetroot pomace extract for 7 days prior to being exposed to 2 mL∙kg∙bm−1 of carbon-tetrachloride (CCl4), a well-established carcinogen and RONS generator. After CCI4 administration, liver homogenate was removed from rats pre-treated with the beetroot extracts and those acting as controls (i.e., CCl4 only). Rats treated with beetroot extracts expressed significantly lower levels of lipid peroxidation measured as thiobarbituric acid reactive substances (TBARS). Furthermore, the beetroot extracts appeared to maintain endogenous antioxidant activity (reduced glutathione, glutathione peroxidase and catalase enzymes) at normal cellular concentrations following the oxidative insult. This led the authors to speculate that in response to in vivo cellular attack, beetroot may exhibit indirect antioxidant effects that act to up regulate antioxidant defence mechanisms [23].

 

Table 1

Overview of human and animal studies investigating the effects of beetroot and its derivatives on oxidative stress and inflammation.

Similar antioxidant effects have also been reported with studies using beetroot juice. Providing rats beetroot juice (8 mL∙kg∙bm∙day−1) for 28 days was shown to attenuate lipid peroxidation, protein oxidation and DNA damage following xenobiotic induced liver injury [54]. In a more recent, rats were fed beetroot juice (8 mL∙kg∙bm∙day−1 for 28 days) and treated with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) on day 27 and 28 of the beetroot juice-feeding period study [58]. Several markers of liver damage and inflammation were significantly increased following the DMBA treatment; however, these were markedly reduced in the rats pre-treated with beetroot juice compared to the control group that received water only (see Table 1). There were no differences in DNA damage between the groups. Intriguingly, rats given beetroot juice only (i.e., not treated with DMBA) exhibited increased activity of phase II detoxifying enzymes (GST and NQO1), which play an important role in endogenous antioxidant defense.

The enhanced endogenous antioxidant activity in vivo, by beetroot is a consistent finding in the literature (see Table 1). According to recent in vitro data, such effects might be related, in part, to betanin and its effect on signalling pathways that mediate the transcription of antioxidant genes. Esatbeyoglu et al. [59] found that betanin (extracted from beetroot) dose dependently (5–15 μm) increased the activity of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a transcription factor that activates a gene promoter sequence: the antioxidant response element (ARE) responsible for the transcription of several endogenous antioxidant enzymes [59,60,61]. Krajka-Kuźniak et al. [62] presented similar findings, showing that betanin (2, 10 and 20 μm concentration) activates the Nrf2-ARE binding sequence in non-tumor human hepatic cell lines. Furthermore, this led to increased activity and mRNA expression of several phase II detoxifying enzymes, including glutathione S-tranferases and NAD(P)H:quinone oxidoreductase, which play important roles in host defense against xenobiotics. This raises the possibility that beetroot’s antioxidant potential is not limited to just scavenging and suppressing RONS, but includes the ability to reinforce the endogenous antioxidant network; however, whether such effects translate in vivo, particularly in humans is yet to be investigated.

Given these findings, it should also be considered that other compounds in beetroot (and their downstream metabolites upon ingestion) possess similar effects to betanin on transcriptional activity. Thus, in vivo, these compounds and metabolites could work synergistically to activate the NRF2-ARE pathway, which, in turn, mediates an increase in endogenous antioxidant activity. Such a possibility deserves further attention.

Collectively, the studies to date provide evidence that beetroot is an excellent source of antioxidants that protects cellular components from oxidation in vitro and importantly, in vivo. This would suggest that beetroot supplementation might be a promising adjunct strategy to help manage diseased states propagated by oxidative stress, such as liver injury and cancer. With that said, there is still a lack of well-conducted human trials, which precludes any definitive recommendations for its clinical use. Moreover, data is currently limited to paradigms inducing oxidative stress through exogenous pathways (i.e., xenobiotics) while endogenously generated RONS play a significant role in human disease [66]. In this respect, strenuous physical exercise could serve as a useful model to study the antioxidant potential of beetroot and its constituents in humans. There are several reports documenting that the mechanical and metabolic muscle damage sustained during intense exercise induces short-term oxidative stress, which can persist for several days until redox balance is restored [67,68]. Therefore, it should be considered that application of a beetroot preparation following an exercise task might serve as a useful model to give an insight into beetroot’s efficacy as an antioxidant agent in humans, and most importantly, provide a more in-depth understanding of its potential application in clinical settings.

 

4. Inflammation

Under normal circumstances, inflammation is regarded as a beneficial process, governing our innate response to biological or physical stimuli such as trauma, infection and other pathogens that may cause the organism harm and disrupt homeostasis [69,70,71]. With that said, immune activation may still have undesirable consequences for the host. In the short term, redness, swelling, pain and diminished function may be experienced at the site of inflammation; however, more concerning is the potential long-term implications if inflammation persists, and is unresolved [71,72]. Failure to remove the invading element and restore normal immune function can cause chronic inflammation resulting in long-term cell dysfunction [73]. Chronic inflammation is often implicated in the onset and progression of several clinical disorders such as obesity, liver disease, cancer and heart disease [69,74,75].

Since the 1970’s, traditional treatment for inflammatory disorders has been non-steroidal anti-inflammatory drugs (NSAIDS) [71]. However, these drugs, particularly in chronic doses, may actually have deleterious consequences for health and evoke negative side effects [74,76]. Above all, they have been deemed ineffective in the treatment of many inflammatory related conditions [74]. Consequently, focus has shifted towards the anti-inflammatory effects of natural food sources and their potential use as alternatives to synthetic NSAID treatments [72].

Betalains and beetroot extracts have emerged as potent anti-inflammatory agents. At least part of their anti-inflammatory effects seems to be mediated by interfering with pro-inflammatory signalling cascades and are summarised in Figure 4. The most important of these is the Nuclear Factor-Kappa B (NF-κB) cascade, as it directly activates and transcribes most gene targets that regulate and amplify the inflammatory response (i.e., cytokines, chemokines, apoptotic and phagocytic cells) [43]. Consequently, NF-κB activity plays a central role in the inflammatory processes that manifest in chronic disease [43]. In a recent study [65], NF-κB DNA-binding activity was dose-dependently attenuated in nephrotoxic rats administered a beetroot extract for 28 days (250 mg or 500 mg∙kg∙bm−1). Furthermore, kidney homogenates from the beetroot treated rats had lower concentrations of immune cells (TNF-α, IL-6 and MPO) and reduced signs of oxidative damage (MDA), which could be directly related to the blunting of the NF-κB pathway. These effects are likely to be mediated, at least in part, by the betalains present in beetroot; recent evidence shows that betanin treatment (25 and 100 mg∙kg∙bm−1 for 5 days) significantly inhibits NF-κB DNA-binding activity in rats induced with acute renal damage [77]. Betalains have also been shown to markedly supress cyclooxygenase-2 (COX-2) expression in vitro, which is an important precursor molecule for pro-inflammatory arachidonic acid metabolites known as prostaglandins [3,26,50]. Reddy et al. [50] who found that betanin (IC50 value 100 μg·mL−1) inhibited cyclooxygenase-2 (COX-2) enzyme activity by 97%, first illustrated this. It is interesting to note that although a slightly higher concentration of betanin was required, its COX-2 inhibitory effects were comparable or greater than several phenolic compounds (cyanidin-3-O-glucoside, lycopene, chlorophyll, b-carotene, and bixin) and anti-inflammatory drugs (Ibuprofen, Vioxx and Celebrex). This raises the possibility that betanin rich beetroot supplements, in sufficient doses, could exhibit anti-inflammatory effects to rival synthetic drugs.

 

Figure 4

Illustration of the inflammatory cascade in response to cellular attack and possible pathways where betalains may exhibit inhibitory effects. PGF2, Prostaglandin F2; PGE2, Prostaglandin E2; COX ½, Cyclooxygenase 1 and 2; LOX, lipoxygenase; LOX-5, ...

A recent study from Vidal et al. [26] provided further support for the anti-inflammatory effects of betalains. As well as supressing COX-2 synthesis, betanidin, extracted from beetroot, dose dependently inhibited (to 9% of control activity), lipoxygenase (LOX), a catalytic enzyme vital for the synthesis of pro-inflammatory leukotriene molecules [70]. Interestingly, these inhibitory effects appeared to be mediated by a blocking action on membrane binding activity, indicating that betalains target cell signalling pathways at the molecular level, acting in a similar fashion to selective COX-2 inhibitor drugs [26,70].

There are a limited number of studies demonstrating that beetroot supplements have anti-inflammatory effects in vivo. Pietrzkowski et al. [27] showed that therapeutic administration of betalain-rich oral capsules made from beetroot extracts alleviated inflammation and pain in osteoarthritic patients. After 10 days of supplementation (100, 70 or 35 mg per day), the pro-inflammatory cytokines; tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), had decreased from baseline by 8.3%–35% and 22%–28.3%, respectively. The activity of two chemokines; regulated oncogene-alpha (GRO-alpha) and regulated upon activation normal T cell growth (RANTES) were also markedly inhibited by the beetroot treatment. Furthermore, the moderated inflammatory response coincided with a significant reduction in self-reported pain on the McGill Pain Questionnaire. Krajka-Kuźniak et al. [64] examined the protective effect of beetroot juice (8 mL∙kg∙bm∙day−1 for 28 days) on markers of liver injury and inflammation induced by the toxic chemical N-nitrosodiethylamine (NDEA) in rats. Compared to an untreated control, the beetroot juice conferred significant hepatic protection against a range of inflammatory markers induced by NDEA administration; lactate dehydrogenase, aspartate aminotransferase, gamma glutamyl transferase and alanine aminotransferase were all shown to be markedly attenuated. In a more recent study, El Gamal et al. [65] fed rats either water (control) or oral doses of beetroot ethanol extract (250 or 500 mg∙kg∙bm∙day−1) for 28 days; from day 20–28 they were treated with the nephrotoxic drug gentamicin (85 mg∙kg∙bm∙day−1). After 28 days, kidney homogenates were removed from both groups and analysed for several markers of renal damage. They found that the beetroot-treated rats had significantly lower concentrations of several pro-inflammatory mediators, including Il-6, TNF-α, myeloperoxidase (representing neutrophil infiltration) and the transcription factor NF-κB. They also found reduced oxidative stress (MDA, uric acid) and increased endogenous antioxidant activity (CAT, non protein sulfhydryl; NP-SH) in the homogenates removed from the rats pre-treated with beetroot. Taken together, these studies support the notion that beetroot supplementation offers anti-inflammatory protection in vivo; however, well designed, long-term clinical trials are clearly required to elucidate whether such a strategy would assist in the management of inflammatory-related disorders.

The antioxidant and anti-inflammatory activity of beetroot has also led to interest in its potential use in diseases characterized by aberrant immune cell function. Indeed, chronic inflammation is increasingly being implicated in the development of malignant tumours and evidence is accumulating to suggest betalain extracts obtained from beetroot may suppress these effects. For instance, Lechener et al. [78] examined whether long term (35 weeks) treatment with a betacyanin containing extract (78 μg∙mL∙day−1 of E162, red food colour prepared from beetroot) would inhibit tumour incidence in rats exposed to a potent tumour promoter (N-nitrosomethylbenzylamine). In comparison to control, the beetroot extracts markedly inhibited cell proliferation, angiogenesis and tumorgenesis in oesophageal lesions, effects largely attributed to its radical scavenging and anti-inflammatory activity. This was evidenced by the significantly reduced number of inflammatory lymphocytes present in the oesophageal tumours of the beetroot-treated rats only. The chemo-preventive effects of betacyanin extracts have also been observed in lung, skin and liver cancer cells in animal models, and recently, in human prostate, skin, breast and pancreatic tumour cells [28,29,30,31]. Furthermore, co-administration of beetroot extracts with doxorubicin, an effective but highly toxic chemotherapy drug, significantly reduces its cytotoxicity, probably by modulating the drug’s induction of tumour promoting RONS [28,31]. Although far from conclusive, these initial findings in animal and human cell lines suggest beetroot supplementation holds promise as a future strategy to at least help manage some of the symptoms of inflammation in cancer.

 

5. Endothelial Function

As described earlier, nitrate delivered via a beetroot source is metabolised to nitrite, which can be further reduced to produce NO [4,13]. The conversion of nitrite to NO can be catalysed by a number of molecules with reductase potential (i.e., electron donors), and to date, several proteins (i.e., deoxymyoglobin, xanthine oxioreducatse) and antioxidants (i.e., vitamin C) have been reported to facilitate this reduction [4,79,80]. One of the most important functions of endogenous NO is to maintain endothelial function [13,81]. The endothelium plays a critical role in the regulation of vascular homeostasis by maintaining thrombotic activity, platelet function, vascular tone and the delicate balance between the release of vasodilating (i.e., NO, prostacyclin) and vasoconstricting agents (i.e., endothelin-1, thromboxane) [81,82]. Because NO mediates many of the endothelium’s functions, a depletion in NO availability, as seen with aging, has been singled out as the principal cause of endothelial dysfunction [81]. Endothelial dysfunction is proposed as a primary risk factor for several cardiovascular disorders and has been implicated in the pathogenesis of hypertension and atherosclerosis [16,83]. Therefore, beetroot, as a natural NO donor, has been explored as a nutritional approach to preserve or restore endothelial function.

Webb et al. [10] were the first to investigate the effects of a beetroot supplement on endothelial function in healthy participants. They measured brachial artery (BA) endothelial function using the flow mediated dilation technique (FMD), which involved calculating BA dilation before and after a 20 min ischemic insult. The ischemic procedure (BA occlusion) was effective at inducing endothelial dysfunction, as evidenced by the 60% decrease from pre to post BAFMD response. However, when participants were pre-treated 2 h prior with a single serving of beetroot juice (500 mL; 23 mmol of nitrate) the BAFMD response was maintained at pre-ischemic levels, suggesting that beetroot juice acted to preserve endothelial function.

Hobbs et al. [12] extended these findings, examining the acute intake of a novel beetroot enriched bread (100 g beetroot, nitrate; 1.1 mmol) on micro vascular function and peripheral arterial stiffness in young healthy males. Although arterial stiffness, assessed by pulse wave velocity and augmentation index, was unaffected by the intervention, the beetroot bread increased micro vascular vasodilation, as measured by changes in cutaneous perfusion using laser doppler imaging (LDI). Endothelium-independent vasodilation (perfusion units) was ~343% greater in the 6 h after ingesting the beetroot enriched bread compared to the control bread. Importantly, this study provided evidence that even a small nitrate load (1.1 mmol) can augment marked improvements in intravascular function. Similar vascular effects were also reported in a study with older populations. Using apparently healthy but slightly obese, older participants (~61 years), Joris and Mensink, [83] investigated whether beetroot juice supplementation would prevent postprandial impairments in BAFMD. In a randomized crossover design, BAFMD response fell by ~1.6% in the control condition, whereas after beetroot juice (140 mL, nitrate; 500 mg) the impairment was only ~0.4%, indicative of a beetroot-mediated protective effect on postprandial endothelial function.

Replicating the aforementioned findings in volunteers with an increased risk of endothelial dysfunction has proved more difficult. For instance, Kenjale et al. [84] reported that acute beetroot juice intake (500 mL) had no influence on endothelial function in peripheral arterial disease (PAD) patients, as assessed by BAFMD response (% arterial dilation) to a hyperaemic stimulus. In another study, type 2 diabetic volunteers were given either beetroot juice (250 mL·day−1; nitrate: 7.5 mmol) or a nitrate depleted placebo but otherwise nutritionally matched beetroot juice for 14 days [85]. After the treatment period, BAFMD response was similar between control and beetroot juice groups (4.94% vs. 4.97% change in vessel diameter, respectively) and no differences in micro vascular vasodilation, as measured by LDI, could be detected between the two conditions. However, perhaps the lack of an effect in these studies is not surprising, given that unlike the studies with healthy cohorts, most volunteers were already receiving vasoactive medications for their respective conditions. It is conceivable that these medications diminished or confounded any potential vascular response afforded by nitrate. Perhaps a more profound response to beetroot supplementation could be elicited in pre-clinical patients, i.e., those not yet requiring prescription medications that interfere with vascular function. An alternative explanation for the findings in the latter study is that bioactive constituents (i.e., betalains, caffeic acid) other than nitrate (likely present in both the nitrate depleted placebo and nitrate rich beetroot juice) could have mediated a dilatory response. Indeed, there is evidence that other antioxidants such as flavonoids possess dilatrory effects in humans [86]. If betalains or indeed other antioxidants present in beetroot exerted similar effects in this study then BAFMD and micro vascular function would not be expected to differ between the two conditions. However, the dilatory effects of these compounds and consequences for endothelial function are yet to be investigated and therefore such an effect can only be speculated at present.

It is also important to note that several disease states, including type 2 diabetes, are characterised by persistent inflammation and an excess production of RONS [87,88]. This could limit the efficacy of nitrate supplementation, because RONS such as superoxide (O2-) directly react with NO, diminishing its bioavailability [85]. Therefore, any benefits of the surplus NO generated by ingesting dietary nitrate could be reduced in the presence of oxidative stress. To counter this, perhaps these pathologies would benefit from higher doses of beetroot juice, not only to increase the amount of ingested nitrate (and NO generation) but also to provide a stronger antioxidant defence (i.e., betalains) against RONS. Such an approach might help enhance nitrate mediated NO bioavailability. Further research is required to establish the role of beetroot supplementation in endothelial dysfunction.

 

6. Cognitive Function

Cognitive function deteriorates with age and one of the key pathological events that precedes its development is reduced cerebral blood flow [89,90,91]. Indeed, an age related decrease or impairment in cerebral perfusion has been implicated in several neurological disorders associated with poor cognitive ability, such as brain damage, clinical dementia and Alzheimer’s disease [92,93]. One of the major triggers and risk factors for the onset and development of cerebral hypo-perfusion is a disruption in neurovascular function; an effect that is, in part, mediated by impaired NO activity [92,93]. A diminished capacity to generate NO can impair the normal function of cerebral energy metabolism (i.e., glucose delivery) and neuronal activity (i.e., cellular communication), which over a chronic period might induce neurodegeneration and cognitive deficits [90,91,92]. Therefore, it is conceivable that a NO generator like beetroot has the potential to improve cerebrovascular blood flow and challenge detriments in cognitive function.

Two recent human studies examined the influence of dietary nitrate on cerebral blood flow. Presley et al. [6] measured cerebral perfusion after providing a group of older adults (~75 years) a high nitrate diet (~12 mmol) including beetroot juice, or a nitrate depleted diet (~0.09 mmol) for 24 h. Magnetic resonance imaging (MRI) revealed that the high nitrate diet stimulated a substantial and preferential increase in frontal cortex perfusion, a region of the brain responsible for essential cognitive processes such as executive function, working memory and task-switching. Further work by Bond et al. [89] supports these conclusions, showing a decrease in cerebrovascular arterial resistance (indicative of increased cerebral blood flow) following a single serving of nitrate rich beetroot juice (500 mL). However, it is important to note that the subjects in this study were young (~21 years), asymptomatic and apparently disease-free, limiting the application of these findings in elderly and diseased populations.

Although long-term clinical trials are yet to be conducted, two recent preliminary studies explored the influence of acute beetroot supplementation on age-related cognitive function. In one of these studies, older (~67 years), type 2 diabetics, supplemented with 250 mL of beetroot juice (nitrate: 7.5 mmol) for 14 days, experienced a significant improvement in simple reaction time compared to a control group [5]. However, no effects were evident in other cognitive tests associated with decision-making, rapid processing, shape and spatial memory [5]. Another study from the same group [94] investigated the effects of a beetroot juice supplement (140 mL·day−1: nitrate: 9.6 mmol) on cognitive function in healthy, older adults (~63 years). After 3 days of supplementation, they failed to detect any changes in cognitive performance for concentration, memory, attention and information processing ability between the beetroot juice and control condition. Furthermore, a range of brain metabolites associated with neuronal functioning (N-acetylaspartate, creatine, choline and myo-Inositol) were not, as hypothesized, upregulated after taking beetroot juice. The somewhat contrasting results between these two studies may be partly explained by differences in participant cohort (type 2 diabetics vs. healthy older adults), cognitive tests employed (Kelly et al. [94] did not use a simple reaction time test) and dose duration. With regards to the latter posit, perhaps the cerebrovascular response required to elicit measurable changes in cognitive function can only be achieved with longer term dosing strategies that have the potential to induce sustained modifications to cerebrovascular function [94]. Nonetheless, the beetroot-mediated increases in simple reaction time reported by Gilchrist et al. [5] is worthy of further investigation, given the potential benefits for clinical populations.

A recent addition to the literature investigated whether acute beetroot juice supplementation could augment cerebral oxygenation status and subsequently aid cognition during a fatiguing exercise task [95]. Beetroot juice (500 mL, nitrate: 5 mmol) ingested 90 min before exercise reduced cerebral deoxygenation status, as measured by near infrared spectroscopy, but failed to improve reaction time and information processing at rest, while cycling at 50%, 70% and 90% of VO2 peak and upon completion of the exercise task. As with the previously mentioned study [94], a single serving of beetroot juice may not have been sufficient to induce measurable changes in cognitive performance. This is the only study that has examined beetroot in this fashion; consequently, there is scope for further research to investigate the potential role of beetroot juice in cognitive function at rest and during exercise.

 

7. Conclusions

Based on the available data, beetroot appears to be a powerful dietary source of health promoting agents that holds potential as therapeutic treatment for several pathological disorders. The powerful antioxidant, anti-inflammatory and vascular-protective effects offered by beetroot and its constituents have been clearly demonstrated by several in vitro and in vivo human and animal studies; hence its increasing popularity as a nutritional approach to help manage cardiovascular disease and cancer. In the human studies to date, beetroot supplementation has been reported to reduce blood pressure, attenuate inflammation, avert oxidative stress, preserve endothelial function and restore cerebrovascular haemodynamics. Furthermore, although beyond the scope of this review, several studies have now established beetroot supplementation as an effective means of enhancing athletic performance [96,97].

 

8. Future Directions

While the precise mechanisms by which beetroot exerts these beneficial effects are yet to be fully elucidated, the present status quo dictates that the cardio-protective, physiological and metabolic effects are mediated by nitrate and its subsequent conversion to NO, while the anti-oxidative and anti-inflammatory effects are mediated by betalains and other phenolics. However, it is important to recognise that the relative contribution of each compound is far more complex and that additive and synergistic effects cannot be ruled out.

The present data indicates that the bioactive constituents in beetroot, especially nitrate, appear to be well absorbed and bioavailable in humans. While an optimal dosing strategy does not currently exist, the available data suggests that the multitude of beneficial health effects offered by beetroot can be realised with amounts easily achievable in the diet or with a supplement such as beetroot juice; although at present, there is insufficient evidence regarding the efficacy, and above all, safety of beetroot supplementation to recommend a long-term strategy. Indeed, potentially detrimental health effects arising from excessive intake of beetroot and supplemental derivatives have not been adequately explored. The historical belief that nitrite is a carcinogenic substance in humans still reverberates and concerns have been raised over the uncontrolled use or excessive intake of nitrate rich substances [98,99]. However, natural beetroot juice supplements, as opposed to sodium nitrite salts for instance, are unlikely to pose a significant health risk, at least in the short term [99,100]. While there is presently no anticipated negative health outcomes associated with other constituents of beetroot, consumers should be aware that some supplements (i.e., juices) could have a relatively high sugar content, which might need to be taken into consideration by some individuals (i.e., diabetics). Future studies are still required to evaluate the long term safety of a dietary beetroot intervention particularly in clinical settings. In this respect, beetroot supplementation could be easily administered, and of course, there would be economic and practical benefits of such an approach.

A variety of beetroot based supplements including juices and capsules are now widely available and relatively inexpensive, particularly in comparison to synthetically manufactured drugs. It is therefore critical that future studies focus on the long-term effects (≥4 weeks) of beetroot supplementation.

Although the results so far are promising, most studies tend to use healthy cohorts as participants which limit the applicability of their findings to clinical populations. Furthermore, the overwhelming majority of human studies have focused on the ergogenic or cardio-protective effects of beetroot supplementation, while little attention has been given to the potential anti-oxidative and anti-inflammatory effects. Consequently, there is a great deal of scope to explore the influence of beetroot supplementation in human disorders characterised by chronic inflammation and oxidative stress (i.e., cancers, arthritis, inflammatory bowel disease etc.).

Nevertheless, beetroot supplementation is a new and exciting area of research that to date has been shown to induce favourable effects in several facets of health and disease. This indicates that beetroot supplementation holds promise as an economic, practical and importantly natural dietary intervention in clinical settings. Because of beetroot’s high biological activity, there are still several unexplored areas in which supplementation might confer health benefits. This includes but is not limited to; pain reduction, cognitive function, vascular function, insulin resistance, cancer and inflammation, especially in older and diseased populations.

Effects of Beetroot Juice Supplementation on Cardiorespiratory Endurance in Athletes

Abstract

Athletes use nutritional supplementation to enhance the effects of training and achieve improvements in their athletic performance. Beetroot juice increases levels of nitric oxide (NO), which serves multiple functions related to increased blood flow, gas exchange, mitochondrial biogenesis and efficiency, and strengthening of muscle contraction. These biomarker improvements indicate that supplementation with beetroot juice could have ergogenic effects on cardiorespiratory endurance that would benefit athletic performance. The aim of this literature review was to determine the effects of beetroot juice supplementation and the combination of beetroot juice with other supplements on cardiorespiratory endurance in athletes. A keyword search of DialNet, MedLine, PubMed, Scopus and Web of Science databases covered publications from 2010 to 2016. After excluding reviews/meta-analyses, animal studies, inaccessible full-text, and studies that did not supplement with beetroot juice and adequately assess cardiorespiratory endurance, 23 articles were selected for analysis. The available results suggest that supplementation with beetroot juice can improve cardiorespiratory endurance in athletes by increasing efficiency, which improves performance at various distances, increases time to exhaustion at submaximal intensities, and may improve the cardiorespiratory performance at anaerobic threshold intensities and maximum oxygen uptake (VO2max). Although the literature shows contradictory data, the findings of other studies lead us to hypothesize that supplementing with beetroot juice could mitigate the ergolytic effects of hypoxia on cardiorespiratory endurance in athletes. It cannot be stated that the combination of beetroot juice with other supplements has a positive or negative effect on cardiorespiratory endurance, but it is possible that the effects of supplementation with beetroot juice can be undermined by interaction with other supplements such as caffeine.

Keywords: nutrition, sport, exercise, nitric oxide, physical activity

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1. Introduction

Cardiorespiratory endurance is defined as a health-related component of physical fitness that relates to the ability of the circulatory and respiratory systems to supply fuel during sustained physical activity and to eliminate fatigue products after supplying fuel [1]. Cardiorespiratory endurance is a performance factor in all sports in which adenosine triphosphate (ATP) is resynthesized, mainly by aerobic metabolism or oxidative processes that produce energy. In these sports, the expended effort typically lasts longer than five minutes, primarily depending on the metabolic level of the oxidative processes involved [2]. Factors that limit performance in this type of endurance patterns include maximum oxygen uptake (VO2max), ventilatory thresholds (first and second ventilatory threshold) and energy efficiency or economy [3,4,5].

In competitive sports, 0.5%–1.5% improvements in performance are considered a critical difference [6]. In order to enhance the effects of training and improve performance, athletes often turn to nutritional supplements [7]. According to the American College of Sports Medicine (ACSM), adequate selection of nutrients and supplements, adjusting intake according to the exercise performed, is necessary for optimal performance in athletes [8]. However, not all supplements have been shown to produce a positive effect on performance. The Australian Institute of Sport [9], classified supplements to which athletes have access, with the goal of categorizing nutritional supplements based on the level of evidence for impact on an athlete's performance (Table 1). However, the effectiveness of supplements also depends on dosage and type of effort, because the potential ergogenic effect may differ by the specific type of sport [10].

 

Table 1

Classification of nutritional supplements, based on performance effect. Adapted from Australian Institute of Sport [9] and Burke [11].

Beetroot juice is used as a supplement because of its high inorganic nitrate (NO3−) content, a compound found naturally in vegetables and in processed meats, where it is used as a preservative [12].

Once ingested, the NO3− is reduced to nitrite (NO2−), by anaerobic bacteria in the oral cavity by the action of nitrate reductase enzymes [13] and then to nitric oxide (NO) in the stomach [14]. This physiological mechanism depends on the entero-salivary circulation of inorganic nitrate without involving NOS activity. Once in the acidic stomach, nitrite is instantly decomposed to convert to NO and other nitrogen oxides performing determinant physiological functions (Figure 1). Nitrate and remaining nitrite is absorbed from the intestine into the circulation, which can become bioactive NO in tissues and blood [14] under physiological hypoxia.

 

Figure 1

Pathway of nitric oxide (NO) production from beetroot juice supplementation. Nitrate (NO3−) is reduced to nitrite (NO2−) by anaerobic bacteria in the oral cavity and then to NO in the stomach. NO3− and remaining NO2− are ...

NO induces several physiological mechanisms that influences O2 utilization during contraction skeletal muscle. Physiological mechanisms for NO2− reduction are facilitated by hypoxic conditions, therefore, NO (vasodilator) is produced in those parts of muscle that are consuming or in need of more O2. This mechanism would allow local blood flow to adapt to O2 requirement providing within skeletal muscle an adequate homogeneous distribution. This physiological response could be positive in terms of muscle function, although it would not explain a reduced O2 cost during exercise [15]. Another probable mechanism is related to NO2− and NO as regulators of cellular O2 utilization [15].

In addition, a potent signaling molecule that affects cell function in many body tissues, NO is endogenously produced by synthesizing nitric oxide from l-arginine oxidation. The molecule has important hemodynamic and metabolic functions [16,17], being a major vasodilator that can increase blood flow to muscles [18] and promote oxygen transfer in the muscle. Additional physiological benefits of NO include improved mitochondrial efficiency and glucose uptake in muscle [19] and enhanced muscle contraction and relaxation processes [20]. Other researchers have reported that NO can act as an immunomodulator [21] and stimulates gene expression and mitochondrial biogenesis [22]. Given the positive effects of beetroot juice, which are induced by means of NO, this supplement has been proposed as part of the therapeutic approach in people with chronic obstructive pulmonary disease [23], hypertension [24], heart failure [25] and insulin resistance [26].

These findings reflect the importance of supplementation with NO3− or nitrate salts to increase the bioavailability of NO in order to influence muscle function improving exercise performance, mainly in aerobic metabolism [27]. Therefore, supplementation with beetroot juice may have an ergogenic effect in athletes [9], especially with respect to cardiorespiratory endurance. However, the assumption that the beetroot juice supplementation improves performance in cardiorespiratory endurance under hypoxic conditions, and the combination of beetroot juice supplementation with other supplements, as caffeine, has a positive effect on cardiorespiratory endurance is controversial.

The objective of the present literature review was to analyze the effects of beetroot juice supplementation on cardiorespiratory endurance in several conditions (normoxia, hypoxia and beetroot juice with other supplements) and determine the appropriate dosage to enhance the potential ergogenic effects on performance. The focus of the article is mainly on the influence of beetroot juice of the acute and chronic responses on trained endurance athletes.

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2. Methodology

A keyword search for articles published in English or Spanish since 2010 was carried out in the DialNet, MedLine, PubMed, Scopus and Web of Science databases on 8 June 2016. The search terms included beet, beetroot, nitrate, nitrite, supplement, supplementation, nutrition, “sport nutrition” and “ergogenic aids”. The 210 selected articles included at least one of those search terms, in combination with endurance, exercise, sport or athlete.

Exclusion criteria were the following: literature reviews and meta-analyses, animal studies, population other than endurance athletes, and inadequate assessment of cardiorespiratory endurance, specifically defined as <VO2max testing or no test lasting more than 5 min to determine how long the subject can maintain the lowest intensity at which VO2max was achieved [28]. Therefore, 23 articles were selected for the present review (Figure 2).

 

Figure 2

Flowchart of article selection.

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3. Results and Discussion

The selected studies on the effects of beetroot juice supplementation on cardiorespiratory endurance are summarized in Table 2.

 

Table 2

Summary of studies that have evaluated the performance or metabolic responses after supplementation protocol with beet juice.

In Table 2, 23 articles were examined regarding beetroot juice supplementation in normoxic conditions, hypoxic conditions and beetroot juice combined with caffeine supplementation: 11 of those articles were related to trained athletes, four of them to cyclists-triathletes, three to cyclists trained, two to trained kayakers, one to trained runners, one to trained swimmers, and one to healthy physically active people. Twenty-one of these articles assessed respiratory parameters including VO2 at several intensities (approximately 60%–100% VO2max, VT1)

Briefly, in trained athletes men and women in normoxia conditions appeared that beetroot juice supplementation enhances aerobic performance by a decrease in VO2 at several intensities (60%–100% VO2max, VT1) increasing the economy during exercise. In kayak studies, a decrease of VO2 at the same intensity in kayakers supplemented with beetroot juice compared to a placebo group was found. In trained swimmers, a decrease in energy expenditure in the experimental condition of beetroot juice supplementation was observed.

Regarding the supplementation with beetroot juice in hypoxic conditions, five studies were selected. The hypothesis that beetroot improves cardiorespiratory performance in hypoxic conditions is controversial.

Two studies evaluated the effect of the combination of beetroot juice and caffeine in men and women trained cyclists-triathletes, and one study evaluated the same supplementation in trained men athletes. The studies did not determine that the effects of beetroot juice combined with caffeine increase the cardiorespiratory performance regarding caffeine supplementation.

3.1. Acute Effects of Beetroot Juice Supplementation on Performance in Cardiorespiratory Endurance

Several studies have shown a positive effect of acute beetroot juice intake on various parameters of performance improvement associated with the cardiovascular and respiratory system. Economy is a parameter that expresses the relationship between oxygen consumption (VO2) and power generated or the distance traveled by an athlete [29], regarded as a performance factor in cardiorespiratory endurance [3,4,5]. Improved economy is due to achieving higher output power with the same VO2 level [30]. Another improvement attributed to beetroot juice supplementation is related to the increased blood flow, favoring the supply of oxygen to the mitochondria [50], which has the side effect of stimulating oxidative metabolism. In addition, supplementation with NO3− could improve the processes of muscle contraction and relaxation [31].

A study in trained cyclists found that beetroot juice supplementation improves performance by 0.8% in a 50-mile test [32]. Significant increases in efficiency, measured as watts (W) per liter of VO2 (W/VO2) were observed in the last 10 miles; these improvements were associated with a decrease in time required to travel this distance. Another study [33] aimed to assess efficiency on a 40-min test at submaximal intensity (20 min at 50% VO2max followed by 20 min at 70% VO2max). A decrease in VO2 and improved efficiency was also observed after beetroot juice supplementation, but did not reach statistical significance. After supplementation and immediately after the submaximal 40-min test, the time-to-exhaustion at an intensity of 90% VO2max improved as much as 16% in the trained cyclists. These findings make us suspect that beetroot juice might have an ergogenic effect, increasing performance in prolonged cycling events that require alternations in relative intensity, from moderate to high VO2max, which is very characteristic of the stages of cycling races.

In a time trial of 16.1 km, supplementation with beetroot juice improved the performance of trained cyclists diminishing a completion time in a 2.7% and by 2.8% in a 4-km time trial [27]. Although, the protocol test used in this study had a high ecological validity, providing an accurate simulation of the physiological responses during competition, it is unclear that beetroot supplementation can increase the performance by this magnitude in elite cyclist [27].

This increased performance was also associated with W/VO2 improvements of 7% in a time trial of 16.1 km and 11% in 4-km time trial [27]. The observed improvements in efficiency match those found in high-performance kayakers when paddling at 60% relative VO2max intensity or in a 4-min test [34].

Response to a submaximal VO2 test at constant load is very important to cardiorespiratory endurance in athletic performance. In this type of test, VO2 increases disproportionately during the first 3 min because of an increase at the respiratory center to meet the exercise-induced increase in energy demand [51]. At an intensity below VT1 60% VO2max efforts, approximately stabilization of VO2 is observed from the 3-min point until the end of the effort [52]. Nonetheless, at intensities greater than VT1 a progressively greater recruitment of type II motor units occurs [53], which have a lower oxidative potential than type I [54], and therefore a progressive increase in VO2 is observed from the third minute until the end of the exercise. This has been called the slow component of VO2 [55], which has been identified as one of the main factors limiting performance in endurance exercise of moderate and/or high intensity [4], because the increase in the slow component of VO2 attains values of VO2max at submaximal intensity, causing fatigue [56].

In experienced athletes, the effect of supplementation with beetroot juice (8.2 mmol nitrate) on time-to-exhaustion was tested at intensities of 60%, 70%, 80% and 100% peak power [35]. Athletes were able to maintain an intensity of 60% (Beetroot: 696 ± 120 vs. Placebo: 593 ± 68 s), 70% (Beetroot: 452 ± 106 vs. Placebo: 390 ± 86 s) and 80% (Beetroot: 294 ± 50 vs. Placebo: 263 ± 50 s) peak power significantly longer during exercise with supplementation, and there was a trend toward increased endurance at 100% peak power. The study results might reflect a lower VO2 response at submaximal intensities, which would reduce the increase in the slow component, delaying the time when the athletes reached VO2max and therefore became fatigued. This would allow a longer sustained effort.

On the other hand, trained runners participating in a 5000-m test showed no significant overall improvement with beetroot juice supplementation, although they ran 5% faster in the later part of the race, particularly the last 1.1 miles [12]. The lack of significance could be related to the timing of the supplementation. Participants took the supplement 90 min before exercise; in the other studies cited, beetroot juice was provided 150–180 min before the effort [27,32,33,34,35] and ergogenic effects of supplementation with beetroot juice were observed at 150 min after ingestion [35].

3.2. Effects of Chronic Supplementation with Beetroot Juice on Cardiorespiratory Endurance

In addition to increasing blood flow and improving muscle contraction and relaxation, beetroot juice supplementation may improve the efficiency of mitochondrial respiration [50] and oxidative phosphorylation [57]. It seems, however, that acute supplementation is insufficient to produce mitochondrial biogenesis, suggesting that these adaptations may require longer supplementation protocols. In trained athletes, acute supplementation with beetroot juice for five days reduces VO2 as much as 3% at an intensity of 70% VO2max. The test was performed at 50% VO2max for 10 min, followed by 10 min at 70% VO2max [31]. Another study in trained cyclists confirmed that supplementation for a period of six days reduces VO2 in a 60-min test. The protocol consisted of 30 min at 45% VO2max followed by another 30 min at 65% VO2max. In addition, riders were able to improve their 10-km time trial performance immediately following the submaximal test [30].

These studies clarify the benefits that could result from supplementation with beetroot juice in longer intake protocols of about six days, as was the case in the time-to-exhaustion test at submaximal intensities following acute supplementation [33,35]. Time-to-exhaustion improved at intensities of 70% of VO2max, between VT1 and VO2max [37]. In trained swimmers, Pinna et al. [38] also corroborated the progressive ergogenic benefits of beetroot juice during an incremental test. At anaerobic threshold intensity, workload increased and aerobic energy expenditure decreased.

In another study, in healthy subjects physically active but not highly trained in any particular sport, Vanhatalo et al. [36] evaluated the acute and chronic (15-day) effects of dietary supplementation with NO3− on VO2 in a constant load test at an intensity of 90% of the gas exchange threshold (GET), similar to the anaerobic threshold, and in a progressive incremental ergometric cycle test, compared to controls. The peak power in the incremental test and the ratio of work rate to GET intensity were increased in the group that received the dietary NO3− supplementation. The findings indicated that dietary supplementation reduces NO3− oxygen consumption at submaximal exercise, and these effects can last for 15 days if supplementation is maintained.

Potential improvements observed in the anaerobic or lactate threshold intensity is especially important for athletes in various forms of endurance sports, because the level achieved in this parameter does not depend on motivation as it occurs when VO2max is determined [58]. This threshold is considered a factor that better discriminates between cardiorespiratory endurance capacities than does VO2max [2,58]. One of the physiological parameters that conditions improvement in the anaerobic threshold is increased mitochondrial population [59]. If the beetroot juice supplementation can promote mitochondrial biogenesis, we might assume that chronic supplementation with beetroot juice would decrease oxygen consumption at anaerobic threshold intensity as an adaptation to exercise.

It has also been suggested that additional beetroot juice supplementation may improve the muscle contraction functions. A study by Whitfield et al. [31] found that VO2 reduction after a constant load test at 70% VO2max occurred without any changes in markers of mitochondrial efficiency such as adenine nucleotide translocase (ANT) and uncoupling protein 3 (UCP3). Similarly, other researchers have suggested that supplementation may positively affect the interaction of actin and myosin bridges [60] by modulating the release of calcium that occurs after the action potential [61]. The effects described by these authors indicate that supplementation with beetroot juice, whether acute or chronic, could improve performance in sports that are characterized either by a predominantly aerobic or anaerobic metabolism [38]. This could explain the positive effects on effort with a high prevalence of anaerobic metabolism observed in a 500-m kayak test [29] or in the contractile force developed by mice [62].

3.3. Effects of Beetroot Juice Supplementation on Performance in Cardiorespiratory Endurance under Hypoxic Conditions

Many competitions, such as the mountain stages in cycling, are held at high altitudes [39], where cardiorespiratory endurance is decreased relative to sea level [63]. Among the factors that could be responsible for this decrease, we would highlight decreased supply of oxygen to muscles, due to a partial reduction in oxygen pressure.

It is known that NO has an important role in the adaptation processes under hypoxic conditions; higher levels of NO2− have been observed in Tibetans [18]. In a study of acute response to hypoxia, people who live at sea level who climb to high altitudes and show decreased NO levels have symptoms of acute altitude sickness [64,65]. The vasodilatory effects of NO may favor oxygen delivery [66], and supplementation with beetroot juice could be effective in reducing the ergolytic effects of hypoxia on cardiorespiratory endurance [39].

A recent study evaluated the effects of supplementation with acute and chronic beetroot juice on a 15-min test at an intensity of 50% VO2max and a 10-km test carried out at a simulated altitude of 2500 m [40]. The test could not verify any positive effect of acute or chronic supplementation on any of the performance variables analyzed. In addition, studies have shown supplementation with beetroot juice did not improve performance in runners with a high level of training in an incremental intensity test or in a 10-km race [41] or in a 1500-m test or tests at various submaximal intensities (50%, 65% and 80% VO2max) [42]. The results in the latter study are also in line with those reported by McLeod [40]; in these two studies, beetroot juice was administered 90 and 120 min, respectively, before exercise. This may be an insufficient time interval for athletes to reach peak NO2− levels in their bloodstream.

The results presented above conflict with other reports [39,43]. Kelly et al. [43] tested the effect of beetroot juice supplementation for three days on performance in a 5-min test at 80% VT1, followed by a test to the point of exhaustion at an intensity at 75% of VT1 and VO2max and a simulated altitude of 2500 m. The results show that supplementation with beetroot juice reduced VO2 to 80% VT1 and there was a statistical trend to improvement in higher intensity exercise (p = 0.07). Improved efficiency was accompanied by a longer time-to-exhaustion in a test at 75% between VT1 and VO2max. In another study that simulated an altitude of 2500 m, supplementation with beetroot juice again reduced the VO2 during a 15-min test at 60% of VO2max and increased the speed achieved in a 16.1-km time trial involving trained cyclists [39]. The results observed in the time trial were consistent with the improvements (2.8%) reported from a 4-km time trial after a protocol of acute supplementation with beetroot juice [27].

Masschelein et al. [44] found that six days of supplementation with beetroot juice can reduce VO2 at rest by 8%, and by 4% at 45% VO2max intensity at a simulated altitude of 5000 m. Although the cited study is not directly generalizable to performance in various types of cardiorespiratory endurance, as competitions are unlikely to take place above an altitude of 2500 m, other parameters such as arterial oxygen saturation (SPO2) and deoxyhemoglobin (HHb) in muscle tissue were analyzed. The results showed that reductions in VO2 were accompanied by greater SPO2 and lower HHb after supplementation with beetroot juice, indicating decreased oxygen extraction by the muscle, which coincides with increased mechanical pedaling efficiency and lower levels of lactate in the blood.

Although the literature shows contradictory data, it is possible that supplementation with beetroot juice may effectively improve performance when hypoxia is present, because oxygenation would improve at the muscular level, reducing the ergolytic effects of hypoxia on aerobic performance.

3.4. Effects of the Combination of Beetroot Juice Supplementation with Other Supplements on Cardiorespiratory Endurance

Caffeine supplementation has become increasingly common among athletes [67]. Among its positive effects is increased stimulation of the central nervous system due to the antagonism of adenosine [68], increased catecholamines and contractility of skeletal muscle [69] that improves calcium output from the sarcoplasmic reticulum through the action potential [70], and a decrease in the subjective perception of pain and the regulation of thermoregulation [71]. Thus, caffeine supplementation has proven ergogenic effects on various modalities of cardiorespiratory endurance [72] and team sports [73,74]. A plateau effect occurs in performance improvement, at doses ranging from 3 to 6 mg/kg of caffeine [75]. To test whether the combined supplementation of beetroot juice (8 mmol of NO3−) and caffeine (5 mg/kg) had a greater effect than each supplement separately, researchers tested the corresponding study groups of cyclists tested for 30 min at 60% VO2max, followed by a test to exhaustion at 80% VO2max [45]. Although the combined supplementation improved time to exhaustion VO2max 80% by 46% compared to placebo, the improvement was insignificant. Furthermore, the additive effect of taking both supplements did not improve performance to a greater extent than separate supplementation with each one [46,47].

In a study that simulated the characteristics of an Olympic cycling time trial, the effect of supplementation in both men and women cyclists was tested using beetroot juice (8.2 mmol of NO3−) and caffeine (3 mg/kg) and the combination of both [47]. The only proven effects were that caffeine supplementation in combination with beetroot juice was effective in improving mean power and time trial results.

In a later study of trained cyclists and triathletes, performance was improved only in the athletes who received a caffeine supplement (3 mg/kg) [46]. No differences were observed in VO2. However, lactate concentration in the blood was increased when athletes received caffeine supplementation. Performance improvement was likely due to an increased anaerobic metabolism after caffeine intake; therefore, it is possible that the effects of supplementation with beetroot juice can be undermined by interaction with other supplements such as caffeine, which interferes with the effects of each supplement taken separately.

3.5. Dosage

Peak NO2− concentration in blood is obtained within 2–3 h of NO3− supplementation [76] and the ergogenic effects of supplementation with beetroot juice can be observed at 150 min after ingestion [36]. Oral antiseptic rinses should not be taken with beetroot juice supplementation, as these can prevent the desired increase in NO2− levels after NO3− ingestion [77]. Although the majority of studies show ergogenic effects of beetroot juice at a supplementation dose of 6–8 mmol NO3− (Table 2), it is possible that high performance athletes might require a slightly higher dose. For example, in high performance kayakers, the ergogenic effect of supplementation with beetroot juice was 1.7% in a 500-m test after ingestion of 9.6 mmol of NO3− but a 4.8 mmol dose did not significantly improve results in a 1000-m test [29].

Practical Considerations

It appears that acute supplementation with beetroot juice increases the power output with the same VO2 levels [30]. This is an interesting finding for athletes as there is evidence that the economy is a key factor to improve cardiorespiratory performance increasing energy efficiency in endurance sports modalities. In addition, time to exhaustion at several intensities (60%–100% VO2max, MAP or VT1) is another usual performance parameter that is improved with acute beetroot supplementation [33,35]. However, not all studies show a positive effect to acute beetroot supplementation indicating that the efficacy of acute nitrate supplementation will be attributed to several factors such as the age, diet, physiological and training status, and other parameters as the intensity, duration, endurance modality and environment conditions [78]. Although most of the studies determine a supplementation dose of 6–8 mmol NO3−, it is unclear that this supplementation dose can be effective to improve cardiorespiratory performance in sports modalities such as kayaking or rowing. The dose should possibly be increased in sports modalities where muscular groups of upper limbs are implicated. Endurance athletes should take the dose of NO3−, approximately 90 min before the competition without oral antiseptic. Acute supplementation with beetroot juice is not sufficient to induce mitochondrial biogenesis, suggesting that mitochondrial adaptations could only occur after longer supplementation protocols. In chronic supplementations with beetroot juice, it appears that the benefits in cardiorespiratory performance might be produced in longer intake protocols of about six days [33,35]. Time-to-exhaustion at several intensities (between 70% and 100% VO2max, VT1) and the load at anaerobic threshold could be enhanced while aerobic energy expenditure could be diminished. Longer-term beetroot supplementation (15 or more days) could be effective, although it would be necessary other studies analyzing the mitochondrial biogenesis to corroborate whether mitochondrial adaptations depend on endurance training and/or beetroot supplementation. To date, this assumption is unknown.

The scientific literature shows discrepancies regarding the improvement of the cardiorespiratory performance induced by the supplementation of beetroot juice under hypoxic conditions. NO3− could mitigate the ergolytic effects of hypoxia on cardiorespiratory in endurance athletes [39].

We cannot assert that the combination of beetroot juice with other supplements has a positive or negative effect on cardiorespiratory endurance. It is possible that the effects of supplementation with beetroot juice can be undermined by interaction with other supplements such as caffeine. More work is needed to confirm the results of these investigations.

 

4. Conclusions

  • Acute supplementation with beetroot juice may have an ergogenic effect on reducing VO2 at less than or equal to VO2max intensity, while improving the relationship between watts required and VO2 level, mechanisms that make it possible to enable increase time-to-exhaustion at less than or equal to VO2max intensity.

  • In addition to improving efficiency and performance in various time trials or increasing time-to-exhaustion at submaximal intensities, chronic supplementation with beetroot juice may improve cardiorespiratory performance at the anaerobic threshold and VO2max intensities.

  • Apparently, the effects of supplementation with beetroot juice might not have a positive interaction with caffeine supplementation, mitigating the effects of beetroot juice intake on cardiorespiratory performance, however, more work is needed to confirm the results of these investigations because the number of studies analyzing the effects of the combination of beetroot juice with other supplements, such as caffeine, is limited.

  • Intake of beetroot juice should be initiated within 90 min before athletic effort, since the peak value of NO3− occurs within 2–3 h after ingestion. At least 6–8 mmol of NO3− intake is required, which can be increased in athletes with a high level of training.

 

 

References for The Potential Benefits of Red Beetroot Supplementation in Health and Disease

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