Numerous studies in recent years have investigated the effects of dietary nitrate (NO3−) on the physiological responses to, and/or performance during, exercise. This interest stems from the fact that dietary NO3− is an important source of nitric oxide (NO) via the “reverse” NO3− → nitrite (NO2−) → NO enterosalivary pathway (Kharti et al., 2017). NO3− induced increases in NO bioavailability have been found to enhance both endurance exercise performance (Jones et al., 2018; Van De Walle & Vukovich, 2018) as a result of alterations in oxygen supply, demand, and/or cellular energetics (Bailey et al., 2010), and sprint performance (Rimer et al., 2016) as a result of improvements in muscle contractile properties, that is, in maximal muscle speed and power (Coggan et al., 2015b, 2018b). For example, Lansley et al. (2011a) observed a 2.7–2.8% improvement in mean power during simulated 4- and 16.1-km cycling time trials, whereas Rimer et al. (2016) reported a 6.5% increase in maximal power during an inertial load sprint cycling test. Positive effects of BRJ seem to be most apparent in untrained or moderately trained individuals (Van De Walle & Vukovich, 2018) or patient groups, (Coggan et al., 2015a, 2018a) and less evident or even absent in highly trained athletes (e.g., Bescós et al., 2012). Notably, however, some studies (Hoon et al., 2015; Oskarsson & McGawley, 2018) have yielded negative results for reasons that do not seem to be related to differences in subject selection or other aspects of the experimental design.
Most studies of dietary NO3− and exercise have used beetroot juice (BRJ) as a source. This is because beets are relatively rich in NO3− (Santamaria, 2006) and are readily juiced. Consequently, lay publications frequently contain articles extolling the virtues of BRJ supplementation as an ergogenic aid, and a large number of BRJ supplements in various forms (i.e., powders, mixed drinks, concentrates, bulk juice) are now marketed to athletes. However, the NO3− content of beets or BRJ depends heavily on a number of factors, including the growing conditions, the variety of beet, and the method of preparation (i.e., fresh vs. processed; Corleto et al., 2018; dos Santos Baião et al., 2016; Wruss et al., 2015). Furthermore, most BRJ supplements are not labeled with their NO3− content, which in any case has only rarely been independently tested. Even some scientific studies have not verified the NO3− content of the BRJ supplement used and/or measured any biomarkers of NO bioavailability (e.g., plasma NO3− or NO2−, breath NO) to ensure that an adequate dose of NO3− has been provided (e.g., Hoon et al., 2015; Oskarsson & McGawley, 2018). Athletes and/or their support staff have therefore generally relied on blind faith that any BRJ supplement contains sufficient NO3− to provide physiological benefits.
The purpose of this study was therefore to determine the NO3− (and NO2−) content of various commercial BRJ supplements. The results may be useful to athletes, coaches, and scientists contemplating which (if any) BRJ product to use and may also offer some insight into factors contributing to variability in the literature with respect to the effects of BRJ on exercise performance.
Methods
To aid interpretation of the data, we chose to test products that (a) contained BRJ as the primary ingredient, (b) did not contain other ingredients (e.g., L-arginine) intended to alter NO bioavailability, and (c) were marketed toward athletes and/or athletic performance or were available from sources readily accessible by athletes. Products were purchased from local grocers, large online retailers, specialized “health food” websites, or directly from the producer. Our initial goal was to obtain samples from three different lots of each product, but in many instances, infrequent production runs limited us to testing just one or two different lots of a given product. We ultimately obtained samples from 45 different lots of 24 different BRJ products (i.e., n = 6 each of powders, mixed beverages, concentrates, and bulk juice) produced by 21 different companies. All samples were purchased between September 2017 and June 2018, stored for ≤2 weeks at room temperature or under refrigeration as recommended for that product, and tested before the declared expiration date.
Powdered products were first reconstituted by thoroughly mixing one serving, that is, an entire packet or scoop, with 100 ml of NO3−- and NO2−-free, double-distilled water. A small aliquot of each reconstituted powder or original liquid product was then diluted 1,000-fold and the NO3− and NO2− concentration measured by injecting 10 µl into a dedicated high-performance liquid chromatography analyzer (ENO-30; Eicom USA, San Diego, CA). This analyzer has previously been described in detail (Coggan et al. 2015a; Troutman et al., 2018). The high-performance liquid chromatography analyzer was calibrated prior to each use by injecting known standards. The NO3− and NO2− content of each product (i.e., mmol/serving) was calculated from these concentration measurements based on either (a) the manufacturer’s recommended serving size or (b) for BRJ products sold in bulk containers, a volume of 500 ml as used in previous studies of BRJ and athletic performance (e.g., Bailey et al., 2010).
Because of the limited number of samples available per product, no attempt was made to compare different products statistically. However, the mean NO3− concentration of different types of BRJ products (i.e., powders vs. mixed beverages vs. concentrates vs. bulk juices) was compared using one-way analysis of variance. A p < .05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism (version 7.02; GraphPad Software, La Jolla, CA).
Results
Data for NO3− and NO2− concentration (i.e., per g or ml) and content (i.e., per recommended serving) for each sample are shown in Figures 1 and 2, whereas average values for content are provided in Table 1. The NO3− concentration of powders (i.e., 174 ± 63 µmol/g; mean ± SD) was significantly higher (i.e., p < .001) than that of concentrates (70 ± 39 µmol/ml), which in turn was higher (p < .05) than that of mixed drinks (13 ± 5 µmol/ml) or bulk juices (18 ± 11 µmol/ml). However, regardless of the type of product, there was considerable variability in NO3− concentration/content between products and often even between samples of the same product. Specifically, there was almost a 50-fold range in NO3− content between different products (albeit partially driven by the assumed 500 ml serving size for bulk juices), whereas the coefficient of variation for the NO3− concentration or content of samples of the same product averaged 30% ± 26% (range 2–83%).
—NO3− concentration (a) and content (b) of BRJ supplements. Data for each sample tested are shown (circles). In addition, the minimal dose apparently required to increase exercise performance (i.e., 5 mmol) (dashed line), based on a recent review by Jones et al. (2018), is also shown. NO3− = nitrate; BRJ = beetroot juice.
Citation: International Journal of Sport Nutrition and Exercise Metabolism 29, 4; 10.1123/ijsnem.2018-0223
—NO2− concentration (a) and content (b) of BRJ supplements. Data for each sample tested are shown (circles). NO2− = nitrite; BRJ = beetroot juice.
Citation: International Journal of Sport Nutrition and Exercise Metabolism 29, 4; 10.1123/ijsnem.2018-0223
BRJ Products Tested
| Company | Product | Serving size | Claimed NO3−content (mmol/serving) | Measured NO3− content (mmol/serving) | Measured NO2− content (mmol/serving) |
|---|---|---|---|---|---|
| Powders | (g) | ||||
| AIM International | RediBeets | 4 | – | 0.43 | 0.03 |
| HumanN | Superbeets | 5 | – | 1.03 | 0.14 |
| HealthySkoop | Endurance Beets | 11 | – | 1.08 | 0.00 |
| Nutrigardens | BeetBoost | 11 | – | 1.78 | 0.00 |
| HumanN | BeetElite | 10 | – | 2.16 ± 0.28 | 0.22 ± 0.01 |
| PureClean | PureClean Powder | 10 | – | 2.56 ± 0.32 | 0.00 ± 0.00 |
| Mixed drinks | (ml) | ||||
| H2Bev | Beet-Power | 250 | – | 1.02 ± 0.62 | 0.00 ± 0.00 |
| CAJ Foods | Beet Performer w/ B12 | 250 | – | 3.01 | 0.00 |
| Unbeetable | Unbeetable Fizz | 250 | 4.08 | 3.52 | 0.00 |
| BluePrint Organic | Beet Blast | 355 | – | 3.96 | 0.06 |
| CAJ Foods | Beet Performer w/Passion Fruit | 250 | – | 3.97 ± 1.47 | 0.02 ± 0.01 |
| Makomas | Ginger Beet Juice | 355 | – | 6.67 | 0.06 |
| Concentrates | (ml) | ||||
| Brownwood Acres | (beet juice concentrate) | 30 | – | 1.05 | 0.01 |
| AIM International | Red Rush | 74 | 8.06 | 2.39 ± 1.97 | 0.01 ± 0.01 |
| Red Ace Organics | Beet Performance Supplement | 60 | – | 2.76 ± 1.54 | 0.00 |
| CherryActive | BeetActive | 30 | 3.69 | 3.93 | 0.06 |
| James White Drinks | Beet It Organic Beetroot Shot | 70 | 4.84 | 5.93 ± 0.45 | 0.00 ± 0.00 |
| James White Drinks | Beet It Sport Pro-Elite Shot | 70 | 6.45 | 6.41 ± 0.60 | 0.00 ± 0.00 |
| Bulk beet juice | (ml) | ||||
| Love Beets | (beet juice) | 500a | – | 3.35 | 0.01 |
| CAJ Foods | Biotta beet juice | 500a | – | 4.81 ± 2.16 | 0.00 ± 0.00 |
| Pomona Organic | (beet juice) | 500a | – | 7.11 | 0.04 |
| James White Drinks | Beet It beet juice | 500a | – | 7.55 ± 1.61 | 0.00 ± 0.00 |
| Knudsen and Sons | (beet juice) | 500a | – | 12.54 ± 0.25 | 0.00 ± 0.00 |
| Lakewood Organic | (beet juice) | 500a | – | 18.77 ± 1.59 | 0.02 ± 0.02 |
Note. BRJ = beetroot juice; NO3− = nitrate; NO2− = nitrite.
aAssumed serving size.
Although traces of NO2− were detectable in all products, the content was generally very low (i.e., ≤0.06 mmol/serving) except for two products (i.e., Superbeets and BeetElite, both from HumanN), which on average contained 0.14 and 0.22 mmol/serving, respectively. The potential significance of the higher NO2− content of the latter two products is considered in the Discussion section.
Discussion
Based on studies demonstrating that BRJ ingestion can enhance exercise performance (Jones et al., 2018), a large number of BRJ-based supplements are now marketed to athletes. The NO3− concentration of beets, however, varies markedly (dos Santos Baião et al., 2016; Wruss et al., 2015), and few BRJ products have been independently tested to determine their NO3− content. Athletes and/or their support staff must therefore generally rely on claims made by producers to determine whether such supplements contain sufficient NO3− to impact physiological function.
To help fill this knowledge gap, we measured the NO3− (and NO2−) content of a wide variety of BRJ-based products, including powders, mixed drinks, concentrates, and bulk juices. Our data demonstrated that there can be significant variation between products and even between samples of the same product. Furthermore, only five of the products consistently contained more than ≥5 mmol of NO3−/serving, which based on previous research seems to be the minimal dose required to enhance exercise performance in most individuals (cf. Jones et al., 2018). These findings are in line with the results of a previous study that included a smaller number of BRJ products sold primarily in Europe (Wruss et al., 2015). Although individuals can always choose to consume larger-than-recommended amounts, potential disadvantages to doing so include increased cost, greater volume to ingest, and higher intake of oxalate. The present data are therefore likely to be highly useful to athletes and their support staff in guiding selection of BRJ supplements containing adequate amounts of NO3− (i.e., ≥5 mmol/serving). They may also help explain some of the negative and null findings in the scientific literature with respect to the effects of BRJ supplementation on exercise performance (Hoon et al., 2015; Oskarsson & McGawley, 2018). Indeed, given the possible variation even between samples of the same product, scientists should measure the NO3− content of any BRJ supplement used or at least provide the lot or batch number.
An important assumption of the present study is that any differing effects in vivo would be solely, or at least primarily, due to differences in the NO3− content of the supplements tested. In fact, beets and therefore BRJ-based products contain numerous other potentially biologically active compounds, including carotenoids, betalains, bioflavonoids, and ascorbic acid (Georgiev et al., 2010; Wruss et al., 2015). Indeed, there is some evidence that BRJ may be more effective than simple NO3− salts at reducing muscle pain after eccentric exercise (Clifford et al., 2017) or in enhancing training-induced improvements in peak oxygen consumption (Thompson et al., 2018). However, Lansley et al. (2011b) demonstrated that supplementation with NO3−-depleted BRJ does not alter metabolism or performance during exercise, indicating that the effects of BRJ are mediated primarily if not exclusively by NO3−. Furthermore, by studying only liquid BRJ products, or BRJ powders likely to be consumed as liquids, we avoided any interpretative difficulties that may have arisen had we compared liquid versus solid supplements (e.g., McDonagh et al., 2018) or included products in which BRJ was not the primary ingredient. Still, it is possible that the in vivo efficacy of the various products that we tested may differ due to differences in factors other than their NO3− content.
Along with NO3−, we also measured the NO2− concentration and hence content of the various BRJ supplements. In general, NO2− levels were quite low except for two products (from the same company) containing ∼0.2 mmol NO2−/serving. It is difficult to predict the impact that this higher NO2− content might have on in vivo responses to ingestion of these products. Previous studies of higher doses of NO2−, that is, ∼2 to ∼4 mmol, provided in the form of sodium salt, have demonstrated improvements in various measures of physical function in older individuals (Justice et al., 2015). It is not known, however, whether the smaller amount found in the present products would be sufficient to elicit comparable effects. It might be expected that these two products would have a more rapid onset of action as a result of bypassing the NO3− reduction step of the enterosalivary pathway of NO production. In line with this, McDonagh et al. (2018) recently reported that plasma NO2− levels peaked within 30 min of ingestion of one of these products (i.e., Superbeets) versus 2–3 hr after ingestion of other BRJ supplements. Unlike the other BRJ products studied, however, Superbeets did not result in a significant reduction in either systolic or mean arterial blood pressure. This is presumably due to the lower dose of NO3− provided (i.e., ∼1 vs. ∼6 mmol), which failed to sustain the initial elevation in NO2−.
In summary, we have measured the NO3− and NO2− concentration and hence content of numerous BRJ supplements. Our data reveal marked variation between different products and often even between different samples of the same product. These data should be beneficial to athletes and/or their support staff interested in implementing BRJ supplementation. They also illustrate the importance of measuring the NO3− content of any BRJ supplements used in scientific research or the putative active ingredient(s) of any nutritional supplement.
Acknowledgments
E. J. Gallardo was supported by the Diversity Scholars Research Program of the
References
Bailey, S.J., Fulford, J., Vanhatalo, A., Winyard, P.G., Blackwell, J.R., DiMenna, F.J., … Jones, A.M. (2010). Dietary nitrate supplementation enhances muscle contractile efficiency during knee-extensor exercise in humans. Journal of Applied Physiology, 109, 135–148. PubMed ID: 20466802 doi:10.1152/japplphysiol.00046.2010
Bescós, R., Feerrer-Roca, V., Galilea, P.A., Roig, A., Drobnic, F., Sureda, A., … Pons, A. (2012). Sodium nitrate supplementation does not enhance endurance performance of endurance athletes. Medicine & Science in Sports & Exercise, 44, 2400–2409. PubMed ID: 22811030 doi:10.1249/MSS.0b013e3182687e5c
Clifford, T., Howatson, G., West, D.J., & Stevenson, E.J. (2017). Beetroot juice is more beneficial than sodium nitrate for attenuating muscle pain after strenuous eccentric-bias exercise. Applied Physiology, Nutrition, and Metabolism, 42, 1185–1191. PubMed ID: 28719765 doi:10.1139/apnm-2017-0238
Coggan, A.R., Broadstreet, S.R., Mahmood, K., Mikhalkova, D., Madigan, M., Bole, I., … Peterson, L.R. (2018a). Dietary nitrate increases VO2peak and performance but does not alter ventilation or efficiency in patients with heart failure with reduced ejection fraction. Journal of Cardiac Failure, 24, 65–73. PubMed ID: 28916479 doi:10.1016/j.cardfail.2017.09.004
Coggan, A.R., Broadstreet, S.R., Mikhalkova, D., Bole, I., Leibowitz, J.L., Kadkhodayan, A., … Peterson, L.R. (2018b). Dietary nitrate-induced increases in human muscle power: High versus low responders. Physiological Report, 6, 13575. PubMed ID: 29368802 doi:10.14814/phy2.13575
Coggan, A.R., Leibowitz, J.L., Anderson Spearie, C., Kadkhodayan, A., Thomas, D.P., Ramamurthy, S., … Peterson, L.R. (2015a). Acute dietary nitrate intake improves muscle contractile function in patients with heart failure: A double-blind, placebo-controlled, randomized trial. Circulation: Heart Failure, 8, 914–920. PubMed ID: 26179185 doi:10.1161/CIRCHEARTFAILURE.115.002141
Coggan, A.R., Leibowitz, J.L., Kadkhodayan, A., Thomas, D.T., Ramamurthy, S., Spearie, C.A., … Peterson, L.R. (2015b). Effect of acute dietary nitrate intake on knee extensor speed and power in healthy men and women. Nitric Oxide, 48, 16–21. PubMed ID: 25199856 doi:10.1016/j.niox.2014.08.014
Corleto, K.A., Singh, J., Jayaprakasha, G.K., & Patil, B.S. (2018). Storage stability of dietary nitrate and phenolic compounds in beetroot (Beta vulgaris) and arugula (Eruca sativa) juices. Journal of Food Science, 83, 1237–1248. PubMed ID: 29660828 doi:10.1111/1750-3841.14129
dos Santos Baião, D., Conte-Junior, C.A., Paschoalin, V.M.F., & Alvares, T.S. (2016). Quantitative and comparative contents of nitrate and nitrite in Beta vulgaris L. by reversed-phase high-performance liquid chromatography fluorescence. Food Analytical Methods, 9, 1002–1008.
Georgiev, V.G., Weber, J., Kneschke, E.M., Denev, P.M., Bley, T., & Pavlov, A.I. (2010). Antioxidant activity and phenolic content of betalain extracts from intact plants and hair root cultures of the red beetroot Beta vulgaris cv. Detroit Dark Red. Plants Foods for Human Nutrition, 65, 105–111. PubMed ID: 20195764 doi:10.1007/s11130-010-0156-6
Hoon, M.W., Fornusek, C., Chapman, P.G., & Johnson, N.A. (2015). The effect of nitrate supplementation on muscle contraction in healthy adults. European Journal of Sport Science, 8, 712–719. PubMed ID: 26681629 doi:10.1080/17461391.2015.1053418
Jones, A.M., Thompson, C., Wylie, L.J., & Vanhatalo, A. (2018). Dietary nitrate and physical performance. Annual Review of Nutrition, 38, 303–328. PubMed ID: 30130468 doi:10.1146/annurev-nutr-082117-051622
Justice, J.N., Johnson, L.C., DeVan, A.E., Cruickshank-Quinn, C., Reisdorph, N., Bassett, C.J., … Seals, D.R. (2015). Improved motor and cognitive performance with sodium nitrite supplementation is related to small metabolite signatures: A pilot trial in middle-aged and older adults. Aging, 7, 1004–1021. PubMed ID: 26626856 doi:10.18632/aging.100842
Kharti, J., Mills, C.E., Maskell, P., Odongerel, C., & Webb, A.J. (2017). It is rocket science—Why dietary nitrate is hard to ‘beet’! Part I: Twists and turns in the realization of the nitrate–nitrite–NO pathway. British Journal of Clinical Pharmacology, 83, 129–139. PubMed ID: 26896747 doi:10.1111/bcp.12913
Lansley, K.E., Winyard, P.G., Bailey, S.J., Vanhatalo, A., Wilkerson, D.P., Blackwell, J.R., … Jones, A.M. (2011a). Acute dietary nitrate supplementation improves cycling time trial performance. Medicine & Science in Sports & Exercise, 43, 1125–1131. PubMed ID: 21471821 doi:10.1249/MSS.0b013e31821597b4
Lansley, K.E., Winyard, P.G., Fulford, J., Vanhatalo, A., Bailey, S.J., Blackwell, J.R., … Jones, A.M. (2011b). Dietary nitrate supplementation reduces the O2 cost of walking and running: A placebo-controlled study. Journal of Applied Physiology, 110, 591–600. PubMed ID: 21071588 doi:10.1152/japplphysiol.01070.2010
McDonagh, S.T.J., Wylie, L.J., Webster, J.M.A., Vanhatalo, A., & Jones, A.M. (2018). Influence of dietary nitrate food forms on nitrate metabolism and blood pressure in healthy normotensive adults. Nitric Oxide, 72, 66–74. PubMed ID: 29223585 doi:10.1016/j.niox.2017.12.001
Oskarsson, J., & McGawley, K. (2018). No individual or combined effects of caffeine and beetroot-juice supplementation during submaximal or maximal running. Applied Physiology, Nutrition, and Metabolism, 43, 697–703. PubMed ID: 29444414 doi:10.1139/apnm-2017-0547
Rimer, E.G., Peterson, L.R., Coggan, A.R., Martin, J.C. (2016). Increases in maximal cycling power with acute dietary nitrate supplementation. International Journal of Sports Physiology and Performance, 11, 715–720. PubMed ID: 26641379 doi:10.1123/ijspp.2015-0533
Santamaria, P. (2006). Nitrate in vegetables: Toxicity, content, intake and EC regulation. Journal of the Science of Food and Agriculture, 86, 10–17. doi:10.1002/jsfa.2351
Thompson, C., Vanhatalo, A., Kadach, S, Wylie, L.J., Fulford, J., Ferguson, S.K., … Jones, A.M. (2018). Discrete physiological effects of beetroot juice and potassium nitrate supplementation following 4-wk sprint interval training. Journal of Applied Physiology, 124, 1519–1528. PubMed ID: 29494294 doi:10.1152/japplphysiol.00047.2018
Troutman, A.D., Gallardo, E.J., Brown, M.B., & Coggan, A.R. (2018). Measurement of nitrate and nitrite in biopsy-sized muscle samples using HPLC. Journal of Applied Physiology. Advance online publication. doi:10.1152/japplphysiol.00625.2018
Van De Walle, G.P., & Vukovich, M.D. (2018). The effect of nitrate supplementation on exercise tolerance and performance: A systematic review and meta-analysis. The Journal of Strength and Conditioning Research, 32, 1796–1808. PubMed ID: 29786633 doi:10.1519/JSC0000000000002046
Wruss, J., Waldenburger, G., Huemer, S., Uygun, P., Lanzerstorfer, P., Müller, U., … Weghuber, J. (2015). Composition characteristics of commercial beetroot products and beetroot juice prepared from seven beetroot varieties grown in Upper Austria. Journal of Food Composition and Analysis, 42, 46–55.
