Supplementation with Nitrates and Physical Activity.

Summary

The sources of nitrates that we obtain through common foods such as spinach, arugula, celery, and beetroot, or through the intake of dietary supplements, are currently considered by athletes from various specialties. It has been shown that nitrate supplementation, provided in the form of beetroot juice, gels, and other formats, improves the bioavailability of nitric oxide, thus playing an important role in modulating skeletal muscle function.

This article summarizes the most relevant aspects of nitrate-nitrite-nitric oxide metabolism, also analyzes the different dietary sources of nitrate, including supplements, and provides recommendations on how to use and when to consider nitrate supplementation in physically active people according to their performance level and the characteristics of the predominant efforts in the sports discipline.

Acute performance benefits are observed within 2-3 hours after ingestion of 6 to 16 mmol of nitrate. Supplementation protocols lasting up to 15 days and using > 8 to 16 mmol or up to 25 mmol also seem to be beneficial for performance, particularly for well-trained endurance athletes, where improvements in exercise from nitrate intake are more difficult to achieve.

The available evidence indicates very few side effects or limitations related to nitrate supplementation, including gastrointestinal discomfort in susceptible athletes. No negative effects related to possible toxicity in physically active and healthy individuals have been demonstrated. A daily intake of up to ~ 16 mmol is considered safe and well tolerated.

Introduction

The consumption of foods rich in inorganic nitrate (N0₃^–) such as beetroot, spinach, arugula, or amaranth leaves has been associated with health benefits and beneficial effects in the treatment of diseases (e.g., hypertension).¹ A higher intake of N0₃^– increases the concentration of nitrite (N0₂^–) in the blood, which leads to greater production of nitric oxide (NO·), a molecule that induces smooth muscle relaxation,² promotes vasodilation, and improves oxygen supply to tissues. The increase in NO· production has been associated with improved physical work efficiency, modulation of skeletal muscle function, calcium homeostasis, improvements in mitochondrial respiration, and mitochondrial biogenesis.³ In fact, studies in physically active individuals suggest that increasing dietary intake of N0₃^– through vegetables or supplements significantly benefits exercise-induced adaptations (e.g., endurance, hypertrophy, etc.) by improving muscle activation, glucose metabolism, and myoblast differentiation.⁴

Summary of nitrate-nitrite-nitric oxide metabolism

The N0₃^– exogenous is inert, but after being ingested, it is rapidly absorbed by the stomach and small intestine and then enters the bloodstream. Once in the salivary glands, the N0₃^– re-enters the oral cavity through protein transporters and concentrates in the saliva. On the dorsal part of the tongue, an anaerobic bacterium reduces about 20 to 25% of the salivary N0₃^– in N0₂^– that is used for bacterial respiration⁵.A part of the N0₂^– is converted into NO· and other reactive nitrogen species in the acidic environment of the stomach, but the remaining N0₂^– enters the small intestine to be absorbed into the blood, where it can be transported to organs and tissues such as the liver or skeletal muscle⁶ for storage or conversion into NO· that circulates in the blood if necessary.⁵ The plasma concentration of N0₃^– and N0₂^– reaches its maximum peak after 1 to 2 h or 2 and 3 h, respectively, after ingestion, then gradually decreases and returns to baseline values in approximately 24 h.² The factors that interfere with the metabolism of salivary N0₃^–, such as the use of mouthwashesantibacterial mouthwashes to reduce the levels of bacteria in the mouth can significantly limit the availability of N0₂^– in the plasma and consequently attenuate its physiological effects.⁷

Parallel to the exogenous pathway (N0₃^– → N0₂^– → NO·), NO· is also generated endogenously by the reaction catalyzed by the enzyme nitric oxide synthase (eNOS), which uses L-Arginine as its main substrate. NO· production is accentuated when tissue oxygen availability is low, and it is possible that reliance on the exogenous pathway may increase during exercise (since eNOS requires oxygen to be active)². In addition, it has been shown that the ingestion of a non-essential amino acid, L-citrulline, promotes NO· production by converting into L-arginine in the kidneys (from novel arginine synthesis).⁸ However, both L-arginine and NO production were not affected by supplementation with arginine alone.⁹ On the other hand, the effectThe synergistic effect of L-citrulline intake to maximize physical performance is due to its function as an anabolic pharmaco-nutrient that promotes protein synthesis, enhances athletic performance, reduces muscle damage caused by intense and prolonged exercise, and improves recovery.⁸

Dietary sources of nitrate

As mentioned above, N0₃^– is abundant in leafy green vegetables (Figure 1). The best sources of N0₃^– are those that provide more than 1000 (high) or 2500 (very high) mg per kg of fresh vegetable (e.g., beet juice, arugula, and amaranth leaves), while those that provide between 500 and 1000 mg, such as chard or beetroot, and less than 500 mg (e.g., cucumber) are considered moderate or low sources.¹⁰

Figure 1. Inorganic nitrate concentration in plant sources. Adapted from the references. ^10,11

Notes: N0₃^– conversion from mg to mmol: 1 mmol = 62.5 mg; 8 mmol = 500 mg; 16 mmol = 1000 mg

The content of N0₃^– from a specific plant source can vary considerably from one plant to another and will depend on factors such as climate, soil conditions, and the time elapsed since harvest. The average dietary intake in adults in the U.S., Europe, and Australia is ~60-120 mg per day (1-2 mmol), with vegetables providing around 80% of the total daily intake of N0₃^–. People who follow a plant-based diet (e.g., vegetarians) are likely to consume higher amounts of nitrate.¹²

Although arugula and amaranth leaves contain high amounts of N0₃^–, the most recognized natural source of N0₃^– is beetroot (Beta vulgaris), which also includes betaine and betanins (betacyanins). Betanins are responsible for the red color of beetroot.¹³ However, as shown in Figure 1, other plants can contribute. Thus, vegetables such as green spinach contain N0₃^–, betaine, and polyphenols (mainly flavonoids); in these cases, it is worth noting that the concentration of flavonoids and vitamin C is inversely proportional to the content of N0₃^–.¹³

Although the recommended daily intake of N0₃^– could be obtained through vegetables with high or very high concentration (Figure 1), it seems that the preparation of beetroot-based sources, such as cooked vegetables, seasonings, and juices, would not allow reaching a sufficiently high nitrate dose to improve sports performance.¹⁴ Therefore, the intake of supplements that provide a higher concentration of N0₃^– alone or combined with synergistic ingredients such as L-citrulline is a common practice among many athletes.¹⁴

Supplementation with nitrates in physically active people.

It has been shown that supplementation with inorganic N0₃^– amplifies some of the effects of NO·, even in healthy people. Although the International Society of Sports Nutrition considered N0₃^– administered in the form of beetroot juice or sodium nitrate within the group of supplements with limited or mixed scientific evidence for improving sports performance, ³ the International Olympic Committee included supplementation with N0₃^– within the category of supplements “With good to strong evidence that directly improve sports performance when used in specific scenarios.” ¹⁵ In addition, the Australian Institute of Sport categorizes supplementation with N0₃^– particularly through the intake of beetroot juice in Category A, includingthe supplements or ingredients that “may support or improve athletic performance.”¹⁶

Current research supports the benefits of oral supplementation with N0₃^– to improve performance during events lasting more than 3 minutes¹⁷, strength, power, and speed exercises⁵, as well as in sports characterized by intermittent efforts (e.g., team sports).¹⁸ Additionally, due to the ability of N0₃^– to promote cerebral blood flow, its administration appears to acutely improve cognitive function (e.g., memory capacity) in humans.¹⁹

The benefits of supplementation with N0₃^– in endurance sports have been proven in people who are lightly (VO₂peak < 45 ml/kg/min) to moderately trained (VO₂peak > 45 to < 60 ml/kg/min), mainly males.²⁰ However, this ergogenic effect seems to be reduced in well-trained athletes (VO₂peak > 60 ml/kg/min).²⁰ In this regard, it is worth noting that when analyzing the results of studies using protocols in which participants are required to cover a distance as quickly as possible, the small improvements observed, although not statistically significant (~0,8 %), could still be relevant in a contextsports, especially in high performance where the differences between winners and losers competing at an international level (for example, the Olympic Games) are generally no greater than 1%.²¹

Nitrate supplementation: format and administration protocols

Beet juice is the most common source of N0₃^– used in supplements.^16,22 The ergogenic effects of N0₃^– ingested through natural dietary sources, supplements like beet juice, or salts such as sodium or potassium were observed after a single intake (2-3 h before exercise) as well as after a supplementation period with a daily dose for 2 to 15 days.Currently, > 6 mmol (> 375 mg) of N0₃^– are accepted as the minimum necessary dose to improve health and optimize performance in physically active people.⁵ However, in athletes with higher performance levels, doses of 8 to 16 (500 to 1000 mg) mmol are recommended acutely or 4 to 16 (250 to 1000 mg) mmol per day regularly ingested (with the final dose taken 2-3 h before exercise or competition).²⁰ Because well-trained athletes have higher basal plasma levels of N0₂^– and N0₃^–, higher doses close to 16 mmol (1000mg)²³ or up to 25 (1562 mg)²³ mmol per day to obtain the desired ergogenic effects. The antioxidants (flavonoids, betalains, and ascorbic acid) present in beet juice compared to salts could influence the efficacy of supplementation.²³ Most N0₃^– supplements designed from beetroot are presented in the form of gel, tablets, juice, chips, cereal bars, and, more recently, as chewables. Regardless of the formulation, all N0₃^– supplements must provide concentrations that allow achieving the desired effects.For example, 9,4 mmol (600 mg) of dietary N0₃^– can be obtained with the intake of a 32 g gel, while lower doses of approximately 6 mmol could be consumed with a 200 ml dose of beetroot juice, or cereal bars.²⁴ It is worth mentioning that gel-form supplements are easier to drink and tolerate for athletes before, during, and after exercise.²²

According to the available scientific evidence, the following supplementation protocols are recommended for physical performance level and sports discipline.

• For healthy young people, recreationally trained in all forms of exercise or sports disciplines, the recommended daily dose is > 6 to 16 mmol.²⁰
• For well-trained endurance athletes, the recommended daily dose is > 8 to 16 or exceptionally up to 25 mmol (600-1000 up to 1550 mg).^20,25
• Supplements should be taken 2 to 3 hours before training or competitions at least one day (acute effect); 2 to 6 days (short-term protocols) or for 7 to 15 days (accumulated effects).²⁰

Possible adverse effects of nitrate supplementation

Although increasing the intake of N0₃^– from regular foods or supplements could be beneficial in the context of physical activity, the acceptable daily intake (the maximum amount that can be consumed daily over a lifetime without increasing the risk of adverse events) remains relatively low, 3,7 mg/kg of body weight (300 mg for a person weighing 81 kg).¹³ This amount can easily be exceeded with a diet rich in vegetables or with the ingestion of nitrate supplements, which is common among endurance athletes.²⁰ However, the only side effects reported so far in healthy and physically active individuals are the following:

• Pink or red coloration (beeturia) of urine and feces. This is a harmless and temporary side effect.
• Possibility of intolerance or gastrointestinal discomfort in susceptible athletes.
• The production of nitrosamines from dietary sources of N0₃^– – N0₂^– is very unlikely!

Despite some concerns related to the possible toxicity of N0₃^–, a daily intake of up to ~ 16 mmol has been considered safe and well tolerated.²⁰

Author

Dr. Fernando Naclerio
Professor in Strength Training and Sports Nutrition
Centre for Exercise Activity and Rehabilitation
Institute for Lifecourse Development
School of Human Sciences
University of Greenwich
Contact email: f.j.naclerio@gre.ac.uk

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