by Monica Mollica Iron Magazine
Nitric oxide (NO) boosting “pre-workout” supplements based on L-arginine (aka arginine) are currently in the rage among many athletes, particularly bodybuilders and strength athletes. While it’s true that arginine is a nitric oxide (NO) precursor and NO is a potent vasodilator 1, 2, most studies in healthy adults have not unequivocally supported the marketing hype that arginine supplementation increases muscle blood flow and/or performance in healthy folks 3-5. In my previous article “The L-Arginine Paradox” I explained why.
In this article I will cover the less well known, albeit highly significant, NO generating process, the nitrate-nitrite-NO pathway. This “new” NO producing pathway holds a lot of promise and supplements that target it will probably will replace the current arginine based NO boosters in the near future….The nitrate-nitrite-NO pathway is especially interesting in that it not only has performance enhancing effects in non-diseased people, but also offers cardiovascular protection.
The nitrate–nitrite–NO pathway
Nitric oxide production from the arginine-eNOS-NO pathway is dependent on the activity of the eNOS enzyme, which itself is controlled by other factors (which I will cover in an upcoming article). In contrast to the arginine-eNOS-NO pathway, which forms the basis of current NO boosting supplements, nitrate-nitrite-NO pathway produces NO by a mechanism that does not involve the eNOS enzyme.
NO production via the nitrate-nitrite-NO pathway starts by conversion of nitrate to nitrite, which is carried out by bacteria in the oral cavity. Once nitrite is formed, several pathways exist (deoxygenated hemoglobin/myoglobin, xanthine oxidase, respiratory chain enzymes etc) which further metabolize nitrite to NO 6, 7.
Many of the biochemical reactions that drive the nitrate-nitrite-NO pathway are greatly accelerated under hypoxic and acidic conditions, such as those occurring during high intensity exercise 8-10. This is an important point because hypoxic conditions reduce the activity of the eNOS enzyme 11, which catalyzes the production of NO from aginine. The importance of the nitrate-nitrite-NO pathway is further underscored by studies showing that nitrite supplementation restores NO supply and is cardioprotective in mice lacking the arginine-eNOS-NO pathway 12.
Thus, the nitrate-nitrite-NO pathway is complementing the better known arginine-eNOS-NO pathway for the generation of NO when the arginine-eNOS-NO pathway isn’t running smootlhy. And as recent studies are showing, the nitrate-nitrite-NO pathway appears to be of more importance to healthy people looking for a performance enhancing edge, than the arginine-eNOS-NO pathway.
Effects of nitrate supplementation on physical performance
Several studies have shown that nitrate supplementation reduces the oxygen cost for a given exercise intensity 13-18. This has practical implications because exercise economy affects tolerance to high-intensity exercise 19. Three recent studies confirm that nitrate supplementation increases tolerance to high-intensity exercise by enhancing muscle contractile efficiency and reducing muscle metabolic perturbations 13, 16, 17. Mechanistically, a central target for the effects of nitrate and its metabolites seems to be the mitochondrion 20.
In one study, healthy men were given 500 ml of a nitrate-rich beetroot juice providing 316 mg of nitrate, or placebo, 6 consecutive days. On the last 3 days they subjects completed low-intensity and high-intensity “step” exercise tests. On days 4-6, the nitrate-rich beetroot juice supplement resulted in a significant increase in blood nitrite levels that was more than double that seen with placebo. During low-intensity exercise, the nitrate-rich beetroot juice supplement attenuated the reduction in muscle phospho-creatine concentration by 35% and reduced the exercise associated oxygen cost with 25%. During high-intensity exercise, time to exhaustion was 12 min 14 sec in the nitrate supplemented group, compared to 9 min 46 sec in placebo, which corresponds to a 25% increased performance. The total ATP turnover rate was significantly less for both low-intensity and high-intensity exercise in the nitrate supplemented group, compared to placebo. It was concluded that the reduced oxygen cost of exercise following nitrate-rich beetroot juice supplementation is due to a reduced ATP cost of muscle force production 16, and that the reduced muscle metabolic perturbation (as reflected in the extent to which the muscle phospho-creatine reserve is depleted over time) allowed high-intensity exercise to be tolerated for a greater period of time 16.
Most studies also show that nitrate supplementation, when taken in amounts that sufficiently elevate blood nitrite levels, boosts performance in high-intensity exercise time trials 21-28. More specifically, a study that investigated the effects of a single dose dietary nitrate supplementation on power output and performance during 2.5 and 10 mile cycling time trials23. Club-level competitive male cyclists were assigned to consume 17 oz of beetroot juice (providing 384 mg nitrate) or 17 oz of nitrate-depleted beetroot juice as placebo, 2.5 h before the cycling trials. The results showed that the nitrate supplementation significantly increased power output during both the 2.5 and 10 mile cycling time trials, and improved 2.5 mile performance by 2.8% and 10 mile performance by 2.7%. The performance increase was attributed to an improved cycling economy, as demonstrated by a higher power output for the same oxygen consumption 23.
Another study tested whether 6 days of nitrate supplementation would improve competitive time-trial performance in trained cyclists, as it does after a single dose. Male cyclists ingested 140 ml/d of concentrated beetroot juice (496 mg nitrate per day) or a placebo (nitrate-depleted beetroot juice) for 6 d. After supplementation on day 6, subjects performed 60 min of submaximal cycling (2×30 min at 45% and 65% Wmax, respectively), followed by a 6.2 mile time trial. It was found that the nitrate supplementation lowered sub-maximal oxygen consumption, and increased time-trial performance with 12 seconds (1.2%) and power output by 2 % 22.
Intake of 200 mg baked beetroot (providing 500 mg nitrate) has been shown in healthy fit men and women to increase running velocity by 5 % during the last 1.1 miles (1.8 km) of the 5-km treadmill run, and also reduce ratings of perceived exertion during that same exercise segment 27. This finding lends credit to the mechanistic basis that the the nitrate-nitrite-NO pathway kicks in when that arginine-eNOS-NO pathway flakes out.
As of this writing, there are yet no studies on the potential effects of nitrate supplementation in conjunction with resistance exercise. But since the hypoxia and acidity stimulates the activity of the nitrate-nitrite-NO pathway, there are good reasons to believe that it will benefit anaerobic resistance exercise as well.
Nitrate intake – health promoting cardiovascular effects
It’s always exciting when performance enhancing substances also confer health benefits. Nitrate, which is found in most vegetables and is particularly abundant in leafy greens and beetroot, has emerged as one major mediating component for the cardiovascular and metabolic health benefits associated with high vegetable consumption 20, 29-38.
Nitrite, the conversion product of nitrate, is more abundant in meant (see below). While most studies on nitrite appear to show that its effects occur via generation of NO, there are also indications that nitrite itself may act as a signaling molecule and regulator of gene expression 39, and be an endocrine mediator of NO-based cellular signaling 40, 41. Nitrite itself can also perform many actions that were previously attributed to NO 42.
The most notable and widely applicable cardiovascular protection effect seen after nitrate and nitrite intake is its well documented salutary impact on blood pressure and vascular function 7, 37, 43-48. Elevated blood pressure is an established risk factor for coronary artery disease, stroke, kidney disease, all-cause mortality, and shortened life expectancy 49, 50. Furthermore, the relation between blood pressure and health consequences is progressive throughout the non-hypertensive range 51. Given these considerations, and the high prevalence of both hypertension and pre-hypertension (30% and 66% respectively, pre-hypertension defined as 120-139/80-89) 52, 53, even a small decrease in blood pressure may have a major health effect. It is notable that two thirds of the American population have pre-hypertension, and that over a third of those are undiagnosed 52. This is an important issue since hypertension causes premature aging of endothelial function and thereby accelerates development of age-related cardiovascular disease 54. Thus, dietary modification and/or supplementation to increase nitrate and nitrite intake in order to ramp up the nitrate-nitrite-NO pathway could benefit the vast majority of people, of all ages (more on that below).
In addition, dietary nitrite and nitrate have been shown to reduce inflammation, restore endothelial function, lower C-reactive protein levels, and protect from heart attack, stroke and type-2 diabetes 20, 55, 56. Nitrate intake, in a dose that corresponds to a rich intake of vegetables, has also been shown to reverse features of metabolic syndrome 38. Long term nitrate ingestion, in addition also reduces abdominal (visceral) fat accumulation and blood levels of triglycerides 38.
Nitrate versus Nitrite – dietary sources
Our diet is the main contributor to the body pool of nitrate and nitrate, with vegetables accounting for 60-85% of daily nitrate intake 30, 36. Meats (both processed and un-processed), the second food source, contain more nitrite than vegetables. Processed meat contains a little more and nitrite and nitrate than un-processed meat, but the difference is much less than what is often claimed in media 57.
A typical US diet usually provides around 40–100 mg nitrate/d 58, 59 and 1-5 mg nitrite/d 60, 61. On the basis of a conservative recommendation to consume 400 g of different fruits and vegetables per day at average nitrate concentrations, the dietary intake of nitrate would be 157 mg/d 30.
Ingested nitrate and nitrite is rapidly and completely be absorbed in the small intestine and taken up in the blood 62-65. About 25% of blood nitrate is then absorbed by the salivary glands, of which the 20% is converted to nitrite by commensal bacteria in the oral cavity 66, 67. This nitrite is then swallowed and re-enters the blood circulation, where it becomes a precursor to NO generation via the nitrate-nitrite-NO pathway 37, 64.
Without this so called entero-salivary circulation of nitrate, and the oral bacteria, nitrate would leave the body unmodified as this chemically stable anion cannot be metabolized by mammalian enzymes. However, this is a round-about-way of getting nitrite from nitrate, and up to 30% of the general population may not have the right oral bacteria to convert nitrate to nitrite 68 (which means they will not get any benefit from nitrate rich foods). Further, the necessary oral bacteria, when present, can easily be removed by anti-bacterial mouthwash, which also would deprive the nitrate-nitrite-NO pathway of NO generating substrate 69. Therefore it’s important to also ingest nitrite in order to feed the nitrate-nitrite-NO pathway. Therefore, nitrate can be considered as a pre-prodrug and nitrite as a prodrug for NO, which a substance with drug-like effects.
Even though nitrate is less toxic than nitrite 70, very large amounts of nitrite (2000-3000 mg) needs to be ingested before toxicity sets in 68. While veggies contain most nitrate and meat most nitrite, in this context intake of veggies should be prioritized because they also provide tons of other health promoting substances, which not only themselves confer health benefits, but also enhance the NO generation from dietary nitrite (regardless of whether this nitrite came from meat or veggies) 33.
As mentioned above, the primary food source of nitrite is meats, and bacterial conversion of vegetable nitrate. I have covered the health effects of meat consumption in detail in a previous article; while un-processed meats have a well deserved place in a health promoting diet, processed meats should be eaten sparingly. Not because of their nitrite content (like media warnings want us to believe), but because meat processing gives rise to other substances with detrimental health effects (e.g. advanced glycemic end products, among others). Thus, the possible processed meat-cancer association 57 has to be investigated in light of all the other compounds present in processed meat, and not unduly blame its nitrite and nitrate content. Therefore, all food sources of nitrate and nitrite are not equal with regard to potential health benefits or risks. Get your nitrate and nitrite from fresh veggies and un-processed meats, and you will reap all the health and performance benefits without having to worry.
Potential health hazards of nitrate and nitrite intake – unfounded public health regulations
Despite the beneficial effects of dietary nitrate, conservative governmental institutions and regulations perceive that dietary sources of nitrite and nitrate are harmful. The purported health risks of exposure to nitrite and nitrate are based on reports about met-hemoglobinemia in infants caused by drinks or food prepared with nitrate-rich (and bacterially contaminated) well water and vegetables such as spinach, celery, and carrots (“blue baby syndrome”), occupational intoxication, increasing nitrate levels in soil and lakes as a result of fertilizer overuse, and the formation of potentially carcinogenic N-nitrosamines. Also, some epidemiological studies have shown a weak association between cured and processed meats, which contain nitrite and nitrate, and cancer 71. This gave nitrite and nitrate a negative image, and as a result efforts have been made to remove as much nitrite and nitrate as possible from our drinking water and food. The Joint Food and Agricultural Organization/World Health Organization has set the Acceptable Daily Intake (ADI) for the nitrate at 1.7 mg/lb body weight (3.7 mg/kg) and for nitrite at 0.03 mg/lb body weight (0.06 mg/kg) 72. Likewise, the Environmental Protection Agency (EPA) has set a daily Reference Dose for nitrate at 3.2 mg/lb body weight (7 mg/kg). This amounts to 222-420 mg nitrate and 3.6 mg nitrite for a 132 lb (60-kg) individual.
All the media scare about the purported hazards if nitrate and nitrite might make it appears as if dietary nitrate and nitrite are unnatural dangerous chemicals added by the industry. This is wrong; while cultivation and fertilization does add nitrate and nitrite to produce, vegetables also naturally contain nitrate and nitrite which enters the ecosystem via the nitrogen cycle 73. Thus, dietary nitrate and nitrite are part of our natural diet, as are vitamins and minerals.
The weak and inconclusive data on the cancer risk of nitrite and nitrate 74, 75 are far outweighed by the well documented health benefits of supplementing the NO pool with dietary nitrate (and to a lesser extent nitrite). Nitrate has actually been show to have beneficial health effects at intakes that traditionally have been considered to be toxic 30.
For example, the well known health promoting and blood pressure-lowering DASH diet can provide over 1200 mg nitrate due to its high vegetable content, which exceeds the World Health Organization’s Acceptable Daily Intake for nitrate by 540% for a 132 lb (60-kg) adult. Further support comes from the traditional Japanese diet, which naturally provides 8.6 mg nitrate per lb body weight per day (1135 mg nitrate per day for a 132 lb individual) 46. If nitrate really was that bad, the traditional Japanese diet wouldn’t be fueling the population with the highest worldwide longevity.
These data call into question the rationale of recommendations to limit nitrate consumption from plant foods. Further, an effect of nitrite intake on cancer also seems less likely because large amounts of nitrite are formed in the body. Fasting saliva contains 2 mg nitrite/L, and after consumption of an amount of nitrate equivalent to 7 oz of spinach (roughly 1400 mg nitrate), the nitrite concentration in saliva may rise as high as 72 mg/L 76. That is much higher than the EPA standard for drinking water of 4.4 mg nitrite/L or the WHO Acceptable Daily Intake of 4.2 mg nitrite/d. Thus, the negative rap surrounding nitrate and nitrite needs a revision in the face of the undisputed health benefits of nitrate-enriched diets.
The nitrate-nitrite-NO pathway: a potential anti-aging tool
The potential health implications of increasing nitrate and nitrite intakes to fuel the nitrate-nitrite-NO pathway is critically important for health and function in older adults, in whom the arginine-eNOS-NO pathway is inefficient and/or dysfunctional 77-83.
Intake of nitrite is especially important in the elderly, who often have altered oral bacteria populations, dry mouth, and who take medications that af
fect stomach acidity and intestinal motility 84. This is underscored by the finding that high-nitrate supplementation might be a requirement to elevate blood levels of nitrate and nitrite in elderly, even among those who consume high-nitrate foods 84.
Aging is also characterized by endothelial dysfunction 80, 85, which is caused by disturbances in the arginine-eNOS-NO pathway and NO bioavailability, among other things 86-95. The importance of nitrite in elderly adults is underscored by the fact that endothelial dysfunction is associated with reduced blood nitrite levels, and that both reduced blood nitrite levels and endothelial dysfunction are correlated with increasing numbers of cardiovascular risk factors 96, 97. Thereby, an increased nitrite intake may provide a first line of defense against cardiovascular disease 8. Moreover, the increased exercise tolerance seen after nitrate supplementation is especially beneficial for elderly, who often cite exhaustion/fatigue as a critical barrier that prevents them from partaking in regular exercise 98, 99 . In this regard, nitrite/nitrate supplementation can indirectly help to prevent sarcopenia and frailty, which are deleterious age related conditions that primarily arise as a consequence from a sedentary lifestyle 100-103.
New nutritional paradigm
Several prominent scientists are actively reevaluating the health and performance effects of food sources of nitrates and nitrites 7, 11, 13, 15-17, 29-37, 43-47, 55, 64, 104-108; hopefully these new results be reflected in upcoming regulatory and public health guidelines for dietary nitrite and nitrate exposures.
There is a major paradigm shift occurring in regards to the health and nutritional value of nitrite and nitrate. In the right context, they can provide enormous benefit to people in all walks of life. It has even been suggested that dietary nitrate and nitrite have a role in the diet as indispensable nutrients that many people are deficient in 30, 109.
Nitrate supplementation has robust NO-like effects, including increased performance, enhanced exercise tolerance, reduced blood pressure and cardiovascular protection. Nitrite can be seen as an NO donor, or storage form of NO (which decomposes in milliseconds) 8, 29, 110, and is now at the forefront of NO biology 42.
The nitrate-nitrite-NO pathway is complementary to the better known L-arginine-NO pathway. One major difference is that production of NO from the nitrate-nitrite-NO pathway is greatly enhanced during hypoxia (low oxygen availability) and low pH, such as during exercise, which is when the L-arginine-NO pathway performs poorly. Thereby it kicks in when we need it the most.
What’s interesting is that the major supply of nitrate and nitrite in our bodies comes from our everyday diet 30. Thus, this alternative NO generating pathway can be harnessed therapeutically for prevention and treatment of diseases, while boosting performance and exercise capacity.
About the Author:
Monica Mollica has a Bachelor’s and Master’s degree in Nutrition from the University of Stockholm, Sweden, and is an ISSA Certified Personal Trainer. She works a dietary consultant, health journalist and writer for www.BrinkZone.com, and is also a web designer and videographer.
Monica has admired and been fascinated by muscular and sculptured strong athletic bodies since childhood, and discovered bodybuilding as an young teenager. Realizing the importance of nutrition for maximal results in the gym, she went for a BSc and MSc with a major in Nutrition at the University.
During her years at the University she was a regular contributor to the Swedish bodybuilding magazine BODY, and she has published the book (in Swedish) “Functional Foods for Health and Energy Balance”, and authored several book chapters in Swedish publications.
It was her insatiable thirst for knowledge and scientific research in the area of bodybuilding and health that brought her to the US. She has completed one semester at the PhD-program “Exercise, Nutrition and Preventive Health” at Baylor University Texas, at the department of Health Human Performance and Recreation, and worked as an ISSA certified personal trainer. Today, Monica is sharing her solid experience by doing dietary consultations and writing about topics related to health, fitness, bodybuilding, anti-aging and longevity.
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