Nitrate Supplementation Research Articles

Acute dietary nitrate supplementation does not change venous volume and compliance in healthy young adults.

In this crossover study, we investigated the influence of inorganic nitrate ([Formula: see text]) supplementation on venous volume and compliance in the resting forearm and calf. Twenty healthy young adults were assigned to receive an [Formula: see text]-rich beverage [beetroot juice (BRJ): 140 mL; ∼8 mmol [Formula: see text]] or an [Formula: see text]-depleted control beverage [prune juice (CON): 166 mL; < 0.01 mmol [Formula: see text]). Two hours after consuming the allocated beverage, each participant rested in the supine position for 20 min. Cuffs were then placed around the right upper arm and right thigh, inflated to 60 mmHg for 8 min, and then decreased to 0 mmHg at a rate of 1 mmHg/s. During inflation and deflation of cuff pressure, changes in venous volume in the forearm and calf were measured by venous occlusion plethysmography. Venous compliance was calculated as the numerical derivative of the cuff pressure-venous volume curve in the limbs. The plasma [Formula: see text] concentration was elevated by intake of BRJ (before, 15.5 ± 5.8 µM; after, 572.0 ± 116.1 µM, P < 0.05) but not by CON (before, 14.8 ± 7.2 µM; after, 15.3 ± 7.4 µM, P > 0.05). On the other hand, there was no significant difference in venous volume or compliance in the forearm or calf between BRJ and CON. These findings suggest that although acute inorganic NO3- supplementation may enhance the activity of nitric oxide (NO) via nitrite → NO pathway, it does not influence venous volume or compliance in the limbs in healthy young adults.

Acute Beetroot Juice Supplementation Enhances Intermittent Running Performance but Does Not Reduce Oxygen Cost of Exercise among Recreational Adults.

Nitrate (NO3−) supplementation has been reported to enhance intermittent exercise performance; however, its impact on oxygen (O2) cost during intermittent running exercise is unclear. The aim of this study was to assess if acute NO3− supplementation would elicit performance benefits in recreationally active individuals during the Yo–Yo intermittent recovery level 1 (Yo-Yo IR1) test, with its potential benefit on O2 consumption (VO2), in a double-blind, randomized, crossover study, 12 recreational males consumed NO3−-rich (NIT; ~12.8 mmol), and NO3−-depleted (PLA; 0.04 mmol) concentrated beetroot juice 3 h before completing the Yo-Yo IR1 test. VO2 was measured at 160, 280 and 440 m (sub-maximal) and when the test was terminated (peak). Performance in the Yo–Yo IR1 was greater with NIT (990 ± 442.25 m) compared to PLA (870 ± 357.4 m, p = 0.007). The VO2 was not significantly different at 160 m (1.92 ± 0.99 vs. 2.1 ± 0.88 L·min−1), 280 m (2.62 ± 0.94 vs. 2.83 ± 0.94 L·min−1), 440 m (3.26 ± 1.04 vs. 3.46 ± 0.98 L·min−1) and peak (4.71 ± 1.01 vs. 4.92 ± 1.17 L·min−1) between NIT and PLA trials (all p > 0.05). The present study has indicated that acute supplementation of NO3− enhanced intermittent running performance but had no effect on VO2 during the Yo–Yo IR1 test in recreational young adults.

Influence of nitrate supplementation on in-vitro methane emission, milk production, ruminal fermentation, and microbial methanotrophs in dairy cows fed at two forage levels

Abstract Modifying the chemical composition of a diet can be a good strategy for reducing methane emission in the rumen. However, this strategy can have adverse effects on the ruminal microbial flora. The aim of our study was to reduce methane without disturbing ruminal function by stimulating the growth and propagation of methanotrophs. In this study, we randomly divided twenty multiparous Holstein dairy cows into 4 groups in a 2×2 factorial design with two forage levels (40% and 60%) and two nitrate supplementation levels (3.5% and zero). We examined the effect of experimental diets on cow performance, ruminal fermentation, blood metabolites and changes of ruminal microbial flora throughout the experimental period (45-day). Additionally, in vitro methane emission was evaluated. Animals fed diet with 60% forage had greater dry matter intake (DMI) and milk fat content, but lower lactose and milk urea content compared with those fed 40% forage diet. Moreover, nitrate supplementation had no significant effect on DMI and milk yield. Furthermore, the interactions showed that nitrate reduces DMI and milk fat independently of forage levels. Our findings showed that nitrate can increase ammonia concentration, pH, nitrite, and acetate while reducing the total volatile fatty acids concentration, propionate, and butyrate in the rumen. With increasing nitrate, methane emission was considerably decreased possibly due to the stimulated growth of Fibrobacteria, Proteobacteria, type II Methanotrophs, and Methanoperedense nitroreducens, especially with high forage level. Overall, nitrate supplementation could potentially increase methane oxidizing microorganisms without adversely affecting cattle performance.

Significance of Inorganic Nitrate Supplement in Cardiovascular Health.

Nitric Oxide (NO) is frequently produced by the enzyme nitric oxide synthase (NOS) and is crucial for the control and effectiveness of the cardiovascular system. However, there is a substantial reduction in NOS activity with aging that can lead to the development of hypertension and other cardiovascular obstacles. Fortunately, NO can also be produced by sequential reduction of inorganic nitrates supplementation. This proves that NO from inorganic nitrate supplements can compensate for inadequate NOS activity, thus providing cardio protective benefits. This review focuses on the general information about nitrous oxide, its types and mechanism of action and the fact that overview of inadequate NOS activity for cardio protective benefits was often studied for cardiovascular treatments. We concluded that the natural plant NO is essential for cardiovascular activity to target site with desired concentration. Moreover, the researchers focused on evidence that suggested that nitrate supplementation could help regulate blood pressure, limit progression of atherosclerosis, and improve myocardial contractility in both healthy individuals and those with cardiovascular diseases.

Dietary Supplements for Athletic Performance in Women: Beta-Alanine, Caffeine, and Nitrate.

Beta-alanine, caffeine, and nitrate are dietary supplements generally recognized by the sport and exercise science community as evidence-based ergogenic performance aids. Evidence supporting the efficacy of these supplements, however, is greatly skewed due to research being conducted primarily in men. The physiological differences between men and women, most notably in sex hormones and menstrual cycle fluctuations, make generalizing male data to the female athlete inappropriate, and potentially harmful to women. This narrative review outlines the studies conducted in women regarding the efficacy of beta-alanine, caffeine, and nitrate supplementation for performance enhancement. Only nine studies on beta-alanine, 15 on caffeine, and 10 on nitrate in healthy women under the age of 40years conducted in normoxia conditions were identified as relevant to this research question. Evidence suggests that beta-alanine may lower the rate of perceived exertion and extend training bouts in women, leading to greater functional adaptations. Studies of caffeine in women suggest the physiological responder status and caffeine habituation may contribute to caffeine's efficacy, with a potential plateau in the dose-response relationship of performance enhancement. Nitrate appears to vary in influence based on activity type and primary muscle group examined. However, the results summarized in the limited literature for each of these three supplements provide no consensus on dosage, timing, or efficacy for women. Furthermore, the literature lacks considerations for hormonal status and its role in metabolism. This gap in sex-based knowledge necessitates further research on these ergogenic supplements in women with greater considerations for the effects of hormonal status.

Sources of rumen enhancers including nitrate, chitosan extract and shrimp shell meal could modulate nutrient degradability and in vitro gas fermentation

ABSTRACT The objective of this research was to investigate the effects of nitrate, chitosan extract or shrimp shell meal on in vitro degradability and gas production kinetics. The study was arranged in a 2 × 7 factorial arrangement in a completely randomized design (CRD). Factor A was two ratios of roughage (R) to concentrate (C) ratio, R:C ratios at 60:40 and 40:60 (R:C), and the other factors were as several kinds and levels of additives (Non: non supplementation; Low-Ni: supplementation with 7 mg of KNO3; High-Ni: supplementation with 14 mg of KNO3; Low-CE: supplementation with 1.6 mg of chitosan extract (CE); High-CE: supplementation with 4.8 mg of chitosan extract; Low-SSM: supplementation with 8 mg of shrimp shell meal (SSM) and High-SSM: supplementation with 24 mg of SSM). Cumulative gas production was significantly decreased in the supplemented group. When increasing the level of a concentrate feed, propionate (C3) and total volatile fatty acid were enhanced, while acetate (C2) and C2 to C3 ratios were reduced (P < 0.05). The C3 and total VFA concentrations were increased with the supplementation of nitrate, chitosan extract and shrimp shell meal. CH4 production by nitrate, chitosan extract and shrimp shell meal addition was found to be lower than that of the control treatment.

Dietary nitrate increases submaximal SERCA activity and ADP transfer to mitochondria in slow-twitch muscle of female mice.

Rapid oscillations in cytosolic calcium (Ca2+) coordinate muscle contraction, relaxation, and physical movement. Intriguingly, dietary nitrate decreases ATP cost of contraction, increases force production, and increases cytosolic Ca2+, which would seemingly necessitate a greater demand for sarcoplasmic reticulum Ca2+ ATPase (SERCA) to sequester Ca2+ within the sarcoplasmic reticulum (SR) during relaxation. As SERCA is highly regulated, we aimed to determine the effect of 7-day nitrate supplementation (1 mM via drinking water) on SERCA enzymatic properties and the functional interaction between SERCA and mitochondrial oxidative phosphorylation. In soleus, we report that dietary nitrate increased force production across all stimulation frequencies tested, and throughout a 25 min fatigue protocol. Mice supplemented with nitrate also displayed an ∼25% increase in submaximal SERCA activity and SERCA efficiency (P = 0.053) in the soleus. To examine a possible link between ATP consumption and production, we established a methodology coupling SERCA and mitochondria in permeabilized muscle fibers. The premise of this experiment is that the addition of Ca2+ in the presence of ATP generates ADP from SERCA to support mitochondrial respiration. Similar to submaximal SERCA activity, mitochondrial respiration supported by SERCA-derived ADP was increased by ∼20% following nitrate in red gastrocnemius. This effect was fully attenuated by the SERCA inhibitor cyclopiazonic acid and was not attributed to differences in mitochondrial oxidative capacity, ADP sensitivity, protein content, or reactive oxygen species emission. Overall, these findings suggest that improvements in submaximal SERCA kinetics may contribute to the effects of nitrate on force production during fatigue.NEW & NOTEWORTHY We show that nitrate supplementation increased force production during fatigue and increased submaximal SERCA activity. This was also evident regarding the high-energy phosphate transfer from SERCA to mitochondria, as nitrate increased mitochondrial respiration supported by SERCA-derived ADP. Surprisingly, these observations were only apparent in muscle primarily expressing type I (soleus) but not type II fibers (EDL). These findings suggest that alterations in SERCA properties are a possible mechanism in which nitrate increases force during fatiguing contractions.

Plasma Nitrate and Nitrite Kinetics after Single Intake of Beetroot Juice in Adult Patients on Chronic Hemodialysis and in Healthy Volunteers: A Randomized, Single-Blind, Placebo-Controlled, Crossover Study

Nitric oxide (NO) contributes to maintaining normal cardiovascular and renal function. This bioactive signalling molecule is generally formed enzymatically by NO synthase in the vascular endothelium. NO bioactivity can also be attributed to dietary intake of inorganic nitrate, which is abundant in our diet, especially in green leafy vegetables and beets. Ingested nitrate is reduced to nitrite by oral commensal bacteria and further to NO systemically. Previous studies have shown that dialysis, by means of removing nitrate and nitrite from the body, can reduce NO bioactivity. Hence, dietary intervention approaches aimed to boost the nitrate–nitrite–NO pathway may be of benefit in dialysis patients. The purpose of this study was to examine the kinetics of plasma nitrate and nitrite after a single intake of nitrate-rich concentrated beetroot juice (BJ) in adult hemodialysis (HD) patients and in age-matched healthy volunteers (HV). Eight HD patients and seven HV participated in this single center, randomized, single-blind, placebo-controlled, crossover study. Each participant received a sequential single administration of active BJ (70 mL, 400 mg nitrate) and placebo BJ (70 mL, 0 mg nitrate) in a random order separated by a washout period of seven days. For the kinetic analysis, blood samples were collected at different time-points before and up to 44 h after BJ intake. Compared with placebo, active BJ significantly increased plasma nitrate and nitrite levels both in HD patients and HV. The area under the curve and the maximal concentration of plasma nitrate, but not of nitrite, were significantly higher in HD patients as compared with HV. In both groups, active BJ ingestion did not affect blood pressure or plasma potassium levels. Both BJs were well tolerated in all participants with no adverse events reported. Our data provide useful information in planning dietary nitrate supplementation efficacy studies in patients with reduced NO bioactivity.

Prior beetroot juice ingestion offsets endothelial dysfunction following prolonged sitting.

Nutritional strategies to prevent endothelial dysfunction following prolonged sitting remain largely unknown. Given that beetroot juice (BRJ) ingestion enhances nitric oxide (NO) bioavailability, we aimed to evaluate whether prior BRJ ingestion would prevent sitting-induced endothelial dysfunction in the leg. Eleven healthy young males (n = 7) and females (n = 4) underwent two experimental trials of prolonged sitting with prior: 1) placebo (PL) ingestion (PL trial) and 2) BRJ ingestion (BRJ trial). All subjects ingested 140 mL of PL or BRJ (∼0.0055 or ∼12.8 mmol of nitrate, respectively) immediately before 3 h of sitting. Pre- and post-sitting measurements of popliteal artery flow-mediated dilation (FMD) and blood pressure, and blood collection were undertaken. During the sitting period, an hourly assessment of popliteal artery diameter and blood velocity, blood pressure, and blood collection were performed. Popliteal artery blood flow and shear rate were significantly and similarly reduced during the sitting period in both trials (P < 0.001). Plasma nitrate and NOx (total nitrite and nitrate) concentrations were significantly increased relative to baseline in the only BRJ trial, and the overall concentrations were significantly higher in the BRJ trial (P < 0.001). Popliteal artery FMD was significantly reduced after the sitting period in the PL trial (P < 0.05), whereas no reduction was observed in the BRJ trial. Therefore, prior BRJ ingestion would prevent sitting-induced leg endothelial dysfunction via enhancing NO bioavailability.NEW & NOTEWORTHY The present study elucidates that beetroot juice ingestion before prolonged sitting offsets sitting-induced leg endothelial dysfunction. Data from the present study provides novel physiological information that enhancing NO bioavailability by dietary nitrate supplementation is an effective tool for prevention of the detrimental vascular effects of prolonged sitting.

Dietary Inorganic Nitrate as an Ergogenic Aid: An Expert Consensus Derived via the Modified Delphi Technique

IntroductionDietary inorganic nitrate is a popular nutritional supplement, which increases nitric oxide bioavailability and may improve exercise performance. Despite over a decade of research into the effects of dietary nitrate supplementation during exercise there is currently no expert consensus on how, when and for whom this compound could be recommended as an ergogenic aid. Moreover, there is no consensus on the safe administration of dietary nitrate as an ergogenic aid. This study aimed to address these research gaps.MethodsThe modified Delphi technique was used to establish the views of 12 expert panel members on the use of dietary nitrate as an ergogenic aid. Over three iterative rounds (two via questionnaire and one via videoconferencing), the expert panel members voted on 222 statements relating to dietary nitrate as an ergogenic aid. Consensus was reached when > 80% of the panel provided the same answer (i.e. yes or no). Statements for which > 80% of the panel cast a vote of insufficient evidence were categorised as such and removed from further voting. These statements were subsequently used to identify directions for future research.ResultsThe 12 panel members contributed to voting in all three rounds. A total of 39 statements (17.6%) reached consensus across the three rounds (20 yes, 19 no). In round one, 21 statements reached consensus (11 yes, 10 no). In round two, seven further statements reached consensus (4 yes, 3 no). In round three, an additional 11 statements reached consensus (5 yes, 6 no). The panel agreed that there was insufficient evidence for 134 (60.4%) of the statements, and were unable to agree on the outcome of the remaining statements.ConclusionsThis study provides information on the current expert consensus on dietary nitrate, which may be of value to athletes, coaches, practitioners and researchers. The effects of dietary nitrate appear to be diminished in individuals with a higher aerobic fitness (peak oxygen consumption [V̇O2peak] > 60 ml/kg/min), and therefore, aerobic fitness should be taken into account when considering use of dietary nitrate as an ergogenic aid. It is recommended that athletes looking to benefit from dietary nitrate supplementation should consume 8–16 mmol nitrate acutely or 4–16 mmol/day nitrate chronically (with the final dose ingested 2–4 h pre-exercise) to maximise ergogenic effects, taking into consideration that, from a safety perspective, athletes may be best advised to increase their intake of nitrate via vegetables and vegetable juices. Acute nitrate supplementation up to ~ 16 mmol is believed to be safe, although the safety of chronic nitrate supplementation requires further investigation. The expert panel agreed that there was insufficient evidence for most of the appraised statements, highlighting the need for future research in this area.Graphical

Genetic Evidence Reveals the Indispensable Role of the rseC Gene for Autotrophy and the Importance of a Functional Electron Balance for Nitrate Reduction in Clostridium ljungdahlii.

For Clostridium ljungdahlii, the RNF complex plays a key role for energy conversion from gaseous substrates such as hydrogen and carbon dioxide. In a previous study, a disruption of RNF-complex genes led to the loss of autotrophy, while heterotrophy was still possible via glycolysis. Furthermore, it was shown that the energy limitation during autotrophy could be lifted by nitrate supplementation, which resulted in an elevated cellular growth and ATP yield. Here, we used CRISPR-Cas12a to delete: (1) the RNF complex-encoding gene cluster rnfCDGEAB; (2) the putative RNF regulator gene rseC; and (3) a gene cluster that encodes for a putative nitrate reductase. The deletion of either rnfCDGEAB or rseC resulted in a complete loss of autotrophy, which could be restored by plasmid-based complementation of the deleted genes. We observed a transcriptional repression of the RNF-gene cluster in the rseC-deletion strain during autotrophy and investigated the distribution of the rseC gene among acetogenic bacteria. To examine nitrate reduction and its connection to the RNF complex, we compared autotrophic and heterotrophic growth of our three deletion strains with either ammonium or nitrate. The rnfCDGEAB- and rseC-deletion strains failed to reduce nitrate as a metabolic activity in non-growing cultures during autotrophy but not during heterotrophy. In contrast, the nitrate reductase deletion strain was able to grow in all tested conditions but lost the ability to reduce nitrate. Our findings highlight the important role of the rseC gene for autotrophy, and in addition, contribute to understand the connection of nitrate reduction to energy metabolism.

The effects of dietary nitrate supplementation in post‐menopausal women: Does timing matter?

There is a need for low risk, non‐estrogen therapies for cardiovascular disease prevention in post‐menopausal women, a population expected to reach 1.2 billion worldwide by 2030. Consumption of beetroot juice and other nitrate‐rich products (green leafy vegetables, supplements) is a promising non‐pharmaceutical strategy for increasing circulating nitric oxide levels and improving vascular function in post‐menopausal women. Whether time since menopause (reproductive age) influences the vasoprotective effects (arterial stiffness and endothelial function) of dietary nitrate supplementation has not been established.PURPOSETo determine the vascular protective mechanisms and efficacy of a nitric oxide boosting supplement (beetroot juice) in women at two post‐ menopausal stages (early‐ vs late‐post menopause).METHODSEarly‐ (1‐6 years following their final menstrual cycle, n=5) and late‐ (n=10) postmenopausal women consumed nitrate‐rich (300 mg/70 mL) and nitrate‐depleted beet root juice acutely and for 7 days. Percent brachial artery flow‐mediated dilation (FMD) was measured at baseline, acutely (90 minutes post‐BRJ consumption), and 15‐minutes and 30‐minutes after 20‐minute upper arm ischemia‐reperfusion (IR) injury for each drink. Brachial‐ankle pulse wave velocity (baPWV) was measured pre‐ and post‐ 7‐day supplementation.RESULTSDue to the nature of this double‐blind trial, data presented are from the baseline (pre‐juice consumption) visit only. Significant (p&lt;0.05) group differences were observed for baseline PWV (early = 1158+115 cm/s; late = 1469+296 cm/s) and FMD at the 30‐minute post‐IR injury timepoint (early = 8.4+4.6%; late = 5.8+2.7%). No group differences were observed in FMD responses at rest (p=0.2) or 15 minutes post‐IR injury (p=0.4).CONCLUSIONThese preliminary results suggest that time since menopause negatively influences arterial stiffness and may prolong recovery from endothelial IR injury in women.

Dietary nitrate supplementation attenuates the mechanoflex in older women with early menopause: A pilot study

Women who reached early menopause (EM, 40‐44 yrs) have a greater risk for cardiovascular disease than women with normal menopause (NM, 50‐54 yrs), which may be attributed to a longer duration of estrogen loss and a greater reduction in nitric oxide bioavailability in both the peripherals and center. Previous studies have shown that dietary nitrate supplementation improves cardiovascular function in older women, but how autonomic neural control changes in daily living, such as during exercise, has been involved is unclear. We examined the hypothesis that 2wks continuous use of dietary nitrate supplements would attenuate the exercise pressor reflex in older women with EM.Six EM (65±4 [SD] yrs) and 8 NM (64±2 yrs) women participated in this randomized, placebo‐controlled, double‐blind, cross‐over pilot study. All subjects underwent 2 wks of beetroot juice (BRJ; 140 ml containing ~800 mg nitrate per day) or placebo (140 ml nitrate‐depleted BRJ, daily) intervention with ≥14 days of wash‐out. Before and after each intervention period, subjects performed SHG using the dominant hand at 40% of maximum voluntary contraction (MVC) until fatigue, followed by 2 min of PECA with an upper arm cuff inflated to 250 mmHg. Heart rate (HR; ECG), blood pressure (BP; finger photoplethysmography), muscle sympathetic nerve activity (MSNA; microneurography), and respiration (capnography) were continuously measured. In addition, 24‐h ambulatory BP standard deviation (SD) was monitored before and after each intervention (an indication of BP variability).There were no differences in baseline HR, BP and MSNA, MVC, and the time to fatigue between the EM and NM groups before and after intervention (all p&gt;0.05). At the first 30 s of SHG, the EM group showed significant decreases in MSNA burst frequency (DBF; ‐4.5±6.5 in EM vs. 2.5±6.2 bursts/min in NM, p=0.065) and burst incidence (DBI; ‐10.6±12.6 vs. 2.6±8.4 bursts/100 heartbeats, p=0.037) after BRJ intervention compared with the NM group (Figure1A and B). However, there were no differences between groups at fatiguing exercise and during PECA after the intervention. The interventional changes in 24‐h diastolic BP SD had a positive correlation with ΔMSNA BF (r=0.55, p=0.053) and BI (r=0.58, p=0.036) at the first 30 s of SHG in all subjects and EM individuals were in the left‐lower area (Figure2 A and B).These results suggest that dietary nitrate supplementation may attenuate and mechanoreflex, but not the metaboreflex, in older women with EM during static hand grip, which has a relation with BP variability during ambulation. Certainly, these preliminary observations need to be verified in more older women in future studies.

Effect of nitrate supplementation on skeletal muscle motor unit activity during isometric blood flow restriction exercise

BackgroundNitrate (NO3−) supplementation has been reported to lower motor unit (MU) firing rate (MUFR) during dynamic resistance exercise; however, its impact on MU activity during isometric and ischemic exercise is unknown.PurposeTo assess the effect of NO3− supplementation on knee extensor MU activities during brief isometric contractions and a 3 min sustained contraction with blood flow restriction (BFR).MethodsSixteen healthy active young adults (six females) completed two trials in a randomized, double-blind, crossover design. Trials were preceded by 5 days of either NO3− (NIT) or placebo (PLA) supplementation. Intramuscular electromyography was used to determine the M. vastus lateralis MU potential (MUP) size, MUFR and near fibre (NF) jiggle (a measure of neuromuscular stability) during brief (20 s) isometric contractions at 25% maximal strength and throughout a 3 min sustained BFR isometric contraction.ResultsPlasma nitrite (NO2−) concentration was elevated after NIT compared to PLA (475 ± 93 vs. 198 ± 46 nmol L−1, p < 0.001). While changes in MUP area, NF jiggle and MUFR were similar between NIT and PLA trials (all p > 0.05), MUP duration was shorter with NIT compared to PLA during brief isometric contractions and the sustained ischemic contraction (p < 0.01). In addition, mean MUP duration, MUP area and NF jiggle increased, and MUFR decreased over the 3 min sustained BFR isometric contraction for both conditions (all p < 0.05).ConclusionsThese findings provide insight into the effect of NO3− supplementation on MUP properties and reveal faster MUP duration after short-term NO3− supplementation which may have positive implications for skeletal muscle contractile performance.

Nitrate supplementation at two forage levels in dairy cows feeding: milk production and composition, fatty acid profiles, blood metabolites, ruminal fermentation, and hydrogen sink

Abstract Nitrate may reduce the ruminal methane emission by competing methanogenesis to achieve more hydrogen. For this purpose, twenty Holstein lactating cows were examined using a 2×2 factorial design in 4 groups for 60 days with two forage levels (40% and 60%) and supplemental nitrate 0% (F40 and F60) and 3.5% (F40N and F60N) of diet dry matter (DM). Then, the effect of nitrate and forage levels on cow performance, ruminal fermentation, methane emission, and metabolic hydrogen sink were evaluated. The nitrate supplementation did not significantly affect milk yield and ECM/DMI, while milk urea nitrogen was increased. Lowest quantity of milk vitamins (A and E) was observed in nitrate groups. The nitrate supplementation increased c9-C18:1, unsaturated fatty acids, and n-6/n-3 contents of the milk. Blood parameters were affected by nitrate supplementation. Blood met-Hb concentration was increased, while blood glucose was decreased in nitrate groups. High forage and nitrate fed animals (F60N) had higher ruminal acetate and lower propionate concentration, and higher acetate+butyrate to propionate ratio than other groups. Nitrite and NH3-N concentrations were higher in the rumen of nitrate fed animals. Nitrate supplementation inhibited gas volume and methane emission without affecting volatile fatty acids at 12 and 24 h of incubation. The H2 balance, H2 production and consumption, and recovery percentage were significantly lower in F60N group. In conclusion, nitrate supplementation can be employed as an alternative strategy for improving ruminal fermentation, milk quality and methane inhibition.

A single dose of dietary nitrate supplementation protects against endothelial ischemia-reperfusion injury in early postmenopausal women.

The onset of menopause and accompanying changes to ovarian hormones often precedes endothelial dysfunction in women. In particular, accelerated impairments in macrovascular and microvascular function coincide with the loss of estrogen, as does impaired endothelial responses to ischemia-reperfusion (IR) injury. In healthy, early postmenopausal women (n=12; 3.9± 1.5 years since menopause) we tested the hypothesis that acute dietary nitrate (NO3-) supplementation would improve endothelial function and attenuate the magnitude of endothelial dysfunction following whole-arm IR in comparison with placebo. In this randomized, double-blind, placebo-controlled, crossover study we tested participants before and after NO3--rich (BRnitrate) and NO3--depleted (BRplacebo) beetroot juice (BR) consumption, as well as following IR injury, and 15min after IR to assess recovery. Analyses with repeated-measures general linear models revealed a condition× time interaction for brachial artery flow-mediated dilation (FMD; P=0.04), and no interaction effect was found for the near-infrared spectroscopy-derived reperfusion slope (P=0.86). Follow-up analysis showed a significant decline in FMD following IR injury with BRplacebo in comparison with all other timepoints (all, P<0.05), while this decline was not present with BRnitrate (all, P>0.05). Our findings demonstrate that a single dose of dietary NO3- minimizes IR-induced macrovascular endothelial dysfunction in healthy, early postmenopausal women, but does not improve resting macrovascular and microvascular function. Trial registration number: NCT03644472. Novelty: In healthy, early postmenopausal women, a single dose of NO3--rich BR can protect against IR-induced endothelial dysfunction. This protection may be due to nitric oxide bioactivity during IR rather than improved endothelial function prior to the IR protocol per se.

Nitrate consumption preserves HFD-induced skeletal muscle mitochondrial ADP sensitivity and lysine acetylation: A potential role for SIRT1

Dietary nitrate supplementation, and the subsequent serial reduction to nitric oxide, has been shown to improve glucose homeostasis in several pre-clinical models of obesity and insulin resistance. While the mechanisms remain poorly defined, the beneficial effects of nitrate appear to be partially dependent on AMPK-mediated signaling events, a central regulator of metabolism and mitochondrial bioenergetics. Since AMPK can activate SIRT1, we aimed to determine if nitrate supplementation (4 mM sodium nitrate via drinking water) improved skeletal muscle mitochondrial bioenergetics and acetylation status in mice fed a high-fat diet (HFD: 60% fat). Consumption of HFD induced whole-body glucose intolerance, and within muscle attenuated insulin-induced Akt phosphorylation, mitochondrial ADP sensitivity (higher apparent Km), submaximal ADP-supported respiration, mitochondrial hydrogen peroxide (mtH2O2) production in the presence of ADP and increased cellular protein carbonylation alongside mitochondrial-specific acetylation. Consumption of nitrate partially preserved glucose tolerance and, within skeletal muscle, normalized insulin-induced Akt phosphorylation, mitochondrial ADP sensitivity, mtH2O2, protein carbonylation and global mitochondrial acetylation status. Nitrate also prevented the HFD-mediated reduction in SIRT1 protein, and interestingly, the positive effects of nitrate ingestion on glucose homeostasis and mitochondrial acetylation levels were abolished in SIRT1 inducible knock-out mice, suggesting SIRT1 is required for the beneficial effects of dietary nitrate. Altogether, dietary nitrate preserves mitochondrial ADP sensitivity and global lysine acetylation in HFD-fed mice, while in the absence of SIRT1, the effects of nitrate on glucose tolerance and mitochondrial acetylation were abrogated.

Effects of dietary nitrate on the O2 cost of submaximal exercise: Accounting for “noise” in pulmonary gas exchange measurements

ABSTRACT Dietary nitrate (NO3 –) supplementation can reduce the oxygen cost of submaximal exercise, but this has not been reported consistently. We hypothesised that the number of step transitions to moderate-intensity exercise, and corresponding effects on the signal-to-noise ratio for pulmonary O2, may be important in this regard. Twelve recreationally active participants were assigned in a randomised, double-blind, crossover design to supplement for 4 days in three conditions: 1) control (CON; water); 2); PL (NO3 –-depleted beetroot juice); and 3) BR (NO3 –-rich beetroot juice). On days 3 and 4, participants completed two 6-min step transitions to moderate-intensity cycle exercise. Breath-by-breath O2 data were collected and O2 kinetic responses were determined for a single transition and when the responses to 2, 3 and 4 transitions were ensemble-averaged. Steady-state O2 was not different between PL and BR when the O2 response to one-, two- or three-step transition was compared but was significantly lower in BR compared to PL when four-step transitions was considered (PL: 1.33 ± 0.34 vs. BR: 1.31 ± 0.34 L·min−1, P < 0.05). There were no differences in pulmonary O2 responses between CON and PL (P > 0.05). Multiple step transitions may be required to detect the influence of NO3 − supplementation on steady-state O2.

Dietary Nitrate Supplementation Is Not Helpful for Endurance Performance at Simulated Altitude Even When Combined With Intermittent Normobaric Hypoxic Training.

IntroductionTraining intensity and nutrition may influence adaptations to training performed in hypoxia and consequently performance outcomes at altitude. This study investigates if performance at simulated altitude is improved to a larger extent when high-intensity interval training is performed in normobaric hypoxia and if this is potentiated when combined with chronic dietary nitrate (NO3−) supplementation.MethodsThirty endurance-trained male participants were allocated to one of three groups: hypoxia (13% FiO2) + NO3−; hypoxia + placebo; and normoxia (20.9% FiO2) + placebo. All performed 12 cycling sessions (eight sessions of 2*6 × 1 min at severe intensity with 1 min recovery and four sessions of 4*6*10 s all-out with 20 s recovery) during a 4-week period (three sessions/week) with supplementation administered 3–2.5 h before each session. An incremental exhaustion test, a severe intensity exercise bout to exhaustion (Tlim) and a 3 min all-out test (3AOT) in hypoxia (FiO2 = 13%) with pulmonary oxygen uptake (O2), O2 kinetics, and changes in vastus lateralis local O2 saturation (SmO2) measured were completed by each participant before and after training.ResultsIn all tests, performance improved to the same extent in hypoxia and normoxia, except for SmO2 after Tlim (p = 0.04, d = 0.82) and 3AOT (p = 0.03, d = 1.43) which were lower in the two hypoxic groups compared with the normoxic one. Dietary NO3− supplementation did not bring any additional benefits.ConclusionPerformance at simulated altitude was not improved to a larger extent when high-intensity interval training was undertaken in normobaric hypoxic conditions, when compared with normoxic training. Additionally, dietary NO3− supplementation was ineffective in further enhancing endurance performance at simulated altitude.

The Importance of Branched-chain Amino Acids and Nitrate in Sports Performance and Health

Branched-chain amino acids (BCAA) and nitrate have become increasingly popular mainly for their potential effect on individuals' health and secondary as ergogenic aids. The purpose of this narrative review was to incorporate the current scientific evidence of BCAA and nitrate supplementation on athletic performance and health. The current recommendations of BCAA and nitrate supplementation are discussed, as well as possible health complications associated with its intake. Pubmed, Scopus, and Web of Science were searched for articles on the effects of BCAA and nitrate supplementation in humans. The positive effect of BCAAs supplementation on athletic performance does not appear to be fully established. BCAAs supplementation seems to be recommended for all athletes who exercise vigorously daily, as it enhances their recovery after causing exercise-induced muscle damage. BCAAs supplementation reduces the feeling of delayed muscle pain, which follows because of muscle damage. Limited scientific data suggest a potentially beneficial effect of BCAAs on reducing central fatigue. Several clinical conditions could benefit from BCAAs' consumption. However, there are no reliable markers to evaluate or quantify their requirements. There are no exact consumption protocols and absolute recommendations, mainly because BCAAs are also obtained through the consumption of animal protein. Dietary nitrates lower blood pressure, reduce the cost of exercise oxygen, and, at least sometimes, enhance exercise capacity. Taking supplements for 2-6 days (or up to 15 days) can increase athletic performance during high-intensity exercise. The duration of continuous maximum exercise for which nitrates appear to be ergogenic is between 5-30 min. There is limited evidence that nitrates are beneficial in prolonged exercise performance (40 min), at least when administered short term. Supplementation of approximately 5-9 mmol of nitrate/day for 1-15 days may have beneficial effects on normal exercise responses, although the exact dose-response relationship has not yet been established. Five to 9 mmol of nitrates can be easily consumed in the normal diet and there is currently no evidence to adequately document that taking additional nitrates produces greater benefits. The effectiveness of acute nitrate supplementation is likely to depend on many factors, such as sex, health, hypoxia, diet, and level of fitness/training experience of the subjects. Nitrate needs are most likely met by ingesting approximately 250-500 g of leafy and root vegetables per day; however, dietary supplements might represent a more convenient and accurate way of covering an athlete's nitrate needs.