Physiological and performance effects of dietary nitrate and N-acetylcysteine supplementation during prolonged heavy-intensity cycling.

The purpose of this study was to investigate effects of concurrent and independent administration of dietary nitrate (NO3-), administered as NO3--rich beetroot juice (BR; ~12.4 mmol of NO3-), and N-acetylcysteine (NAC; 70 mg·kg-1) on physiological responses during prolonged exercise and subsequent high-intensity exercise tolerance. Sixteen recreationally active males supplemented with NO3--depleted beetroot juice (PL) or BR for 6 days and ingested an acute dose of NAC or maltodextrin (MAL) 1 h prior to performing 1 h of heavy-intensity cycling exercise immediately followed by a severe-intensity time-to-exhaustion (TTE) test in four conditions: 1) PL+MAL, 2) PL+NAC, 3) BR+MAL and 4) BR+NAC. Pre-exercise plasma [NO3-] and nitrite ([NO2-]) were elevated following BR+NAC  and BR+MAL (both P < 0.01) compared with PL+NAC and PL+MAL; plasma [cysteine] was increased in PL+NAC  and BR+NAC (both P < 0.01) compared to PL+MAL. Muscle excitability declined over time during the prolonged cycling bout in all conditions  but was better preserved in PL+NAC  compared to BR+NAC (P < 0.01) and PL+MAL (P < 0.05). There was no effect of supplementation on subsequent TTE . These findings indicate that co-ingestion of BR and NAC does not appreciably alter physiological responses during prolonged heavy-intensity cycling or enhance subsequent exercise tolerance.

High-Throughput Griess Assay of Nitrite and Nitrate in Plasma and Red Blood Cells for Human Physiology Studies under Extreme Conditions.

The metabolism of nitric oxide plays an increasingly interesting role in the physiological response of the human body to extreme environmental conditions, such as underwater, in an extremely cold climate, and at low oxygen concentrations. Field studies need the development of analytical methods to measure nitrite and nitrate in plasma and red blood cells with high requirements of accuracy, precision, and sensitivity. An optimized spectrophotometric Griess method for nitrite-nitrate affords sensitivity in the low millimolar range and precision within ±2 µM for both nitrite and nitrate, requiring 100 µL of scarcely available plasma sample or less than 50 µL of red blood cells. A scheduled time-efficient procedure affords measurement of as many as 80 blood samples, with combined nitrite and nitrate measurement in plasma and red blood cells. Performance and usefulness were tested in pilot studies that use blood fractions deriving from subjects who dwelt in an Antarctica scientific station and on breath-holding and scuba divers who performed training at sea and in a land-based deep pool facility. The method demonstrated adequate to measure low basal concentrations of nitrite and high production of nitrate as a consequence of water column pressure-triggered vasodilatation in deep-water divers.

Marching to the Beet: The effect of dietary nitrate supplementation on high altitude exercise performance and adaptation during a military trekking expedition.

PURPOSE: The aim was to investigate the effect of dietary nitrate supplementation (in the form of beetroot juice, BRJ) for 20 days on salivary nitrite (a potential precursor of bioactive nitric oxide), exercise performance and high altitude (HA) acclimatisation in field conditions (hypobaric hypoxia). METHODS: This was a single-blinded randomised control study of 22 healthy adult participants (12 men, 10 women, mean age 28 ± 12 years) across a HA military expedition. Participants were randomised pre-ascent to receive two 70 ml dose per day of either BRJ (~12.5 mmol nitrate per day; n = 11) or non-nitrate calorie matched control (n = 11). Participants ingested supplement doses daily, beginning 3 days prior to departure and continued until the highest sleeping altitude (4800 m) reached on day 17 of the expedition. Data were collected at baseline (44 m altitude), at 2350 m (day 9), 3400 m (day 12) and 4800 m (day 17). RESULTS: BRJ enhanced the salivary levels of nitrite (p = 0.007). There was a significant decrease in peripheral oxygen saturation and there were increases in heart rate, diastolic blood pressure, and rating of perceived exertion with increasing altitude (p=<0.001). Harvard Step Test fitness scores significantly declined at 4800 m in the control group (p = 0.003) compared with baseline. In contrast, there was no decline in fitness scores at 4800 m compared with baseline (p = 0.26) in the BRJ group. Heart rate recovery speed following exercise at 4800 m was significantly prolonged in the control group (p=<0.01) but was unchanged in the BRJ group (p = 0.61). BRJ did not affect the burden of HA illness (p = 1.00). CONCLUSIONS: BRJ increases salivary nitrite levels and ameliorates the decline in fitness at altitude but does not affect the occurrence of HA illness.

Pharmacokinetics of Nitrate and Nitrite Following Beetroot Juice Drink Consumption.

BACKGROUND: Nitrate (NO3 -)-rich beetrAs BR juice can naturally contain both NO3 In four separate treatments, 11 healthy adults consumed 250 mL of BR containing one of the following: (i) high NO3 Ingestion of the HL and MM BR increased plasma [NO2 Inorganic NO3 - consumptio

Inorganic nitrate supplementation attenuates conduit artery retrograde and oscillatory shear in older adults.

Aging causes deleterious changes in resting conduit artery shear patterns and reduced blood flow during exercise partially attributable to reduced nitric oxide (NO). Inorganic nitrate increases circulating NO bioavailability and may, therefore, improve age-associated changes in shear rate as well as exercise hyperemia. Ten older adults (age: 67 ± 3 yr) consumed 4.03 mmol nitrate and 0.29 mmol nitrite (active) or devoid of both (placebo) daily for 4 wk in a randomized, double-blinded, crossover fashion. Brachial artery diameter (D) and blood velocity (Vmean) were measured via Doppler ultrasound at rest for the characterization of shear profile as well as during two handgrip exercise trials (4 and 8 kg) for calculation of forearm blood flow (Vmean × cross-sectional area, FBF) and conductance [FBF/mean arterial pressure, forearm vascular conductance (FVC)]. Plasma [nitrate] and [nitrite] increased following active (P < 0.05 for both) but not placebo (P = 0.68 and 0.40, respectively) supplementation. Neither mean nor antegrade shear rate changed following either supplement (beverage-by-time P = 0.14 and 0.21, respectively). Retrograde (-13.4 ± 7.0 to -9.7 ± 6.8·s-1) and oscillatory (0.20 ± 0.08 to 0.15 ± 0.09 A.U., P < 0.05 for both) shear decreased following active, but not placebo (P = 0.81 and 0.70, respectively), supplementation. The FBF response (Δ from rest) to neither 4-kg nor 8-kg trials changed following either supplement (beverage-by-time P = 0.53 and 0.11, respectively). Similarly, no changes were observed in FVC responses to 4-kg or 8-kg trials (beverage-by-time P = 0.23 and 0.07, respectively). These data indicate that inorganic nitrate supplementation improves conduit artery shear profiles, but not exercise hyperemia, in older adults.NEW & NOTEWORTHY We report for the first time, to our knowledge, that 4 wk of inorganic nitrate supplementation attenuates retrograde and oscillatory shear in the brachial artery of older adults. However, this was not associated with greater hyperemic or vasodilatory responses to exercise. In sum, these data highlight favorable changes in shear patterns with aging, which may reduce the risk of atherosclerotic cardiovascular disease.

No thermoregulatory or ergogenic effect of dietary nitrate among physically inactive males, exercising above gas exchange threshold in hot and dry conditions.

The aim of this study was to determine the effect of five days dietary nitrate (NO3-) consumption on exercise tolerance and thermoregulation during cycling in hot, dry conditions. In a double-blind, randomised crossover design, 11 healthy males participated in an exercise tolerance test (Tlim) in the heat (35°C, 28% relative humidity), cycling above the thermoneutral gas exchange threshold, after five days of dietary supplementation, with either NO3-rich beetroot juice (BR; ∼ 9.2 mmol NO3-) or placebo (PLA). Changes in plasma [NO3-] and nitrite [NO2-], core and mean skin temperatures, mean local and whole-body sweat rates, heart rate, perceptual ratings and pulmonary gas exchange were measured during exercise, alongside calorimetric estimations of thermal balance. Mean arterial pressures (MAP) were recorded pre-Tlim. There were no differences in Tlim between conditions (BR = 22.8 ± 8.1 min; Placebo = 20.7 ± 7.9 min) (P = 0.184), despite increases in plasma [NO3-] and [NO2-] (P < 0.001) and a 3.8% reduction in resting MAP (P = 0.004) in the BR condition. There were no other differences in thermoregulatory, cardio-metabolic, perceptual or calorimetric responses to the Tlim between conditions (P > 0.05). Dietary NO3- supplementation had no effect on exercise tolerance or thermoregulation in hot, dry conditions, despite reductions in resting MAP and increases in plasma [NO3-] and [NO2-]. Healthy, yet physically inactive individuals with no known impairments in vasodilatory and sudomotor function do not appear to require BR for ergogenic or thermolytic effects during exercise in the heat.

The impact of beetroot juice supplementation on muscular endurance, maximal strength and countermovement jump performance.

Purpose: Dietary nitrate has been shown to enhance muscle contractile function and has, therefore, been linked to increased muscle power and sprint exercise performance. However, the impact of dietary nitrate supplementation on maximal strength, performance and muscular endurance remains to be established. Methods: Fifteen recreationally active males (25 ± 4 y, BMI 24 ± 3 kg/m2) participated in a randomized double-blinded cross-over study comprising two 6-d supplementation periods; 140 mL/d nitrate-rich (BR; 985 mg/d) and nitrate-depleted (PLA; 0.37 mg/d) beetroot juice. Three hours following the last supplement, we assessed countermovement jump (CMJ) performance, maximal strength and power of the upper leg by voluntary isometric (30° and 60° angle) and isokinetic contractions (60, 120, 180 and 300°·s-1), and muscular endurance (total workload) by 30 reciprocal isokinetic voluntary contractions at 180°·s-1. Results: Despite differences in plasma nitrate (BR: 879 ± 239 vs. PLA: 33 ± 13 µmol/L, P < 0.001) and nitrite (BR: 463 ± 217 vs. PLA: 176 ± 50 nmol/L, P < 0.001) concentrations prior to exercise testing, CMJ height (BR: 39.3 ± 6.3 vs. PLA: 39.6 ± 6.3 cm; P = 0.39) and muscular endurance (BR: 3.93 ± 0.69 vs. PLA: 3.90 ± 0.66 kJ; P = 0.74) were not different between treatments. In line, isometric strength (P > 0.50 for both angles) and isokinetic knee extension power (P > 0.33 for all velocities) did not differ between treatments. Isokinetic knee flexion power was significantly higher following BR compared with PLA ingestion at 60°·s-1 (P = 0.001), but not at 120°·s-1 (P = 0.24), 180°·s-1 (P = 0.066), and 300°·s-1 (P = 0.36). Conclusion: Nitrate supplementation does not improve maximal strength, countermovement jump performance and muscular endurance in healthy, active males.

Oxidative stress and nitrate/nitrite (NOx) status following citrulline supplementation in type 2 diabetes: a randomised, double-blind, placebo-controlled trial.

BACKGROUND: The pathogenesis of diabetes is accompanied by oxidative stress. Citrulline can be considered as a potent antioxidant. The present study aimed to examine the effects of citrulline supplementation on the status of oxidative stress and serum levels of nitrite/nitrate (NOx) in patients suffering from type 2 diabetes (T2DM). METHODS: The present study comprises a double-blind placebo-controlled randomised clinical trial. The study subjects include 54 patients with T2DM chosen from specialised clinics of Tabriz University of Medical Sciences. The patients were allocated to one of the placebo or intervention groups. The patients in the placebo and the intervention group received one sachet of microcrystalline cellulose or l-citrulline (3 g), respectively, every day for 2 months. Malondialdehyde (MDA), glutathione peroxidase (GPx), total antioxidant capacity (TAC), superoxide dismutase (SOD), serum levels of fasting blood sugar (FBS), citrulline and NOx were assessed before and after the intervention. RESULTS: Forty-five patients completed the trial. Significant decreases were found in serum levels of FBS and MDA. Serum levels of TAC, citrulline and NOx in the intervention group increased compared to those of the placebo group, after adjusting the data for the baseline values and confounders. Differences between and within the two groups were statistically nonsignificant for GPx and SOD at the end of the study. Body mass index and energy intake were not changed significantly after the intervention. CONCLUSIONS: The results of the present study show the positive effects of citrulline supplementation with respect to attenuating FBS levels and oxidative stress, as well as boosting NOx status, in patients with T2DM.

Influence of Dietary Nitrate Supplementation on High-Intensity Intermittent Running Performance at Different Doses of Normobaric Hypoxia in Endurance-Trained Males.

This study investigated whether supplementation with nitrate-rich beetroot juice (BR) can improve high-intensity intermittent running performance in trained males in normoxia and different doses of normobaric hypoxia. Eight endurance-trained males (V˙O2peak, 62 ± 6 ml·kg-1·min-1) completed repeated 90 s intervals at 110% of peak treadmill velocity, from an initial step incremental test, interspersed by 60 s of passive recovery until exhaustion (Tlim). Participants completed the first three experimental trials during days 3, 5, and 7 of BR or nitrate-depleted beetroot juice (PLA) supplementation and completed the remaining experimental visits on the alternative supplement following at least 7 days of washout. The fraction of inspired oxygen during visits 1-3 was either 0.209, 0.182, or 0.157, equivalent to an altitude of 0, 1,200, and 2,400 m, respectively, and this order was replicated on visits 4-6. Arterial oxygen saturation declined dose dependently as fraction of inspired oxygen was lowered (p < .05). Plasma nitrite concentration was higher pre- and postexercise after BR compared with PLA supplementation (p < .05). There was no difference in Tlim between PLA and BR at 0 m (445 [324, 508] and 410 [368, 548] s); 1,200 m (341 [270, 390] and 332 [314, 356] s); or 2,400 m (233 [177, 373] and 251 [221, 323] s) (median and [interquartile range]; p > .05). The findings from this study suggest that short-term BR supplementation does not improve high-intensity intermittent running performance in endurance-trained males in normoxia or at doses of normobaric hypoxia that correspond to altitudes at which athletes typically train while on altitude training camps.

Nitrate-rich beetroot juice offsets salivary acidity following carbohydrate ingestion before and after endurance exercise in healthy male runners.

There have been recent calls for strategies to improve oral health in athletes. High carbohydrate diets, exercise induced dehydration and transient perturbations to immune function combine to increase oral disease risk in this group. We tested whether a single dose of nitrate (NO3-) would offset the reduction in salivary pH following carbohydrate ingestion before and after an exercise bout designed to cause mild dehydration. Eleven trained male runners ([Formula: see text] 53 ± 9 ml∙kg-1∙min-1, age 30 ± 7 years) completed a randomised placebo-controlled study comprising four experimental trials. Participants ingested the following fluids one hour before each trial: (a) 140 ml of water (negative-control), (b) 140 ml of water (positive-control), (c) 140 ml of NO3- rich beetroot juice (~12.4 mmol NO3-) (NO3- trial) or (d) 140 ml NO3- depleted beetroot juice (placebo-trial). During the negative-control trial, participants ingested 795 ml of water in three equal aliquots: before, during, and after 90 min of submaximal running. In the other trials they received 795 ml of carbohydrate supplements in the same fashion. Venous blood was collected before and after the exercise bout and saliva was sampled before and repeatedly over the 20 min following carbohydrate or water ingestion. As expected, nitrite (NO2-) and NO3- were higher in plasma and saliva during the NO3- trial than all other trials (all P<0.001). Compared to the negative-control, salivary-pH was significantly reduced following the ingestion of carbohydrate in the positive-control and placebo trials (both P <0.05). Salivary-pH was similar between the negative-control and NO3- trials before and after exercise despite ingestion of carbohydrate in the NO3- trial (both P≥0.221). Ingesting NO3- attenuates the expected reduction in salivary-pH following carbohydrate supplements and exercise-induced dehydration. NO3- should be considered by athletes as a novel nutritional strategy to reduce the risk of developing acidity related oral health conditions.

Acute beetroot juice supplementation improves exercise tolerance and cycling efficiency in adults with obesity.

BACKGROUND: Exercise training improves health outcomes in individuals with obesity (IO); however, it remains challenging for IO to adhere to exercise. Thus, it is critical to identify novel strategies that improve exercise tolerance (ET) and adherence in IO. Beetroot juice (BRJ), high in inorganic dietary nitrate, consistently improves exercise performance in athletes, individuals with cardiopulmonary diseases, and nonobese lean individuals. These improvements may be explained by reduced oxygen uptake (VO2 ) during exercise, enhanced blood flow, and greater mitochondrial efficiency. To date, we are aware of no studies that have compared the effects of BRJ, sodium nitrate (NaNO3), and nitrate-depleted BRJ (PLA) for improving ET and cardiometabolic health in IO. PURPOSE: Determine if BRJ improves ET, exercise efficiency (EE), and cardiometabolic health in IO and identify possible mechanisms of action. METHODS: Vascular hemodynamic, submaximal- and maximal-exercise VO2 , and time to exhaustion (TTE) were assessed in 16 participants 2.5 hr following consumption of: 1) BRJ, 2) NaNO3 , 3) PLA, or 4) CON. RESULTS: A significant treatment effect was observed for submaximal exercise VO2 (p = .003), and TTE (p < .001). Post hoc analyses revealed lower VO2 during submaximal exercise in BRJ compared to PLA (p = .009) NaNO3 (p = .042) and CON (0.009), equating to an average improvement of ~ 7% with BRJ. TTE was greater for BRJ compared to other treatment arms, PLA (p = .008), NaNO3 (p = .038), and CON (p=<0.001), equating to ~ 15% improvement with BRJ. No significant changes were observed for other outcomes. CONCLUSIONS: Consumption of BRJ improved EE during submaximal exercise by 7%, and TTE by 15% compared to other conditions. These results suggest that BRJ may improve EE and exercise tolerance in IO.

The Effect of Dietary Inorganic Nitrate Supplementation on Cardiac Function during Submaximal Exercise in Men with Heart Failure with Reduced Ejection Fraction (HFrEF): A Pilot Study.

Heart failure with reduced ejection fraction (HFrEF) is a common end point for patients with coronary artery disease and it is characterized by exercise intolerance due, in part, to a reduction in cardiac output. Nitric oxide (NO) plays a vital role in cardiac function and patients with HFrEF have been identified as having reduced vascular NO. This pilot study aimed to investigate if nitrate supplementation could improve cardiac measures during acute, submaximal exercise. Five male participants (61 ± 3 years) with HFrEF (EF 32 ± 2.2%) completed this pilot study. All participants supplemented with inorganic nitrate (beetroot juice) or a nitrate-depleted placebo for ~13 days prior to testing. Participants completed a three-stage submaximal exercise protocol on a recumbent cycle ergometer with simultaneous echocardiography for calculation of cardiac output (Q), stroke volume (SV), and total peripheral resistance (TPR). Heart rate and blood pressure were measured at rest and during each stage. Both plasma nitrate (mean = ~1028%, p = 0.004) and nitrite (mean = ~109%, p = 0.01) increased following supplementation. There were no differences between interventions at rest, but the percent change in SV and Q from rest to stage two and stage three of exercise was higher following nitrate supplementation (all p > 0.05, ES > 0.8). Both interventions showed decreases in TPR during exercise, but the percent reduction TPR in stages two and three was greater following nitrate supplementation (p = 0.09, ES = 0.98 and p = 0.14, ES = 0.82, respectively). There were clinically relevant increases in cardiac function during exercise following supplementation with nitrate. The findings from this pilot study warrant further investigation in larger clinical trials.

A randomized controlled trial of nitrate supplementation in well-trained middle and older-aged adults.

PURPOSE: Nitrate (NO3-), through its conversion to nitrite (NO2-) and nitric oxide, has been shown to increase exercise tolerance in healthy younger adults and older diseased patients. Nitrate's effect in well-trained middle to older-aged adults has not been studied. Therefore, the purpose of this investigation was to examine the effects of a NO3- rich beverage on submaximal constant work rate exercise time in well-trained middle to older-aged adults. METHODS: This was a randomized controlled cross-over trial with 15 well-trained middle to older-aged adults, 41-64 year-old, who received one of two treatments (NO3- rich beverage then placebo or placebo then NO3- rich beverage), after which an exercise test at 75 percent of the subject's maximal work rate was completed. RESULTS: The NO3- rich beverage increased plasma NO3- and NO2- levels by 260 µM and 0.47 µM, respectively (p<0.001). Exercise time was not significantly different (p = 0.31) between the NO3- rich versus placebo conditions (1130±151 vs 1060±132 sec, respectively). Changes in exercise time between the two conditions ranged from a 55% improvement to a 40% decrease with the NO3- rich beverage. Oxygen consumption and rating of perceived exertion were not significantly different between the two conditions. CONCLUSION: In middle to older-aged well-trained adults, NO3- supplementation has non-significant, albeit highly variable, effects on exercise tolerance. ClinicalTrials.gov Identifier: NCT03371966.

Acute ingestion of beetroot juice does not improve short-duration repeated sprint running performance in male team sport athletes.

The effects of acute ingestion of nitrate on short-duration repeated sprint performance (RSP) are unclear. This study investigated the effect of acute ingestion of beetroot juice on a test of RSP in team sport athletes. Sixteen male team sport athletes undertook four trials using a 40 m maximum shuttle run test (MST), which incorporates 10 × 40 m shuttle sprints with 30 s between the start of each sprint. Two familiarisation trials, followed by nitrate-rich beetroot juice (BR; ~6 mmol nitrate) and nitrate-depleted beetroot juice (PLA; ~0.0034 mmol nitrate) trials were completed in a randomised, double-blind manner. Ingestion of beetroot juice 3 h prior to exercise elevated plasma nitrate concentrations ~6-fold in BR (BR, 413 ± 56 µM; PLA, 69 ± 30 µM; P < 0.001). RSP, assessed by sprint performance decrement (Sdec; %), did not differ (P = 0.337) between BR (5.31 ± 2.49%) and PLA (5.71 ± 2.61%). There was no difference between trials for total sprint time (P = 0.806), fastest sprint (P = 0.341), slowest sprint (P = 0.787), or post-exercise blood lactate concentration (BR, 11.8 ± 2.5 mM; PLA, 12.2 ± 2.3 mM; P = 0.109). Therefore, acute ingestion of beetroot juice did not improve a test of short-duration RSP in team sport athletes.

Long-term nitric oxide synthase inhibition prevents 17ß-estradiol-induced suppression of cyclooxygenase-dependent contractions and enhancement of endothelium-dependent hyperpolarization-like relaxation in mesenteric arteries of ovariectomized rats.

Endothelial dysfunction is associated with a reduced bioavailability of nitric oxide (NO). In this study, the effects of 17ß-estradiol supplement on endothelial function were examined in ovariectomized (OVX) rats following long-term inhibition of NO synthases with L-NAME. Female Sprague Dawley rats were ovariectomized at 12 weeks old. They were supplemented with 17ß-estradiol (25 µg/kg/day, intramuscularly) or its vehicle (olive oil) until they were killed. At 18 weeks old, they were administered daily with NO synthase inhibitor L-NAME (60 mg/kg, by gavage) or its vehicle (distilled water) for 6 weeks. Rats were then anesthetized for blood pressure measurement and for isolation of mesenteric arteries and aortae for isometric tension measurement. Long-term L-NAME-treatment, without or with 17ß-estradiol supplement, resulted in reduced plasma nitrite/nitrate level without causing an increase in blood pressure in OVX rats. Acute inhibition of cyclooxygenase (COX) with indomethacin improved relaxations of mesenteric arteries to the calcium ionophore A23187 in OVX rats, and in those with long-term L-NAME-treatment without or with 17ß-estradiol supplement, but not in those with female hormone supplement only. 17ß-estradiol supplement or long-term L-NAME-treatment resulted in a greater endothelium-dependent hyperpolarization-like relaxation in mesenteric arteries. In the quiescent aorta, 17ß-estradiol supplement or long-term L-NAME-treatment unmasked the COX-dependent components of A23187-induced contractions, but prevented that of the smooth muscle contractions to U46619 in OVX rats. In summary, long-term 17ß-estradiol-supplement results in differential effects in different blood vessel types, and its beneficial vascular effects are masked under the conditions with NO synthase inhibition.

A Nitrate-Rich Vegetable Intervention Elevates Plasma Nitrate and Nitrite Concentrations and Reduces Blood Pressure in Healthy Young Adults.

BACKGROUND: Emerging evidence suggests that increasing dietary nitrate intake may be an effective approach to reduce blood pressure. Beetroot juice is often used to supplement dietary nitrate, whereas nitrate intake levels from habitual diet are low. An increase in the habitual intake of nitrate-rich vegetables may represent an alternative to nitrate supplementation. However, the effectiveness and acceptability of a nitrate-rich-vegetables diet remain to be established. OBJECTIVE: The aim was to investigate the effect and feasibility of two different intervention strategies to increase dietary nitrate intake, on plasma nitrate/nitrite concentrations and blood pressure. DESIGN: A randomized, crossover trial was used. PARTICIPANTS: Participants were healthy men and women (both n=15; age: 24±6 years) from the Netherlands. INTERVENTION: Participants were instructed to consume ∼400 mg nitrate at lunch, provided through nitrate-rich vegetables and dietary counseling, or beetroot juice supplementation. Both interventions lasted 1 week, with 1-week washout (January to April 2017). MAIN OUTCOME: Plasma nitrate and nitrite concentrations and resting systolic and diastolic blood pressure were measured in an overnight fasted state (before and after intervention) and ∼2.5 hours after lunch (before and throughout intervention on day 1, 4, and 7). STATISTICAL ANALYSIS: Two-factor (time × treatment) repeated-measures analyses of variance were performed. RESULTS: Mean plasma nitrate concentrations increased with both interventions, with a larger increase in beetroot juice vs nitrate-rich vegetables, both in a fasted state and ∼2.5 hours after lunch (day 1, beetroot juice: 2.31±0.56 mg/dL [373±90 µmol/L] vs nitrate-rich vegetables: 1.71±0.83 mg/dL [277±134 µmol/L]; P<0.001). Likewise, mean plasma nitrite concentrations increased with both interventions, but were higher after lunch in beetroot juice than in nitrate-rich vegetables (day 1: 2.58±1.52 µg/dL [560±331 nmol/L] vs 2.15±1.21 µg/dL [468±263 nmol/L]; P=0.020). Fasting mean systolic and diastolic blood pressure did not change, but mean systolic and diastolic blood pressure assessed ∼2.5 hours after lunch were significantly reduced throughout both intervention periods (P<0.05), with no differences between beetroot juice and nitrate-rich vegetables (day 1, systolic blood pressure: -5.1±9.5 mm Hg and diastolic blood pressure: -5.3±8.9 mm Hg). CONCLUSION: Short-term consumption of dietary nitrate in the form of nitrate-rich vegetables represents an effective means to increase plasma nitrate and nitrite concentrations, and reduces blood pressure to the same extent as beetroot juice supplementation.

The effects of oral arginine on its metabolic pathways in Sprague-Dawley rats.

Oral arginine supplements are popular mainly for their presumed vasodilatory benefit. Arginine is a substrate for at least four enzymes including nitric oxide synthase (NOS) and arginase, but the impact of oral supplements on its different metabolic pathways is not clear. Deficiencies of arginine-metabolising enzymes are associated with conditions such as hyperammonaemia, endothelial dysfunction, central nervous system and muscle dysfunction, which complicate the use of oral arginine supplements. We examined the effect of l-arginine (l-Arg) and d-arginine (d-Arg), each at 500 mg/kg per d in drinking water administered for 4 weeks to separate groups of 9-week-old male Sprague-Dawley rats. We quantified the expression of enzymes and plasma, urine and organ levels of various metabolites of arginine. l-Arg significantly decreased cationic transporter-1 expression in the liver and the ileum and increased endothelial NOS expression in the aorta and the kidney and plasma nitrite levels, but did not affect the mean arterial pressure. l-Arg also decreased the expression of arginase II in the ileum, arginine:glycine amidinotransferase in the liver and the kidney and glyoxalase I in the liver, ileum and brain, but increased the expression of arginine decarboxylase and polyamines levels in the liver. d-Arg, the supposedly inert isomer, also unexpectedly affected the expression of some enzymes and metabolites. In conclusion, both l- and d-Arg significantly affected enzymes and metabolites in several pathways that use arginine as a substrate and further studies with different doses and treatment durations are planned to establish their safety or adverse effects to guide their use as oral supplements.

A randomized pilot study of nitrate supplementation with beetroot juice in acute respiratory failure.

Nitrate rich beetroot juice (BRJ) can enhance nitric oxide signaling, leading to improved physical function in healthy and diseased populations, but its safety and biologic efficacy have not been evaluated in a critically ill population. We randomized 22 previously functional acute respiratory failure patients to either BRJ or placebo daily until day 14 or discharge. We measured blood nitrate and nitrite levels and quantified strength and physical function at intensive care unit (ICU) and hospital discharge. Participants were predominantly male (54%), aged 68.5 years with an APACHE III score of 62. BRJ increased plasma nitrate (mean 219.2 µM increase, p = 0.002) and nitrite levels (mean 0.144 µM increase, p = 0.02). We identified no adverse events. The unadjusted and adjusted effect sizes of the intervention on the short physical performance battery were small (d = 0.12 and d = 0.17, respectively). In this pilot trial, administration of BRJ was feasible and safe, increased blood nitrate and nitrate levels, but had a small effect on physical function. Future studies could evaluate the clinical efficacy of BRJ as a therapy to improve physical function in survivors of critical illness.

Physiologically relevant screening of polyphenol-rich commercial preparations for bioactivity in vascular endothelial cells and application to healthy volunteers: A viable workflow and a cautionary tale.

This study describes the screening of 13 commercially-available plant extracts for pharmacological activity modulating vascular function using an endothelial cell model. A French maritime pine bark extract (FMPBE) was found to have the greatest effect upon nitric oxide availability in control (181% ± 36% of untreated cells) and dysfunctional cells (132% ± 8% of untreated control cells). In healthy volunteers, the FMPBE increased plasma nitrite concentrations 8 h post-consumption compared to baseline (baseline corrected median 1.71 ± 0.38 (25% IQR) and 4.76 (75% IQR) µM, p < 0.05). This was followed by a placebo-controlled, healthy volunteer study, which showed no effects on plasma nitrite. It was confirmed that different batches of extract had been used in the healthy volunteer studies, and this second batch lacked bioactivity, assessed using the in vitro model. No difference in plasma catechin levels was seen at 8 h following supplementation between the studies (252 ± 194 nM versus 50 ± 64 nM, p > 0.05), however HPLC-UV fingerprinting showed that the new batch had a 5-15% in major constituents (including procyanidins A2, B1 and B2) compared to the original batch. This research describes a robust mechanism for screening bioactive extracts for vascular effects. It also highlights batch variability as a significant limitation when using complex extracts for pharmacological activity, and suggests the use of in vitro systems as a tool to identify this problem in future studies.