Relationships between nitric oxide biomarkers and physiological outcomes following dietary nitrate supplementation.

Dietary nitrate (NO3-) supplementation can increase nitric oxide (NO) bioavailability, reduce blood pressure (BP) and improve muscle contractile function in humans. Plasma nitrite concentration (plasma [NO2-]) is the most oft-used biomarker of NO bioavailability. However, it is unclear which of several NO biomarkers (NO3-, NO2-, S-nitrosothiols (RSNOs)) in plasma, whole blood (WB), red blood cells (RBC) and skeletal muscle correlate with the physiological effects of acute and chronic dietary NO3- supplementation. Using a randomized, double-blind, crossover design, 12 participants (9 males) consumed NO3--rich beetroot juice (BR) (∼12.8 mmol NO3-) and NO3--depleted placebo beetroot juice (PL) acutely and then chronically (for two weeks). Biological samples were collected, resting BP was assessed, and 10 maximal voluntary isometric contractions of the knee extensors were performed at 2.5-3.5 h following supplement ingestion on day 1 and day 14. Diastolic BP was significantly lower in BR (-2 ± 3 mmHg, P = 0.03) compared to PL following acute supplementation, while the absolute rate of torque development (RTD) was significantly greater in BR at 0-30 ms (39 ± 57 N m s-1, P = 0.03) and 0-50 ms (79 ± 99 N m s-1, P = 0.02) compared to PL following two weeks supplementation. Greater WB [RSNOs] rather than plasma [NO2-] was correlated with lower diastolic BP (r = -0.68, P = 0.02) in BR compared to PL following acute supplementation, while greater skeletal muscle [NO3-] was correlated with greater RTD at 0-30 ms (r = 0.64, P=0.03) in BR compared to PL following chronic supplementation. We conclude that [RSNOs] in blood, and [NO3-] in skeletal muscle, are relevant biomarkers of NO bioavailability which are related to the reduction of BP and the enhanced muscle contractile function following dietary NO3- ingestion in humans.

Reduction in blood pressure following acute dietary nitrate ingestion is correlated with increased red blood cell S-nitrosothiol concentrations.

Dietary nitrate (NO3-) supplementation can enhance nitric oxide (NO) bioavailability and lower blood pressure (BP) in humans. The nitrite concentration ([NO2-]) in the plasma is the most commonly used biomarker of increased NO availability. However, it is unknown to what extent changes in other NO congeners, such as S-nitrosothiols (RSNOs), and in other blood components, such as red blood cells (RBC), also contribute to the BP lowering effects of dietary NO3-. We investigated the correlations between changes in NO biomarkers in different blood compartments and changes in BP variables following acute NO3- ingestion. Resting BP was measured and blood samples were collected at baseline, and at 1, 2, 3, 4 and 24 h following acute beetroot juice (∼12.8 mmol NO3-, ∼11 mg NO3-/kg) ingestion in 20 healthy volunteers. Spearman rank correlation coefficients were determined between the peak individual increases in NO biomarkers (NO3-, NO2-, RSNOs) in plasma, RBC and whole blood, and corresponding decreases in resting BP variables. No significant correlation was observed between increased plasma [NO2-] and reduced BP, but increased RBC [NO2-] was correlated with decreased systolic BP (rs = -0.50, P = 0.03). Notably, increased RBC [RSNOs] was significantly correlated with decreases in systolic (rs = -0.68, P = 0.001), diastolic (rs = -0.59, P = 0.008) and mean arterial pressure (rs = -0.64, P = 0.003). Fisher's z transformation indicated no difference in the strength of the correlations between increases in RBC [NO2-] or [RSNOs] and decreased systolic blood pressure. In conclusion, increased RBC [RSNOs] may be an important mediator of the reduction in resting BP observed following dietary NO3- supplementation.

15 N-labeled dietary nitrate supplementation increases human skeletal muscle nitrate concentration and improves muscle torque production.

AIM: Dietary nitrate (NO3 - ) supplementation increases nitric oxide bioavailability and can enhance exercise performance. We investigated the distribution and metabolic fate of ingested NO3 - at rest and during exercise with a focus on skeletal muscle. METHODS: In a randomized, crossover study, 10 healthy volunteers consumed 12.8 mmol 15 N-labeled potassium nitrate (K15 NO3 ; NIT) or potassium chloride placebo (PLA). Muscle biopsies were taken at baseline, at 1- and 3-h post-supplement ingestion, and immediately following the completion of 60 maximal intermittent contractions of the knee extensors. Muscle, plasma, saliva, and urine samples were analyzed using chemiluminescence to determine absolute [NO3 - ] and [NO2 - ], and by mass spectrometry to determine the proportion of NO3 - and NO2 - that was 15 N-labeled. RESULTS: Neither muscle [NO3 - ] nor [NO2 - ] were altered by PLA. Following NIT, muscle [NO3 - ] (but not [NO2 - ]) was elevated at 1-h (from ~35 to 147 nmol/g, p < 0.001) and 3-h, with almost all of the increase being 15 N-labeled. There was a significant reduction in 15 N-labeled muscle [NO3 - ] from pre- to post-exercise. Relative to PLA, mean muscle torque production was ~7% greater during the first 18 contractions following NIT. This improvement in torque was correlated with the pre-exercise 15 N-labeled muscle [NO3 - ] and the magnitude of decline in 15 N-labeled muscle [NO3 - ] during exercise (r = 0.66 and r = 0.62, respectively; p < 0.01). CONCLUSION: This study shows, for the first time, that skeletal muscle rapidly takes up dietary NO3 - , the elevated muscle [NO3 - ] following NO3 - ingestion declines during exercise, and muscle NO3 - dynamics are associated with enhanced torque production during maximal intermittent muscle contractions.

Estimating nitrate intake in the Australian diet: Design and validation of a food frequency questionnaire.

BACKGROUND: Dietary nitrates may play a role in mediating several key physiological processes impacting health and/or exercise performance. However, current methods for assessing dietary nitrate (NO3 - ) consumption are inadequate. The present study aimed to examine the dietary nitrate intake in a sample of 50 healthy adults, as well as test the validity of a purposefully developed food frequency questionnaire (FFQ). METHODS: Dietary nitrate intake was estimated over a week using (i) three 24-h dietary recalls; (ii) a short-term (7-day) FFQ; and (iii) a biomarker (urinary nitrate), in conjunction with a nitrate reference database. RESULTS: Daily dietary nitrate intake estimates were 130.94 mg (average of three 24-h recalls) and 180.62 mg (FFQ). The mean urinary NO3 - excretion was 1974.79 µmol day-1 (or 917.9 µmol L-1 ). Despite the difference between the two dietary assessment methods, there was a moderate positive correlation (r = 0.736, ρ < 0.001) between the two tools. There was also a positive correlation between urinary NO3 - and 24-h recall data (r = 0.632, ρ < 0.001), as well as between urinary NO3 - and FFQ (r = 0.579, ρ < 0.001). CONCLUSIONS: The ability to accurately estimate nitrate intakes depends on having suitable reference methods to estimate the concentrations of nitrate in the food supply, coupled with valid and reliable dietary assessment tools. Based on the findings from the present study, at an individual level, dietary recalls or records may be more accurate in estimating intakes of NO3 - . However, given the lower cost and time needed for administration relative to recalls, the FFQ has merit for estimating NO3 - intakes in health interventions, dietary surveys and surveillance programs.

Time course of human skeletal muscle nitrate and nitrite concentration changes following dietary nitrate ingestion.

Dietary nitrate (NO3-) ingestion can be beneficial for health and exercise performance. Recently, based on animal and limited human studies, a skeletal muscle NO3- reservoir has been suggested to be important in whole body nitric oxide (NO) homeostasis. The purpose of this study was to determine the time course of changes in human skeletal muscle NO3- concentration ([NO3-]) following the ingestion of dietary NO3-. Sixteen participants were allocated to either an experimental group (NIT: n = 11) which consumed a bolus of ∼1300 mg (12.8 mmol) potassium nitrate (KNO3), or a placebo group (PLA: n = 5) which consumed a bolus of potassium chloride (KCl). Biological samples (muscle (vastus lateralis), blood, saliva and urine) were collected shortly before NIT or PLA ingestion and at intervals over the course of the subsequent 24 h. At baseline, no differences were observed for muscle [NO3-] and [NO2-] between NIT and PLA (P > 0.05). In PLA, there were no changes in muscle [NO3-] or [NO2-] over time. In NIT, muscle [NO3-] was significantly elevated above baseline (54 ± 29 nmol/g) at 0.5 h, reached a peak at 3 h (181 ± 128 nmol/g), and was not different to baseline from 9 h onwards (P > 0.05). Muscle [NO2-] did not change significantly over time. Following ingestion of a bolus of dietary NO3-, skeletal muscle [NO3-] increases rapidly, reaches a peak at ∼3 h and subsequently declines towards baseline values. Following dietary NO3- ingestion, human m. vastus lateralis [NO3-] expressed a slightly delayed pharmacokinetic profile compared to plasma [NO3-].

Highly Cushioned Shoes Improve Running Performance in Both the Absence and Presence of Muscle Damage.

PURPOSE: We tested the hypotheses that a highly cushioned running shoe (HCS) would 1) improve incremental exercise performance and reduce the oxygen cost (Oc) of submaximal running, and 2) attenuate the deterioration in Oc elicited by muscle damage consequent to a downhill run. METHODS: Thirty-two recreationally active participants completed an incremental treadmill test in an HCS and a control running shoe (CON) for the determination of Oc and maximal performance. Subsequently, participants were pair matched and randomly assigned to one of the two footwear conditions to perform a moderate-intensity running bout before and 48 h after a 30-min downhill run designed to elicit muscle damage. RESULTS: Incremental treadmill test performance was improved (+5.7%; +1:16 min:ss; P < 0.01) in the HCS when assessed in the nondamaged state, relative to CON. This coincided with a significantly lower Oc (-3.2%; -6 mL·kg-1·km-1; P < 0.001) at a range of running speeds and an increase in the speed corresponding to 3 mM blood lactate (+3.2%; +0.4 km·h-1; P < 0.05). As anticipated, the downhill run resulted in significant changes in biochemical, histological, and perceptual markers of muscle damage, and a significant increase in Oc (+5.2%; 10.1 mL·kg-1·km-1) was observed 48 h post. In the presence of muscle damage, Oc was significantly lower in HCS (-4.6%; -10 mL·kg-1·km-1) compared with CON. CONCLUSIONS: These results indicate that HCS improved incremental exercise performance and Oc in the absence of muscle damage and show, for the first time, that despite worsening of Oc consequent to muscle damage, improved Oc in HCS is maintained.

S-nitrosothiols, and other products of nitrate metabolism, are increased in multiple human blood compartments following ingestion of beetroot juice.

Ingested inorganic nitrate (NO3⁻) has multiple effects in the human body including vasodilation, inhibition of platelet aggregation, and improved skeletal muscle function. The functional effects of oral NO3⁻ involve the in vivo reduction of NO3⁻ to nitrite (NO2⁻) and thence to nitric oxide (NO). However, the potential involvement of S-nitrosothiol (RSNO) formation is unclear. We hypothesised that the RSNO concentration ([RSNO]) in red blood cells (RBCs) and plasma is increased by NO3⁻-rich beetroot juice ingestion. In healthy human volunteers, we tested the effect of dietary supplementation with NO3⁻-rich beetroot juice (BR) or NO3⁻-depleted beetroot juice (placebo; PL) on [RSNO], [NO3⁻] and [NO2⁻] in RBCs, whole blood and plasma, as measured by ozone-based chemiluminescence. The median basal [RSNO] in plasma samples (n = 22) was 10 (5-13) nM (interquartile range in brackets). In comparison, the median values for basal [RSNO] in the corresponding RBC preparations (n = 19) and whole blood samples (n = 19) were higher (p < 0.001) than in plasma, being 40 (30-60) nM and 35 (25-80) nM, respectively. The median RBC [RSNO] in a separate cohort of healthy subjects (n = 5) was increased to 110 (93-125) nM after ingesting BR (12.8 mmol NO3⁻) compared to a corresponding baseline value of 25 (21-31) nM (Mann-Whitney test, p < 0.01). The median plasma [RSNO] in another cohort of healthy subjects (n = 14) was increased almost ten-fold to 104 (58-151) nM after BR supplementation (7 × 6.4 mmol of NO3⁻ over two days, p < 0.01) compared to PL. In conclusion, RBC and plasma [RSNO] are increased by BR ingestion. In addition to NO2⁻, RSNO may be involved in dietary NO3⁻ metabolism/actions.

Human skeletal muscle nitrate store: influence of dietary nitrate supplementation and exercise.

KEY POINTS: Nitric oxide (NO), a potent vasodilator and a regulator of many physiological processes, is produced in mammals both enzymatically and by reduction of nitrite and nitrate ions. We have previously reported that, in rodents, skeletal muscle serves as a nitrate reservoir, with nitrate levels greatly exceeding those in blood or other internal organs, and with nitrate being reduced to NO during exercise. In the current study, we show that nitrate concentration is substantially greater in skeletal muscle than in blood and is elevated further by dietary nitrate ingestion in human volunteers. We also show that high-intensity exercise results in a reduction in the skeletal muscle nitrate store following supplementation, likely as a consequence of its reduction to nitrite and NO. We also report the presence of sialin, a nitrate transporter, and xanthine oxidoreductase in human skeletal muscle, indicating that muscle has the necessary apparatus for nitrate transport, storage and metabolism. ABSTRACT: Rodent skeletal muscle contains a large store of nitrate that can be augmented by the consumption of dietary nitrate. This muscle nitrate reservoir has been found to be an important source of nitrite and nitric oxide (NO) via its reduction by tissue xanthine oxidoreductase. To explore if this pathway is also active in human skeletal muscle during exercise, and if it is sensitive to local nitrate availability, we assessed exercise-induced changes in muscle nitrate and nitrite concentrations in young healthy humans, under baseline conditions and following dietary nitrate consumption. We found that baseline nitrate and nitrite concentrations were far higher in muscle than in plasma (∼4-fold and ∼29-fold, respectively), and that the consumption of a single bolus of dietary nitrate (12.8 mmol) significantly elevated nitrate concentration in both plasma (∼19-fold) and muscle (∼5-fold). Consistent with these observations, and with previous suggestions of active muscle nitrate transport, we present western blot data to show significant expression of the active nitrate/nitrite transporter sialin in human skeletal muscle. Furthermore, we report an exercise-induced reduction in human muscle nitrate concentration (by ∼39%), but only in the presence of an increased muscle nitrate store. Our results indicate that human skeletal muscle nitrate stores are sensitive to dietary nitrate intake and may contribute to NO generation during exercise. Together, these findings suggest that skeletal muscle plays an important role in the transport, storage and metabolism of nitrate in humans.

Discrete physiological effects of beetroot juice and potassium nitrate supplementation following 4-wk sprint interval training.

The physiological and exercise performance adaptations to sprint interval training (SIT) may be modified by dietary nitrate ([Formula: see text]) supplementation. However, it is possible that different types of [Formula: see text] supplementation evoke divergent physiological and performance adaptations to SIT. The purpose of this study was to compare the effects of 4-wk SIT with and without concurrent dietary [Formula: see text] supplementation administered as either [Formula: see text]-rich beetroot juice (BR) or potassium [Formula: see text] (KNO3). Thirty recreationally active subjects completed a battery of exercise tests before and after a 4-wk intervention in which they were allocated to one of three groups: 1) SIT undertaken without dietary [Formula: see text] supplementation (SIT); 2) SIT accompanied by concurrent BR supplementation (SIT + BR); or 3) SIT accompanied by concurrent KNO3 supplementation (SIT + KNO3). During severe-intensity exercise, V̇o2peak and time to task failure were improved to a greater extent with SIT + BR than SIT and SIT + KNO3 ( P < 0.05). There was also a greater reduction in the accumulation of muscle lactate at 3 min of severe-intensity exercise in SIT + BR compared with SIT + KNO3 ( P < 0.05). Plasma [Formula: see text] concentration fell to a greater extent during severe-intensity exercise in SIT + BR compared with SIT and SIT + KNO3 ( P < 0.05). There were no differences between groups in the reduction in the muscle phosphocreatine recovery time constant from pre- to postintervention ( P > 0.05). These findings indicate that 4-wk SIT with concurrent BR supplementation results in greater exercise capacity adaptations compared with SIT alone and SIT with concurrent KNO3 supplementation. This may be the result of greater NO-mediated signaling in SIT + BR compared with SIT + KNO3. NEW & NOTEWORTHY We compared the influence of different forms of dietary nitrate supplementation on the physiological and performance adaptations to sprint interval training (SIT). Compared with SIT alone, supplementation with nitrate-rich beetroot juice, but not potassium [Formula: see text], enhanced some physiological adaptations to training.