Dietary NO3- does not enhance endothelial dependent cutaneous vascular conductance in older women.

Prior work has yet to determine whether the reduction of dietary nitrate (NO3-) to NO, via the enterosalivary pathway, may modify cutaneous vascular conductance (CVC) responses to local heating in older women. Changes occurring with the transition to menopause related to hormonal flux, increased adiposity, and/or decreased physical activity may further compound the negative influence of aging on nitric oxide (NO)-dependent CVC. Herein, we characterized changes in NO-dependent CVC following acute ingestion of 140 mL of NO3--rich beetroot juice in 24 older women (age: 65 ± 5 y, BMI: 31.2 ± 3.7 kg/m2). Red blood cell (RBC) flux was measured continuously via laser-Doppler flowmetry on the dorsal aspect of the forearm during local skin heating to 39 °C/44 °C before and 3 h after NO3- ingestion. NO-dependent changes in CVC were calculated as RBC flux/mean arterial blood pressure at 39 °C and normalized as a proportion of maximal CVC at 44 °C (%CVCmax). Changes (Δ) in fractional exhaled NO (FeNO) following NO3- ingestion were used an index of NO bioavailability. Despite increased FeNO (+81 ± 70 %, P < 0.001), %CVCmax at 39 °C was reduced (-16 ± 10 %, P < 0.001) following NO3- ingestion. A greater reduction in %CVCmax was weakly to moderately associated with higher body fat% (r = 0.45 [0.05-0.72], P = 0.029), central adiposity% (r = 0.50 [0.13-0.75], P = 0.012), neutrophil% (r = 0.42 [0.02-0.70], P = 0.041), and higher neutrophil to lymphocyte ratio (r = 0.49 [0.11-0.75], P = 0.016). These findings demonstrate a single dose of dietary NO3- does not promote CVC responses to local heating in sedentary older women with overweight and obesity. Correlation with multiple biomarkers suggest systemic inflammation may be involved.

Short-term beetroot juice supplementation improves muscle speed and power but does not reduce blood pressure or oxidative stress in 65-79 y old men and women.

We have previously demonstrated that acute ingestion of inorganic nitrate (NO3-)-rich beetroot juice (BRJ), a source of nitric oxide (NO) via the NO3- → nitrite (NO2-) → NO pathway, can improve muscle speed and power in older individuals. It is not known, however, whether this effect is maintained or perhaps even enhanced with repeated ingestion, or if tolerance develops as with organic nitrates, e.g., nitroglycerin. Using a double-blind, placebo-controlled, crossover design, we therefore studied 16 community-dwelling older (age 71 ± 5 y) individuals after both acute and short-term (i.e., daily for 2 wk) BRJ supplementation. Blood samples were drawn and blood pressure was measured periodically during each ∼3 h experiment, with muscle function determined using isokinetic dynamometry. Acute ingestion of BRJ containing 18.2 ± 6.2 mmol of NO3- increased plasma NO3- and NO2- concentrations 23 ± 11 and 2.7 ± 2.1-fold over placebo, respectively. This was accompanied by 5 ± 11% and 7 ± 13% increases in maximal knee extensor speed (Vmax) and power (Pmax), respectively. After daily supplementation for 2 wk, BRJ ingestion elevated NO3- and NO2- levels 24 ± 12 and 3.3 ± 4.0-fold, respectively, whereas Vmax and Pmax were 7 ± 9% and 9 ± 11% higher than baseline. No changes were observed in blood pressure or in plasma markers of oxidative stress with either acute or short-term NO3- supplementation. We conclude that both acute and short-term dietary NO3- supplementation result in similar improvements in muscle function in older individuals. The magnitudes of these improvements are sufficient to offset the decline resulting from a decade or more of aging and are therefore likely to be clinically significant.

Skeletal muscle blood flow during exercise is reduced in a rat model of pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is characterized by exercise intolerance. Muscle blood flow may be reduced during exercise in PAH; however, this has not been directly measured. Therefore, we investigated blood flow during exercise in a rat model of monocrotaline (MCT)-induced pulmonary hypertension (PH). Male Sprague-Dawley rats (∼200 g) were injected with 60 mg/kg MCT (MCT, n = 23) and vehicle control (saline; CON, n = 16). Maximal rate of oxygen consumption (V̇o2max) and voluntary running were measured before PH induction. Right ventricle (RV) morphology and function were assessed via echocardiography and invasive hemodynamic measures. Treadmill running at 50% V̇o2max was performed by a subgroup of rats (MCT, n = 8; CON, n = 7). Injection of fluorescent microspheres determined muscle blood flow via photo spectroscopy. MCT demonstrated a severe phenotype via RV hypertrophy (Fulton index, 0.61 vs. 0.31; P < 0.001), high RV systolic pressure (51.5 vs. 22.4 mmHg; P < 0.001), and lower V̇o2max (53.2 vs. 71.8 mL·min-1·kg-1; P < 0.0001) compared with CON. Two-way ANOVA revealed exercising skeletal muscle blood flow relative to power output was reduced in MCT compared with CON (P < 0.001), and plasma lactate was increased in MCT (10.8 vs. 4.5 mmol/L; P = 0.002). Significant relationships between skeletal blood flow and blood lactate during exercise were observed for individual muscles (r = -0.58 to -0.74; P < 0.05). No differences in capillarization were identified. Skeletal muscle blood flow is significantly reduced in experimental PH. Reduced blood flow during exercise may be, at least in part, consequent to reduced exercise intensity in PH. This adds further evidence of peripheral muscle dysfunction and exercise intolerance in PAH.

Nitric oxide and skeletal muscle contractile function.

Nitric oxide (NO) is complex modulator of skeletal muscle contractile function, capable of increasing or decreasing force and power output depending on multiple factors. This review explores the effects and potential mechanisms for modulation of skeletal muscle contractile function by NO, from pharmacological agents in isolated muscle preparations to dietary nitrate supplementation in humans and animals. Pharmacological manipulation in vitro suggests that NO signaling diminishes submaximal isometric force, whereas dietary manipulation in vivo suggest that NO enhances submaximal force. The bases for these different responses are unknown but could reflect dose-dependent effects. Maximal isometric force is unaffected by physiologically relevant levels of NO, which do not induce overt protein oxidation. Pharmacological and dietary manipulation of NO signaling enhances the maximal rate of isometric force development, unloaded shortening velocity, and peak power. We hypothesize that these effects are mediated by post-translational modifications of myofibrillar proteins that modulate thick filament regulation of contraction (e.g., mechanosensing and strain-dependence of cross-bridge kinetics). NO effects on contractile function appear to have some level of fiber type and sex-specificity. The mechanisms behind NO-mediated changes in skeletal muscle function need to be explored through proteomics analysis and advanced biophysical assays to advance the development of small molecules and open intriguing therapeutic and ergogenic possibilities for aging, disease, and athletic performance.

Simultaneous Pharmacokinetic Analysis of Nitrate and its Reduced Metabolite, Nitrite, Following Ingestion of Inorganic Nitrate in a Mixed Patient Population.

PURPOSE: The pharmacokinetic properties of plasma NO3- and its reduced metabolite, NO2-, have been separately described, but there has been no reported attempt to simultaneously model their pharmacokinetics following NO3- ingestion. This report describes development of such a model from retrospective analyses of concentrations largely obtained from primary endpoint efficacy trials. METHODS: Linear and non-linear mixed effects analyses were used to statistically define concentration dependency on time, dose, as well as patient and study variables, and to integrate NO3- and NO2- concentrations from studies conducted at different times, locations, patient groups, and several studies in which sample range was limited to a few hours. Published pharmacokinetic studies for both substances were used to supplement model development. RESULTS: A population pharmacokinetic model relating NO3- and NO2- concentrations was developed. The model incorporated endogenous levels of the two entities, and determined these were not influenced by exogenous NO3- delivery. Covariate analysis revealed intersubject variability in NO3- exposure was partially described by body weight differences influencing volume of distribution. The model was applied to visualize exposure versus response (muscle contraction performance) in individual patients. CONCLUSIONS: Extension of the present first-generation model, to ultimately optimize NO3- dose versus pharmacological effects, is warranted.

What's in Your Beet Juice? Nitrate and Nitrite Content of Beet Juice Products Marketed to Athletes.

Consumption of beetroot juice (BRJ) supplements has become popular among athletes, because beets tend to be rich in nitrate (NO3-), which can enhance exercise performance by increasing nitric oxide production. The NO3- content of beets can vary significantly, however, making it difficult to know how much NO3- any product actually contains. Samples from 45 different lots of 24 different BRJ products from 21 different companies were therefore analyzed for NO3- (and nitrite; NO2-) concentration using high performance liquid chromatography. The NO3- and NO2- content, i.e., amount per serving, was then calculated based on either 1) the manufacturer's recommended serving size (for prepackaged/single dose products) or 2) as used in previous studies, a volume of 500 mL (for BRJ sold in bulk containers). There was moderate-to-large variability in NO3- content between samples of the same product, with a mean coefficient of variation of 30±26% (range 2 to 83%). There was even greater variability between products, with a ~50-fold range in NO3- content between the lowest and highest. Only five products consistently provided ≥5 mmol of NO3- per serving, which seems to be the minimal dose required to enhance exercise performance in most individuals. NO2- contents were generally low (i.e., ≤0.5% compared to NO3-), although two products contained 10 and 14%. The present results may be useful to athletes and their support staff contemplating which (if any) BRJ product to utilize. These data may also offer insight into variability in the literature with respect to the effects of BRJ on exercise performance.

Dietary Nitrate Increases VO2peak and Performance but Does Not Alter Ventilation or Efficiency in Patients With Heart Failure With Reduced Ejection Fraction.

BACKGROUND: Patients with heart failure with reduced ejection fraction (HFrEF) exhibit lower efficiency, dyspnea, and diminished peak oxygen uptake (VO2peak) during exercise. Dietary nitrate (NO3-), a source of nitric oxide (NO), has improved these measures in some studies of other populations. We determined the effects of acute NO3- ingestion on exercise responses in 8 patients with HFrEF using a randomized, double-blind, placebo-controlled, crossover design. METHODS AND RESULTS: Plasma NO3-, nitrite (NO2-), and breath NO were measured at multiple time points and respiratory gas exchange was determined during exercise after ingestion of beetroot juice containing or devoid of 11.2 mmol of NO3-. NO3- intake increased (P < .05-0.001) plasma NO3- and NO2- and breath NO by 1469 ± 245%, 105 ± 34%, and 60 ± 18%, respectively. Efficiency and ventilation during exercise were unchanged. However, NO3- ingestion increased (P < .05) VO2peak by 8 ± 2% (ie, from 21.4 ± 2.1 to 23.0 ± 2.3 mL.min-1.kg-1). Time to fatigue improved (P < .05) by 7 ± 3 % (ie, from 582 ± 84 to 612 ± 81 seconds). CONCLUSIONS: Acute dietary NO3- intake increases VO2peak and performance in patients with HFrEF. These data, in conjunction with our recent data demonstrating that dietary NO3- also improves muscle contractile function, suggest that dietary NO3- supplementation may be a valuable means of enhancing exercise capacity in this population.

Dietary Nitrate Enhances the Contractile Properties of Human Skeletal Muscle.

Dietary nitrate, a source of nitric oxide (NO), improves the contractile properties of human muscle. We present the hypothesis that this is due to nitrosylation of the ryanodine receptor and increased NO signaling via the soluble guanyl cyclase-cyclic guanosine monophosphate-protein kinase G pathway, which together increase the free intracellular Ca concentration along with the Ca sensitivity of the myofilaments themselves.

Sex affects myocardial blood flow and fatty acid substrate metabolism in humans with nonischemic heart failure.

BACKGROUND: In animal models of heart failure (HF), myocardial metabolism shifts from high-energy fatty acid (FA) metabolism toward glucose. However, FA (vs glucose) metabolism generates more ATP/mole; thus, FA metabolism may be especially advantageous in HF. Sex modulates myocardial blood flow (MBF) and substrate metabolism in normal humans. Whether sex affects MBF and metabolism in patients with HF is unknown. METHODS AND RESULTS: We studied 19 well-matched men and women with nonischemic HF (EF ≤ 35%). MBF and myocardial substrate metabolism were quantified using positron emission tomography. Women had higher MBF (mL/g/minute), FA uptake (mL/g/minute), and FA utilization (nmol/g/minute) (P < 0.005, P < 0.005, P < 0.05, respectively) and trended toward having higher FA oxidation than men (P = 0.09). These findings were independent of age, obesity, and insulin resistance. There were no sex-related differences in fasting myocardial glucose uptake or metabolism. Higher MBF was related to improved event-free survival (HR 0.31, P = 0.02). CONCLUSIONS: In nonischemic HF, women have higher MBF and FA uptake and metabolism than men, irrespective of age, obesity, or insulin resistance. Moreover, higher MBF portends a better prognosis. These sex-related differences should be taken into account in the development and targeting of novel agents aimed at modulating MBF and metabolism in HF.

Oximetric angiosome imaging in diabetic feet.

PURPOSE: To develop a noncontrast oximetric angiosome imaging approach to assess skeletal muscle oxygenation in diabetic feet. MATERIALS AND METHODS: In four healthy and five subjects with diabetes, the feasibility of foot oximetry was examined using a 3T clinical magnetic resonance imaging (MRI) scanner. The subjects' feet were scanned at rest and during a toe-flexion isometric exercise. The oxygen extraction fraction of skeletal muscle was measured using a susceptibility-based MRI method. Our newly developed MR foot oximetric angiosome model was compared with the traditional angiosome model in the assessment of the distribution of oxygen extraction fraction. RESULTS: Using the traditional angiosome during the toe-flexion exercise, the oxygen extraction fraction in the medial foot of healthy subjects increased (4.9 ± 3%) and decreased (-2.7 ± 4.4%) in subjects with diabetes (difference = 7.6%; 95% confidence interval = -13.7 ± 1.4; P = 0.02). Using the oximetric angiosome, the percent difference in the areas of oxygen extraction fraction within the 0.7-1.0 range (expected oxygen extraction fraction during exercise) between rest and exercise was higher in healthy subjects (8 ± 4%) than in subjects with diabetes (4 ± 4%; P = 0.02). CONCLUSION: This study demonstrates the feasibility of measuring skeletal muscle oxygen extraction fraction in the foot muscle during a toe-flexion isometric exercise. Instead of assessing oxygen extraction fraction in a foot muscle region linked to a supplying artery (traditional angiosome), the foot oximetric angiosome model assesses oxygen extraction fraction by its different levels in all foot muscle regions and thus may be more appropriate for assessing local ischemia in ulcerated diabetic feet. J. Magn. Reson. Imaging 2016. J. MAGN. RESON. IMAGING 2016;44:940-946.

Dietary Nitrate and Skeletal Muscle Contractile Function in Heart Failure.

Heart failure (HF) patients suffer from exercise intolerance that diminishes their ability to perform normal activities of daily living and hence compromises their quality of life. This is due largely to detrimental changes in skeletal muscle mass, structure, metabolism, and function. This includes an impairment of muscle contractile performance, i.e., a decline in the maximal force, speed, and power of muscle shortening. Although numerous mechanisms underlie this reduction in contractility, one contributing factor may be a decrease in nitric oxide (NO) bioavailability. Consistent with this, recent data demonstrate that acute ingestion of NO3 (-)-rich beetroot juice, a source of NO via the NO synthase-independent enterosalivary pathway, markedly increases maximal muscle speed and power in HF patients. This review discusses the role of muscle contractile dysfunction in the exercise intolerance characteristic of HF, and the evidence that dietary NO3 (-) supplementation may represent a novel and simple therapy for this currently underappreciated problem.

Type 2 diabetes, obesity, and sex difference affect the fate of glucose in the human heart.

Type 2 diabetes, obesity, and sex difference affect myocardial glucose uptake and utilization. However, their effect on the intramyocellular fate of glucose in humans has been unknown. How the heart uses glucose is important, because it affects energy production and oxygen efficiency, which in turn affect heart function and adaptability. We hypothesized that type 2 diabetes, sex difference, and obesity affect myocardial glucose oxidation, glycolysis, and glycogen production. In a first-in-human study, we measured intramyocardiocellular glucose metabolism from time-activity curves generated from previously obtained positron emission tomography scans of 110 subjects in 3 groups: nonobese, obese, and diabetes. Group and sex difference interacted in the prediction of all glucose uptake, utilization, and metabolism rates. Group independently predicted fractional glucose uptake and its components: glycolysis, glycogen deposition, and glucose oxidation rates. Sex difference predicted glycolysis rates. However, there were fewer differences in glucose metabolism between diabetic patients and others when plasma glucose levels were included in the modeling. The potentially detrimental effects of obesity and diabetes on myocardial glucose metabolism are more pronounced in men than women. This sex difference dimorphism needs to be taken into account in the design, trials, and application of metabolic modulator therapy. Slightly higher plasma glucose levels improve depressed glucose oxidation and glycogen deposition rates in diabetic patients.

Effect of acute dietary nitrate intake on maximal knee extensor speed and power in healthy men and women.

Nitric oxide (NO) has been demonstrated to enhance the maximal shortening velocity and maximal power of rodent muscle. Dietary nitrate (NO3(-)) intake has been demonstrated to increase NO bioavailability in humans. We therefore hypothesized that acute dietary NO3(-) intake (in the form of a concentrated beetroot juice (BRJ) supplement) would improve muscle speed and power in humans. To test this hypothesis, healthy men and women (n = 12; age = 22-50 y) were studied using a randomized, double-blind, placebo-controlled crossover design. After an overnight fast, subjects ingested 140 mL of BRJ either containing or devoid of 11.2 mmol of NO3(-). After 2 h, knee extensor contractile function was assessed using a Biodex 4 isokinetic dynamometer. Breath NO levels were also measured periodically using a Niox Mino analyzer as a biomarker of whole-body NO production. No significant changes in breath NO were observed in the placebo trial, whereas breath NO rose by 61% (P < 0.001; effect size = 1.19) after dietary NO3(-) intake. This was accompanied by a 4% (P < 0.01; effect size = 0.74) increase in peak knee extensor power at the highest angular velocity tested (i.e., 6.28 rad/s). Calculated maximal knee extensor power was therefore greater (i.e., 7.90 ± 0.59 vs. 7.44 ± 0.53 W/kg; P < 0.05; effect size = 0.63) after dietary NO3(-) intake, as was the calculated maximal velocity (i.e., 14.5 ± 0.9 vs. 13.1 ± 0.8 rad/s; P < 0.05; effect size = 0.67). No differences in muscle function were observed during 50 consecutive knee extensions performed at 3.14 rad/s. We conclude that acute dietary NO3(-) intake increases whole-body NO production and muscle speed and power in healthy men and women.

Noncontrast skeletal muscle oximetry.

PURPOSE: The objective of this study was to develop a new noncontrast method to directly quantify regional skeletal muscle oxygenation. METHODS: The feasibility of the method was examined in five healthy volunteers using a 3 T clinical MRI scanner, at rest and during a sustained isometric contraction. The perfusion of skeletal muscle of the calf was measured using an arterial spin labeling method, whereas the oxygen extraction fraction of the muscle was measured using a susceptibility-based MRI technique. RESULTS: In all volunteers, the perfusion in soleus muscle increased significantly from 6.5 ± 2.0 mL (100 g min)(-1) at rest to 47.9 ± 7.7 mL (100 g min)(-1) during exercise (P < 0.05). Although the corresponding oxygen extraction fraction did not change significantly, the rate of oxygen consumption increased from 0.43 ± 0.13 to 4.2 ± 1.5 mL (100 g min)(-1) (P < 0.05). Similar results were observed in gastrocnemius muscle but with greater oxygen extraction fraction increase than the soleus muscle. CONCLUSION: This is the first MR oximetry developed for quantification of regional skeletal muscle oxygenation. A broad range of medical conditions could benefit from these techniques, including cardiology, gerontology, kinesiology, and physical therapy.

Beetroot juice supplementation and exercise performance: is there more to the story than just nitrate?

This mini-review summarizes the comparative effects of different sources of dietary nitrate (NO3-), beetroot juice (BRJ) and nitrate salts (NIT), on physiological function and exercise capacity. Our objectives were to determine whether BRJ is superior to NIT in enhancing exercise-related outcomes, and to explore the potential contribution of other putatively beneficial compounds in BRJ beyond NO3-. We conducted a comparative analysis of recent studies focused on the impact of BRJ versus NIT on submaximal oxygen consumption (VO2), endurance performance, adaptations to training, and recovery from muscle-damaging exercise. While both NO3- sources provide benefits, there is some evidence that BRJ may offer additional advantages, specifically in reducing VO2 during high-intensity exercise, magnifying performance improvements with training, and improving recovery post-exercise. These reported differences could be due to the hypothesized antioxidant and/or anti-inflammatory properties of BRJ resulting from the rich spectrum of phytonutrients it contains. However, significant limitations to published studies directly comparing BRJ and NIT make it quite challenging to draw any firm conclusions. We provide recommendations to help guide further research into the important question of whether there is more to the story of BRJ than just NO3-.