Effects of short-term dietary nitrate supplementation on exercise and coronary blood flow responses in patients with peripheral artery disease.

Background: Peripheral arterial disease (PAD) is a prevalent vascular disorder characterized by atherosclerotic occlusion of peripheral arteries, resulting in reduced blood flow to the lower extremities and poor walking ability. Older patients with PAD are also at a markedly increased risk of cardiovascular events, including myocardial infarction. Recent evidence indicates that inorganic nitrate supplementation, which is abundant in certain vegetables, augments nitric oxide (NO) bioavailability and may have beneficial effects on walking, blood pressure, and vascular function in patients with PAD. Objective: We sought to determine if short-term nitrate supplementation (via beetroot juice) improves peak treadmill time and coronary hyperemic responses to plantar flexion exercise relative to placebo (nitrate-depleted juice) in older patients with PAD. The primary endpoints were peak treadmill time and the peak coronary hyperemic response to plantar flexion exercise. Methods: Eleven PAD patients (52-80 yr.; 9 men/2 women; Fontaine stage II) were randomized (double-blind) to either nitrate-rich (Beet-IT, 0.3 g inorganic nitrate twice/day; BRnitrate) or nitrate-depleted (Beet-IT, 0.04 g inorganic nitrate twice/day, BRplacebo) beetroot juice for 4 to 6 days, followed by a washout of 7 to 14 days before crossing over to the other treatment. Patients completed graded plantar flexion exercise with their most symptomatic leg to fatigue, followed by isometric handgrip until volitional fatigue at 40% of maximum on day 4 of supplementation, and a treadmill test to peak exertion 1-2 days later while continuing supplementation. Hemodynamics and exercise tolerance, and coronary blood flow velocity (CBV) responses were measured. Results: Although peak walking time and claudication onset time during treadmill exercise did not differ significantly between BRplacebo and BRnitrate, the diastolic blood pressure response at the peak treadmill walking stage was significantly lower in the BRnitrate condition. Increases in CBV from baseline to peak plantar flexion exercise after BRplacebo and BRnitrate showed a trend for a greater increase in CBV at the peak workload of plantar flexion with BRnitrate (p = 0.06; Cohen's d = 0.56). Conclusion: Overall, these preliminary findings suggest that inorganic nitrate supplementation in PAD patients is safe, well-tolerated, and may improve the coronary hyperemic and blood pressure responses when their calf muscles are most predisposed to ischemia.Clinical trial registration:https://clinicaltrials.gov/, identifier NCT02553733.

Thiol-catalyzed formation of NO-ferroheme regulates intravascular NO signaling.

Nitric oxide (NO) is an endogenously produced signaling molecule that regulates blood flow and platelet activation. However, intracellular and intravascular diffusion of NO are limited by scavenging reactions with several hemoproteins, raising questions as to how free NO can signal in hemoprotein-rich environments. We explore the hypothesis that NO can be stabilized as a labile ferrous heme-nitrosyl complex (Fe2+-NO, NO-ferroheme). We observe a reaction between NO, labile ferric heme (Fe3+) and reduced thiols to yield NO-ferroheme and a thiyl radical. This thiol-catalyzed reductive nitrosylation occurs when heme is solubilized in lipophilic environments such as red blood cell membranes or bound to serum albumin. The resulting NO-ferroheme resists oxidative inactivation, is soluble in cell membranes and is transported intravascularly by albumin to promote potent vasodilation. We therefore provide an alternative route for NO delivery from erythrocytes and blood via transfer of NO-ferroheme and activation of apo-soluble guanylyl cyclase.

Thiol catalyzed formation of NO-ferroheme regulates canonical intravascular NO signaling.

Nitric oxide (NO) is an endogenously produced physiological signaling molecule that regulates blood flow and platelet activation. However, both the intracellular and intravascular diffusion of NO is severely limited by scavenging reactions with hemoglobin, myoglobin, and other hemoproteins, raising unanswered questions as to how free NO can signal in hemoprotein-rich environments, like blood and cardiomyocytes. We explored the hypothesis that NO could be stabilized as a ferrous heme-nitrosyl complex (Fe 2+ -NO, NO-ferroheme) either in solution within membranes or bound to albumin. Unexpectedly, we observed a rapid reaction of NO with free ferric heme (Fe 3+ ) and a reduced thiol under physiological conditions to yield NO-ferroheme and a thiyl radical. This thiol-catalyzed reductive nitrosylation reaction occurs readily when the hemin is solubilized in lipophilic environments, such as red blood cell membranes, or bound to serum albumin. NO-ferroheme albumin is stable, even in the presence of excess oxyhemoglobin, and potently inhibits platelet activation. NO-ferroheme-albumin administered intravenously to mice dose-dependently vasodilates at low- to mid-nanomolar concentrations. In conclusion, we report the fastest rate of reductive nitrosylation observed to date to generate a NO-ferroheme molecule that resists oxidative inactivation, is soluble in cell membranes, and is transported intravascularly by albumin to promote potent vasodilation.

Efficacy and Variability in Plasma Nitrite Levels during Long-Term Supplementation with Nitrate Containing Beetroot Juice.

Long-term consumption of beetroot juice on efficacy of converting dietary nitrate to plasma nitrate and nitrite was investigated. Adults were randomized to consume either beetroot juice with 380 mg of nitrate (BR) or a beetroot juice placebo (PL) for 12-weeks. Plasma nitrate and nitrite were measured before and 90-minutes after consuming their intervention beverage. Percent change in nitrite across the 90 min was greater in BR (273.2 ± 39.9%) vs. PL (4.9 ± 36.9%). Long-term consumption of nitrate containing beetroot juice increased fasting nitrate and nitrite plasma levels compared to baseline.

Effect of Vitamin C and Protein Supplementation on Plasma Nitrate and Nitrite Response following Consumption of Beetroot Juice.

Beetroot juice is a food high in nitrate and is associated with cardiometabolic health benefits and enhanced exercise performance through the production of nitric oxide in the nitrate−nitrite−nitric oxide pathway. Since various food components influence this pathway, the aim of this trial was to study the effect of beetroot juice alone and in conjunction with vitamin C or protein on the acute response to plasma nitrate and nitrite levels in healthy middle- to older-aged adults. In this cross-over trial, each participant received, in a randomized order, a single dose of Beet It Sport® alone; Beet It Sport®, plus a 200 mg vitamin C supplement; and Beet It Sport® plus 15 g of whey protein. Plasma levels of nitrate and nitrite were determined prior to and at 1 and 3 h after intervention. Log plasma nitrate and nitrite was calculated to obtain data that were normally distributed, and these data were analyzed using two-way within-factors ANOVA, with time and treatment as the independent factors. There were no statistically significant differences for log plasma nitrate (p = 0.308) or log plasma nitrite (p = 0.391) values across treatments. Log plasma nitrate increased significantly from pre-consumption levels after 1 h (p < 0.001) and 3 h (p < 0.001), but plasma nitrate was lower at 3 h than 1 h (p < 0.001). Log plasma nitrite increased from pre to 1 h (p < 0.001) and 3 h (p < 0.001) with log values at 3 h higher than at 1 h (p = 0.003). In this cohort, we observed no differences in log plasma nitrate and nitrite at 1 h and 3 h after co-ingesting beetroot juice with vitamin C or a whey protein supplement compared to beetroot juice alone. Further research needs to be undertaken to expand the blood-sampling time-frame and to examine factors that may influence the kinetics of the plasma nitrate to nitrite efficacy, such as differences in fluid volume and osmolarity between treatments employed.

Mycobacterium tuberculosis DosS binds H2S through its Fe3+ heme iron to regulate the DosR dormancy regulon.

Mycobacterium tuberculosis (Mtb) senses and responds to host-derived gasotransmitters NO and CO via heme-containing sensor kinases DosS and DosT and the response regulator DosR. Hydrogen sulfide (H2S) is an important signaling molecule in mammals, but its role in Mtb physiology is unclear. We have previously shown that exogenous H2S can modulate expression of genes in the Dos dormancy regulon via an unknown mechanism(s). Here, we test the hypothesis that Mtb senses and responds to H2S via the DosS/T/R system. Using UV-Vis and EPR spectroscopy, we show that H2S binds directly to the ferric (Fe3+) heme of DosS (KDapp = 5.30 µM) but not the ferrous (Fe2+) form. No interaction with DosT(Fe2+-O2) was detected. We found that the binding of sulfide can slowly reduce the DosS heme iron to the ferrous form. Steered Molecular Dynamics simulations show that H2S, and not the charged HS- species, can enter the DosS heme pocket. We also show that H2S increases DosS autokinase activity and subsequent phosphorylation of DosR, and H2S-mediated increases in Dos regulon gene expression is lost in Mtb lacking DosS. Finally, we demonstrate that physiological levels of H2S in macrophages can induce DosR regulon genes via DosS. Overall, these data reveal a novel mechanism whereby Mtb senses and responds to a third host gasotransmitter, H2S, via DosS(Fe3+). These findings highlight the remarkable plasticity of DosS and establish a new paradigm for how bacteria can sense multiple gasotransmitters through a single heme sensor kinase.

Inorganic nitrate supplementation and blood flow restricted exercise tolerance in post-menopausal women.

Exercise tolerance appears to benefit most from dietary nitrate (NO3-) supplementation when muscle oxygen (O2) availability is low. Using a double-blind, randomized cross-over design, we tested the hypothesis that acute NO3- supplementation would improve blood flow restricted exercise duration in post-menopausal women, a population with reduced endogenous nitric oxide bioavailability. Thirteen women (57-76 yr) performed rhythmic isometric handgrip contractions (10% MVC, 30 per min) during progressive forearm blood flow restriction (upper arm cuff gradually inflated 20 mmHg each min) on three study visits, with 7-10 days between visits. Approximately one week following the first (familiarization) visit, participants consumed 140 ml of NO3- concentrated (9.7 mmol, 0.6 gm NO3-) or NO3-depleted beetroot juice (placebo) on separate days (≥7 days apart), with handgrip exercise beginning 100 min post-consumption. Handgrip force recordings were analyzed to determine if NO3- supplementation enhanced force development as blood flow restriction progressed. Nitrate supplementation increased plasma NO3- (16.2-fold) and NO2- (4.2-fold) and time to volitional fatigue (61.8 ± 56.5 s longer duration vs. placebo visit; p = 0.03). Nitrate supplementation increased the rate of force development as forearm muscle ischemia progressed (p = 0.023 between 50 and 75% of time to fatigue) with non-significant effects thereafter (p = 0.052). No effects of nitrate supplementation were observed for mean duration of contraction or relaxation rates (all p > 0.150). These results suggest that acute NO3- supplementation prolongs time-to-fatigue and speeds grip force development during progressive forearm muscle ischemia in postmenopausal women.

Tri-iodide and vanadium chloride based chemiluminescent methods for quantification of nitrogen oxides.

Nitric Oxide (NO) is an important signaling molecule that plays roles in controlling vascular tone, hemostasis, host defense, and many other physiological functions. Low NO bioavailability contributes to pathology and NO administration has therapeutic potential in a variety of diseases. Thus, accurate measurements of NO bioavailability and reactivity are critical. Due to its short lifetime in vivo and many in vitro conditions, NO bioavailability and reactivity are often best determined by measuring NO congeners and metabolites that are more stable. Chemiluminescence-based detection of NO following chemical reduction of these compounds using the tri-iodide and vanadium chloride methods have been widely used in a variety of clinical and laboratory studies. In this review, we describe these methods used to detect nitrite, nitrate, nitrosothiols and other species and discuss limitations and proper controls.

Mechanistic insights into cell-free hemoglobin-induced injury during septic shock.

Cell-free hemoglobin (CFH) levels are elevated in septic shock and are higher in nonsurvivors. Whether CFH is only a marker of sepsis severity or is involved in pathogenesis is unknown. This study aimed to investigate whether CFH worsens sepsis-associated injuries and to determine potential mechanisms of harm. Fifty-one, 10-12 kg purpose-bred beagles were randomized to receive Staphylococcus aureus intrapulmonary challenges or saline followed by CFH infusions (oxyhemoglobin >80%) or placebo. Animals received antibiotics and intensive care support for 96 h. CFH significantly increased mean pulmonary arterial pressures and right ventricular afterload in both septic and nonseptic animals, effects that were significantly greater in nonsurvivors. These findings are consistent with CFH-associated nitric oxide (NO) scavenging and were associated with significantly depressed cardiac function, and worsened shock, lactate levels, metabolic acidosis, and multiorgan failure. In septic animals only, CFH administration significantly increased mean alveolar-arterial oxygenation gradients, also to a significantly greater degree in nonsurvivors. CFH-associated iron levels were significantly suppressed in infected animals, suggesting that bacterial iron uptake worsened pneumonia. Notably, cytokine levels were similar in survivors and nonsurvivors and were not predictive of outcome. In the absence and presence of infection, CFH infusions resulted in pulmonary hypertension, cardiogenic shock, and multiorgan failure, likely through NO scavenging. In the presence of infection alone, CFH infusions worsened oxygen exchange and lung injury, presumably by supplying iron that promoted bacterial growth. CFH elevation, a known consequence of clinical septic shock, adversely impacts sepsis outcomes through more than one mechanism, and is a biologically plausible, nonantibiotic, noncytokine target for therapeutic intervention.NEW & NOTEWORTHY Cell-free hemoglobin (CFH) elevations are a known consequence of clinical sepsis. Using a two-by-two factorial design and extensive physiological and biochemical evidence, we found a direct mechanism of injury related to nitric oxide scavenging leading to pulmonary hypertension increasing right heart afterload, depressed cardiac function, worsening circulatory failure, and death, as well as an indirect mechanism related to iron toxicity. These discoveries alter conventional thinking about septic shock pathogenesis and provide novel therapeutic approaches.

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

[This corrects the article DOI: 10.1371/journal.pone.0235047.].

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.

Haptoglobin therapy has differential effects depending on severity of canine septic shock and cell-free hemoglobin level.

BACKGROUND: During sepsis, higher plasma cell-free hemoglobin (CFH) levels portend worse outcomes. In sepsis models, plasma proteins that bind CFH improve survival. In our canine antibiotic-treated Staphylococcus aureus pneumonia model, with and without red blood cell (RBC) exchange transfusion, commercial human haptoglobin (Hp) concentrates bound and compartmentalized CFH intravascularly, increased CFH clearance, and lowered iron levels, improving shock, lung injury, and survival. We now investigate in our model how very high CFH levels and treatment time affect Hp's beneficial effects. MATERIALS AND METHODS: Two separate canine pneumonia sepsis Hp studies were undertaken: one with exchange transfusion of RBCs after prolonged storage to raise CFH to very high levels and another with rapidly lethal sepsis alone to shorten time to treat. All animals received continuous standard intensive care unit supportive care for 96 hours. RESULTS: Older RBCs markedly elevated plasma CFH levels and, when combined with Hp therapy, created supraphysiologic CFH-Hp complexes that did not increase CFH or iron clearance or improve lung injury and survival. In a rapidly lethal bacterial challenge model without RBC transfusion, Hp binding did not increase clearance of complexes or iron or show benefits seen previously in the less lethal model. DISCUSSION: High-level CFH-Hp complexes may impair clearance mechanisms and eliminate Hp's beneficial effect during sepsis. Rapidly lethal sepsis narrows the therapeutic window for CFH and iron clearance, also decreasing Hp's beneficial effects. In designing clinical trials, dosing and kinetics may be critical factors if Hp infusion is used to treat sepsis.

Development of Zinc Chelating Resin Polymer Beads for the Removal of Cell-Free Hemoglobin.

Red blood cell (RBC) hemolysis is one of the most common storage lesions in packed RBCs (pRBC). Older units of pRBCs, especially those > 21 days old, have increasing levels of hemolysis leading to increased oxidative stress and premature platelet activation. This effect can mostly be attributed to the increase of cell-free hemoglobin (Hb). Therefore, removal of cell-free Hb from pRBCs prior to transfusion could mitigate these deleterious effects. We propose a new method for the removal of Hb from pRBCs using zinc beads. Prepared Hb solutions and pRBCs were treated with zinc beads using two different protocols. UV-Vis spectrophotometry was used to determine Hb concentrations, before and after treatment. Experiments were run in triplicate and paired t tests were used to determine significant differences between groups. Zinc beads removed on average 94% of cell-free Hb within 15 min and 78% Hb from pRBCs (p < 0.0001), demonstrating a maximum binding capacity ~ 66.2 ± 0.7 mg Hb/mL beads. No differences in RBC morphology or deformability were observed after treatment. This study demonstrates the feasibility of using zinc beads for the rapid and targeted removal of Hb from pRBC units. Further investigation is needed to scale this method for large volume removal.

Effects of acute dietary nitrate supplementation on aortic blood pressures and pulse wave characteristics in post-menopausal women.

PURPOSE: Consumption of nitrate-rich beetroot juice can lower blood pressure in peripheral as well as central arteries and may exert additional hemodynamic benefits (e.g. reduced aortic wave reflections). The specific influence of nitrate supplementation on arterial pressures and aortic wave properties in postmenopausal women, a group that experiences accelerated increases in these variables with age, is unknown. Accordingly, the primary aim of this study was to determine the effect of consuming nitrate-rich beetroot juice on resting brachial and aortic blood pressures (BP) and pulse wave characteristics in a group of healthy postmenopausal women, in comparison to a true (nitrate-free beetroot juice) placebo. METHODS: Brachial (oscillometric cuff) and radial (SphygmoCor) pressures and derived-aortic waveforms were measured during supine rest in thirteen healthy postmenopausal women (63 ±â€¯1 yr) before and 100 min after consumption of 140 ml of either nitrate-rich (9.7 mmol, 0.6 gm NO3-) or nitrate-depleted beetroot juice on randomized visits approximately 10 days apart (cross-over design). Ten young premenopausal women (22 ±â€¯1 yr) served as a reference (non-supplemented) cohort. RESULTS: Brachial and derived-aortic variables showed the expected age-associated differences in these women (all p < 0.05). In post-menopausal women, nitrate supplementation reduced (p < 0.05 vs. placebo visit) brachial systolic BP (BRnitrate -4.9 ±â€¯2.1 mmHg vs BRplacebo +1.1 ± 1.8 mmHg), brachial mean BP (BRnitrate -4.1 ±â€¯1.7 mmHg vs BRplacebo +0.9 ± 1.3 mmHg), aortic systolic BP (BRnitrate -6.3 ±â€¯2.0 mmHg vs BRplacebo +0.5 ± 1.7 mmHg) and aortic mean BP (BRnitrate -4.1 ±â€¯1.7 mmHg vs BRplacebo +0.9 ± 1.3 mmHg), and increased pulse pressure amplification (BRnitrate +4.6 ± 2.0% vs BRplacebo +0.7 ± 2.5%, p = 0.04), but did not alter aortic pulse wave velocity or any other derived-aortic variables (e.g., augmentation pressure or index). CONCLUSIONS: Dietary nitrate supplementation favorably modifies aortic systolic and mean blood pressure under resting conditions in healthy postmenopausal women. Acute supplementation of nitrate does not, however, appear to restore indices of aortic stiffness in this group. Future work should evaluate chronic, long-term effects of this non-pharmacological supplement.

Erythrocytic bioactivation of nitrite and its potentiation by far-red light.

BACKGROUND: Nitrite is reduced by heme-proteins and molybdenum-containing enzymes to form the important signaling molecule nitric oxide (NO), mediating NO signaling. Substantial evidence suggests that deoxygenated hemoglobin within red blood cells (RBCs) is the main erythrocytic protein responsible for mediating nitrite-dependent NO signaling. In other work, infrared and far red light have been shown to have therapeutic potential that some attribute to production of NO. Here we explore whether a combination of nitrite and far red light treatment has an additive effect in NO-dependent processes, and whether this effect is mediated by RBCs. METHODS AND RESULTS: Using photoacoustic imaging in a rat model as a function of varying inspired oxygen, we found that far red light (660 nm, five min. exposure) and nitrite feeding (three weeks in drinking water at 100 mg/L) each separately increased tissue oxygenation and vessel diameter, and the combined treatment was additive. We also employed inhibition of human platelet activation measured by flow cytometry to assess RBC-dependent nitrite bioactivation and found that far red light dramatically potentiates platelet inhibition by nitrite. Blocking RBC-surface thiols abrogated these effects of nitrite and far-red light. RBC-dependent production of NO was also shown to be enhanced by far red light using a chemiluminescence-based nitric oxide analyzer. In addition, RBC-dependent bioactivation of nitrite led to prolonged lag times for clotting in platelet poor plasma that was enhanced by exposure to far red light. CONCLUSIONS: Our results suggest that nitrite leads to the formation of a photolabile RBC surface thiol-bound species such as an S-nitrosothiol or heme-nitrosyl (NO-bound heme) for which far red light enhances NO signaling. These findings expand our understanding of RBC-mediated NO production from nitrite. This pathway of NO production may have therapeutic potential in several applications including thrombosis, and, thus, warrants further study.

Impact of different standard red blood cell storage temperatures on human and canine RBC hemolysis and chromium survival.

BACKGROUND: Storage temperature is a critical factor for maintaining red-blood cell (RBC) viability, especially during prolonged cold storage. The target range of 1 to 6°C was established decades ago and may no longer be optimal for current blood-banking practices. STUDY DESIGN AND METHODS: Human and canine RBCs were collected under standard conditions and stored in precision-controlled refrigerators at 2°C, 4°C, or 6°C. RESULTS: During 42-day storage, human and canine RBCs showed progressive increases in supernatant non-transferrin-bound iron, cell-free hemoglobin, base deficit, and lactate levels that were overall greater at 6°C and 4°C than at 2°C. Animals transfused with 7-day-old RBCs had similar plasma cell-free hemoglobin and non-transferrin-bound iron levels at 1 to 72 hours for all three temperature conditions by chromium-51 recovery analysis. However, animals transfused with 35-day-old RBCs stored at higher temperatures developed plasma elevations in non-transferrin-bound iron and cell-free hemoglobin at 24 and 72 hours. Despite apparent impaired 35-day storage at 4°C and 6°C compared to 2°C, posttransfusion chromium-51 recovery at 24 hours was superior at higher temperatures. This finding was confounded by a preparation artifact related to an interaction between temperature and storage duration that leads to removal of fragile cells with repeated washing of the radiolabeled RBC test sample and renders the test sample unrepresentative of the stored unit. CONCLUSIONS: RBCs stored at the lower bounds of the temperature range are less metabolically active and produce less anaerobic acidosis and hemolysis, leading to a more suitable transfusion product. The higher refrigeration temperatures are not optimal during extended RBC storage and may confound chromium viability studies.

Potential therapeutic action of nitrite in sickle cell disease.

Sickle cell disease is caused by a mutant form of hemoglobin that polymerizes under hypoxic conditions, increasing rigidity, fragility, calcium influx-mediated dehydration, and adhesivity of red blood cells. Increased red cell fragility results in hemolysis, which reduces nitric oxide (NO) bioavailability, and induces platelet activation and inflammation leading to adhesion of circulating blood cells. Nitric Oxide inhibits adhesion and platelet activation. Nitrite has emerged as an attractive therapeutic agent that targets delivery of NO activity to areas of hypoxia through bioactivation by deoxygenated red blood cell hemoglobin. In this study, we demonstrate anti-platelet activity of nitrite at doses achievable through dietary interventions with comparison to similar doses with other NO donating agents. Unlike other NO donating agents, nitrite activity is shown to be potentiated in the presence of red blood cells in hypoxic conditions. We also show that nitrite reduces calcium associated loss of phospholipid asymmetry that is associated with increased red cell adhesion, and that red cell deformability is also improved. We show that nitrite inhibits red cell adhesion in a microfluidic flow-channel assay after endothelial cell activation. In further investigations, we show that leukocyte and platelet adhesion is blunted in nitrite-fed wild type mice compared to control after either lipopolysaccharide- or hemolysis-induced inflammation. Moreover, we demonstrate that nitrite treatment results in a reduction in adhesion of circulating blood cells and reduced red blood cell hemolysis in humanized transgenic sickle cell mice subjected to local hypoxia. These data suggest that nitrite is an effective anti-platelet and anti-adhesion agent that is activated by red blood cells, with enhanced potency under physiological hypoxia and in venous blood that may be useful therapeutically.

Effects of supervised exercise and dietary nitrate in older adults with controlled hypertension and/or heart failure with preserved ejection fraction.

Aerobic exercise training is an effective therapy to improve peak aerobic power (peak VO2) in individuals with hypertension (HTN, AHA/ACC class A) and heart failure patients with preserved ejection fraction (HFpEF). High nitrate containing beetroot juice (BRJ) also improves sub-maximal endurance and decreases blood pressure in both HTN and HFpEF. We hypothesized that combining an aerobic exercise and dietary nitrate intervention would result in additive or even synergistic positive effects on exercise tolerance and blood pressure in HTN or HFpEF. We report results from two pilot studies examining the effects of supervised aerobic exercise combined with dietary nitrate in patients with controlled HTN (n = 26, average age 65 ± 5 years) and in patients with HFpEF (n = 20, average age 69 ± 7 years). All patients underwent an aerobic exercise training regimen; half were randomly assigned to consume a high nitrate-containing beet juice beverage (BRJ containing 6.1 mmol nitrate for the HFpEF study consumed three times a week and 8 mmol nitrate for the HTN study consumed daily) while the other half consumed a beet juice beverage with the nitrate removed (placebo). The main result was that there was no added benefit observed for any outcomes when comparing BRJ to placebo in either HTN or HFpEF patients undergoing exercise training (p ≥ 0.14). There were within-group benefits. In the pilot study in patients with HFpEF, aerobic endurance (primary outcome), defined as the exercise time to volitional exhaustion during submaximal cycling at 75% of maximal power output, improved during exercise training within each group from baseline to end of study, 369 ± 149 s vs 520 ± 257 s (p = 0.04) for the placebo group and 384 ± 129 s vs 483 ± 258 s for the BRJ group (p = 0.15). Resting systolic blood pressure in patients with HFpEF also improved during exercise training in both groups, 136 ± 16 mm Hg vs 122 ± 3 mm Hg for the placebo group (p < 0.05) and 132 ± 12 mm Hg vs 119 ± 9 mm Hg for the BRJ group (p < 0.05). In the HTN pilot study, during a treadmill graded exercise test, peak oxygen consumption (primary outcome) did not change significantly, but time to exhaustion (also a primary outcome) improved in both groups, 504 ± 32 s vs 601 ± 38 s (p < 0.05) for the placebo group and 690 ± 38 s vs 772 ± 95 s for the BRJ group (p < 0.05) which was associated with a reduction in supine resting systolic blood pressure in BRJ group. Arterial compliance also improved during aerobic exercise training in both the HFpEF and the HTN patients for both BRJ and placebo groups. Future work is needed to determine if larger nitrate doses would provide an added benefit to supervised aerobic exercise in HTN and HFpEF patients.

Beet Root Juice: An Ergogenic Aid for Exercise and the Aging Brain.

BACKGROUND: Exercise has positive neuroplastic effects on the aging brain. It has also been shown that ingestion of beet root juice (BRJ) increases blood flow to the brain and enhances exercise performance. Here, we examined whether there are synergistic effects of BRJ and exercise on neuroplasticity in the aging brain. METHODS: Peak metabolic equivalent (MET) capacity and resting-state magnetic resonance imaging functional brain network organization are reported on 26 older (mean age = 65.4 years) participants randomly assigned to 6 weeks of exercise + BRJ or exercise + placebo. RESULTS: Somatomotor community structure consistency was significantly enhanced in the exercise + BRJ group following the intervention (MBRJ = -2.27, SE = 0.145, MPlacebo = -2.89, SE = 0.156, p = .007). Differences in second-order connections between the somatomotor cortex and insular cortex were also significant; the exercise + BRJ group (M = 3.28, SE = 0.167) had a significantly lower number of connections than exercise + placebo (M = 3.91, SE = 0.18, p = .017) following the intervention. Evaluation of peak MET capacity revealed a trend for the exercise + BRJ group to have higher MET capacity following the intervention. CONCLUSIONS: Older adults who exercised and consumed BRJ demonstrated greater consistency within the motor community and fewer secondary connections with the insular cortex compared with those who exercised without BRJ. The exercise + BRJ group had brain networks that more closely resembled those of younger adults, showing the potential enhanced neuroplasticity conferred by combining exercise and BRJ consumption.