The Effect of Dietary Nitrate on the Oral Microbiome and Salivary Biomarkers in Individuals with High Blood Pressure.

BACKGROUND: Green leafy vegetables (GLV) contain inorganic nitrate, an anion with potential prebiotic effects on the oral microbiome. However, it remains unclear whether GLV and pharmacological supplementation [potassium nitrate (PN)] with a nitrate salt induce similar effects on the oral microbiome. OBJECTIVES: This study aimed to compare the effect of GLV with PN supplementation on the oral microbiome composition and salivary biomarkers in individuals with high blood pressure. METHODS: Seventy individuals were randomly allocated to 3 different groups to follow a 5-wk dietary intervention. Group 1 consumed 300 mg/d of nitrate in form of GLV. Group 2 consumed pills with 300 mg/d of PN and low-nitrate vegetables. Group 3 consumed pills with potassium chloride (placebo: PLAC) and low-nitrate vegetables. The oral microbiome composition and salivary biomarkers of oral health were analyzed before and after the dietary intervention. RESULTS: The GLV and PN groups showed similar microbial changes, probably nitrate-dependent, including an increase in the abundance of Neisseria, Capnocytophaga, Campylobacter species, and a decrease in Veillonella, Megasphaera, Actinomyces, and Eubacterium species after the treatment. Increased abundance of Rothia species, and reduced abundance of Streptococcus, Prevotella, Actinomyces, and Mogibacterium species were observed in the GLV group, which could be nitrate-independent. GLV and PN treatments increased salivary pH, but only GLV treatment showed an increase in the salivary buffering capacity and a reduction of lactate. CONCLUSION: The combination of nitrate-dependent and nitrate-independent microbial changes in the GLV group has a stronger effect to potentially improve oral health biomarkers compared with PN.

Control of human energy expenditure by cytochrome c oxidase subunit IV-2.

Resting metabolic rate (RMR) in humans shows pronounced individual variations, but the underlying molecular mechanism remains elusive. Cytochrome c oxidase (COX) plays a key role in control of metabolic rate, and recent studies of the subunit 4 isoform 2 (COX IV-2) indicate involvement in the cellular response to hypoxia and oxidative stress. We evaluated whether the COX subunit IV isoform composition may explain the pronounced individual variations in resting metabolic rate (RMR). RMR was determined in healthy humans by indirect calorimetry and correlated to levels of COX IV-2 and COX IV-1 in vastus lateralis. Overexpression and knock down of the COX IV isoforms were performed in primary myotubes followed by evaluation of the cell respiration and production of reactive oxygen species. Here we show that COX IV-2 protein is constitutively expressed in human skeletal muscle and strongly correlated to RMR. Primary human myotubes overexpressing COX IV-2 displayed markedly (>60%) lower respiration, reduced (>50%) cellular H2O2 production, higher resistance toward both oxidative stress, and severe hypoxia compared with control cells. These results suggest an important role of isoform COX IV-2 in the control of energy expenditure, hypoxic tolerance, and mitochondrial ROS homeostasis in humans.

Effects of antiseptic mouthwash on resting metabolic rate: A randomized, double-blind, crossover study.

The nitrate-nitrite-nitric oxide pathway has emerged as a significant source of nitric oxide (NO) bioactivity. Dietary intake of inorganic nitrate has a number of cardiovascular effects as well as a decrease in oxygen cost during exercise and a reduction in resting metabolic rate (RMR). Oral bacteria have a key role in bioactivation of inorganic nitrate since they catalyse the conversion of salivary nitrate to the more reactive nitrite anion. Recent studies demonstrate that blood pressure increases with the use of an antiseptic mouthwash, indicating that endogenous, NO-synthase derived nitrate is recycled into nitrite and NO, sufficiently to modulate cardiovascular function. Here we tested if also RMR would be affected by an antiseptic mouthwash. Seventeen healthy normotensive female subjects (23 ± 4 y) participated in this randomized, double-blinded, crossover study. During two 3-day periods separated by 28 days the subjects consumed a diet low in nitrate combined with rinsing their mouth three times daily with a chlorhexidine-containing mouthwash (mouthwash) or placebo mouthwash (placebo) with similar taste but no antiseptic properties. Resting metabolic rate (RMR) was measured by indirect calorimetry and 24 h ambulatory blood pressure recordings were obtained after each intervention together with blood, saliva and urine samples. Treatment with chlorhexidine-containing mouthwash effectively reduced oral conversion of nitrate to nitrite but had no effect on plasma levels of these anions or plasma cGMP. RMR and 24 h ambulatory blood pressure were unaffected by the intervention. We conclude that in young healthy females an antiseptic mouthwash was effective in disrupting oral bacterial nitrate conversion to nitrite, but this was not associated with changes in plasma nitrite, RMR or blood pressure.

Hypoxic erythrocytes mediate cardioprotection through activation of soluble guanylate cyclase and release of cyclic GMP.

Red blood cells (RBCs) mediate cardioprotection via nitric oxide-like bioactivity, but the signaling and the identity of any mediator released by the RBCs remains unknown. We investigated whether RBCs exposed to hypoxia release a cardioprotective mediator and explored the nature of this mediator. Perfusion of isolated hearts subjected to ischemia-reperfusion with extracellular supernatant from mouse RBCs exposed to hypoxia resulted in improved postischemic cardiac function and reduced infarct size. Hypoxia increased extracellular export of cyclic guanosine monophosphate (cGMP) from mouse RBCs, and exogenous cGMP mimicked the cardioprotection induced by the supernatant. The protection induced by hypoxic RBCs was dependent on RBC-soluble guanylate cyclase and cGMP transport and was sensitive to phosphodiesterase 5 and activated cardiomyocyte protein kinase G. Oral administration of nitrate to mice to increase nitric oxide bioactivity further enhanced the cardioprotective effect of hypoxic RBCs. In a placebo-controlled clinical trial, a clear cardioprotective, soluble guanylate cyclase-dependent effect was induced by RBCs collected from patients randomized to 5 weeks nitrate-rich diet. It is concluded that RBCs generate and export cGMP as a response to hypoxia, mediating cardioprotection via a paracrine effect. This effect can be further augmented by a simple dietary intervention, suggesting preventive and therapeutic opportunities in ischemic heart disease.

Renal handling of nitrate in women and men with elevated blood pressure.

AIM: The inorganic anions nitrate and nitrite are oxidation products of nitric oxide (NO) that have often been used as an index of NO generation. More than just being surrogate markers of NO, nitrate/nitrite can recycle to bioactive NO again. Nitrate is predominantly eliminated via the kidneys; however, there is less knowledge regarding tubular handling. The aim of this study, as part of a large randomized controlled trial, was to explore potential sex differences in renal nitrate handling during low and high dietary nitrate intake. We hypothesized that renal clearance and excretion of nitrate are higher in men compared to women. METHODS: In prehypertensive and hypertensive individuals (n = 231), nitrate and nitrite were measured in plasma and urine at low dietary nitrate intake (baseline) and after 5 weeks supplementation with nitrate (300 mg potassium nitrate/day) or placebo (300 mg potassium chloride/day). Twenty-four hours ambulatory blood pressure recordings and urine collections were conducted. RESULTS: At baseline, plasma nitrate and nitrite, as well as the downstream marker of NO signalling cyclic guanosine monophosphate, were similar in women and men. Approximately 80% of filtered nitrate was spared by the kidneys. Urinary nitrate concentration, amount of nitrate excreted, renal nitrate clearance (Cnitrate ) and fractional excretion of nitrate (FEnitrate ) were lower in women compared to men. No association was observed between plasma nitrate concentrations and glomerular filtration rate (GFR), nor between FEnitrate and GFR in either sex. After 5 weeks of nitrate supplementation plasma nitrate and nitrite increased significantly, but blood pressure remained unchanged. FEnitrate increased significantly and the sex difference observed at baseline disappeared. CONCLUSION: Our findings demonstrate substantial nitrate sparing capacity of the kidneys, which is higher in women compared to men. This suggests higher tubular nitrate reabsorption in women but the underlying mechanism(s) warrants further investigation.

A randomized clinical trial of the effects of leafy green vegetables and inorganic nitrate on blood pressure.

BACKGROUND: A diet rich in fruits and vegetables is associated with lowering of blood pressure (BP), but the nutrient(s) responsible for these effects remain unclear. Research suggests that inorganic nitrate present in leafy green vegetables is converted into NO in vivo to improve cardiovascular function. OBJECTIVE: In this study, we evaluated the effect of leafy green vegetables on BP in subjects with elevated BP, with the aim of elucidating if any such effect is related to their high nitrate content. DESIGN: We enrolled 243 subjects, 50-70 y old, with a clinic systolic BP (SBP) of 130-159 mm Hg. After a 2-wk run-in period on a nitrate-restricted diet the subjects were randomly assigned to receive 1 of the following 3 interventions daily for 5 wk: low-nitrate vegetables + placebo pills, low-nitrate vegetables + nitrate pills (300 mg nitrate), or leafy green vegetables containing 300 mg nitrate + placebo pills. The primary end point measure was the difference in change in 24 h ambulatory SBP between the groups. RESULTS: A total of 231 subjects (95%) completed the study. The insignificant change in ambulatory SBP (mean ± standard deviation) was -0.6 ± 6.2 mm Hg in the placebo group, -1.2 ± 6.8 mm Hg in the potassium nitrate group, and -0.5 ± 6.6 mm Hg in the leafy green vegetable group. There was no significant difference in change between the 3 groups. CONCLUSIONS: A 5-wk dietary supplementation with leafy green vegetables or pills containing the same amount of inorganic nitrate does not decrease ambulatory SBP in subjects with elevated BP. This trial was registered at clinicaltrials.gov as NCT02916615.

Blood Pressure-Lowering Effect of Orally Ingested Nitrite Is Abolished by a Proton Pump Inhibitor.

Inorganic nitrate and nitrite from dietary and endogenous sources are metabolized to NO and other bioactive nitrogen oxides that affect blood pressure. The mechanisms for nitrite bioactivation are unclear, but recent studies in rodents suggest that gastric acidity may influence the systemic effects of this anion. In a randomized, double-blind, placebo-controlled crossover study, we tested the effects of a proton pump inhibitor on the acute cardiovascular effects of nitrite. Fifteen healthy nonsmoking, normotensive subjects, aged 19 to 39 years, were pretreated with placebo or esomeprazole (3×40 mg) before ingesting sodium nitrite (0.3 mg kg-1), followed by blood pressure monitoring. Nitrite reduced systolic blood pressure by a maximum of 6±1.3 mm Hg when taken after placebo, whereas pretreatment with esomeprazole blunted this effect. Peak plasma nitrite, nitrate, and nitroso species levels after nitrite ingestion were similar in both interventions. In 8 healthy volunteers, we then infused increasing doses of sodium nitrite (1, 10, and 30 nmol kg-1 min-1) intravenously. Interestingly, although plasma nitrite peaked at similar levels as with orally ingested nitrite (≈1.8 µmol/L), no changes in blood pressure were observed. In rodents, esomeprazole did not affect the blood pressure response to the NO donor, DEA NONOate, or vascular relaxation to nitroprusside and acetylcholine, demonstrating an intact downstream NO-signaling pathway. We conclude that the acute blood pressure-lowering effect of nitrite requires an acidic gastric environment. Future studies will reveal if the cardiovascular complications associated with the use of proton pump inhibitors are linked to interference with the nitrate-nitrite-NO pathway.

Profound differences between humans and rodents in the ability to concentrate salivary nitrate: Implications for translational research.

In humans dietary circulating nitrate accumulates rapidly in saliva through active transport in the salivary glands. By this mechanism resulting salivary nitrate concentrations are 10-20 times higher than in plasma. In the oral cavity nitrate is reduced by commensal bacteria to nitrite, which is subsequently swallowed and further metabolized to nitric oxide (NO) and other bioactive nitrogen oxides in blood and tissues. This entero-salivary circulation of nitrate is central in the various NO-like effects observed after ingestion of inorganic nitrate. The very same system has also been the focus of toxicologists studying potential carcinogenic effects of nitrite-dependent nitrosamine formation. Whether active transport of nitrate and accumulation in saliva occurs also in rodents is not entirely clear. Here we measured salivary and plasma levels of nitrate and nitrite in humans, rats and mice after administration of a standardized dose of nitrate. After oral (humans) or intraperitoneal (rodents) sodium nitrate administration (0.1mmol/kg), plasma nitrate levels increased markedly reaching ~300µM in all three species. In humans ingestion of nitrate was followed by a rapid increase in salivary nitrate to >6000µM, ie 20 times higher than those found in plasma. In contrast, in rats and mice salivary nitrate concentrations never exceeded the levels in plasma. Nitrite levels in saliva and plasma followed a similar pattern, ie marked increases in humans but modest elevations in rodents. In mice there was also no accumulation of nitrate in the salivary glands as measured directly in whole glands obtained after acute administration of nitrate. This study suggests that in contrast to humans, rats and mice do not actively concentrate circulating nitrate in saliva. These apparent species differences should be taken into consideration when studying the nitrate-nitrite-nitric oxide pathway in rodents, when calculating doses, exploring physiological, therapeutic and toxicological effects and comparing with human data.