Vikas Kapil

Inorganic Nitrate Supplementation Lowers Blood Pressure in Humans. Role for Nitrite-Derived NO

Ingestion of dietary (inorganic) nitrate elevates circulating and tissue levels of nitrite via bioconversion in the entero-salivary circulation. In addition, nitrite is a potent vasodilator in humans, an effect thought to underlie the blood pressure–lowering effects of dietary nitrate (in the form of beetroot juice) ingestion. Whether inorganic nitrate underlies these effects and whether the effects of either naturally occurring dietary nitrate or inorganic nitrate supplementation are dose dependent remain uncertain. Using a randomized crossover study design, we show that nitrate supplementation (KNO3 capsules: 4 versus 12 mmol [n=6] or 24 mmol of KNO3 (1488 mg of nitrate) versus 24 mmol of KCl [n=20]) or vegetable intake (250 mL of beetroot juice [5.5 mmol nitrate] versus 250 mL of water [n=9]) causes dose-dependent elevation in plasma nitrite concentration and elevation of cGMP concentration with a consequent decrease in blood pressure in healthy volunteers. In addition, post hoc analysis demonstrates a sex difference in sensitivity to nitrate supplementation dependent on resting baseline blood pressure and plasma nitrite concentration, whereby blood pressure is decreased in male volunteers, with higher baseline blood pressure and lower plasma nitrite concentration but not in female volunteers. Our findings demonstrate dose-dependent decreases in blood pressure and vasoprotection after inorganic nitrate ingestion in the form of either supplementation or by dietary elevation. In addition, our post hoc analyses intimate sex differences in nitrate processing involving the entero-salivary circulation that are likely to be major contributing factors to the lower blood pressures and the vasoprotective phenotype of premenopausal women.

Dietary Nitrate Provides Sustained Blood Pressure Lowering in Hypertensive Patients: A Randomized, Phase 2, Double-Blind, Placebo-Controlled Study

Abstract—Single dose administration of dietary inorganic nitrate acutely reduces blood pressure (BP) in normotensive healthy volunteers, via bioconversion to the vasodilator nitric oxide. We assessed whether dietary nitrate might provide sustained BP lowering in patients with hypertension. We randomly assigned 68 patients with hypertension in a double-blind, placebo-controlled clinical trial to receive daily dietary supplementation for 4 weeks with either dietary nitrate (250 mL daily, as beetroot juice) or a placebo (250 mL daily, as nitrate-free beetroot juice) after a 2-week run-in period and followed by a 2-week washout. We performed stratified randomization of drug-naive (n=34) and treated (n=34) patients with hypertension aged 18 to 85 years. The primary end point was change in clinic, ambulatory, and home BP compared with placebo. Daily supplementation with dietary nitrate was associated with reduction in BP measured by 3 different methods. Mean (95% confidence interval) reduction in clinic BP was 7.7/2.4 mm Hg (3.6–11.8/0.0–4.9, P<0.001 and P=0.050). Twenty-four-hour ambulatory BP was reduced by 7.7/5.2 mm Hg (4.1–11.2/2.7–7.7, P<0.001 for both). Home BP was reduced by 8.1/3.8 mm Hg (3.8–12.4/0.7–6.9, P<0.001 and P<0.01) with no evidence of tachyphylaxis over the 4-week intervention period. Endothelial function improved by ≈20% (P<0.001), and arterial stiffness was reduced by 0.59 m/s (0.24–0.93; P<0.01) after dietary nitrate consumption with no change after placebo. The intervention was well tolerated. This is the first evidence of durable BP reduction with dietary nitrate supplementation in a relevant patient group. These findings suggest a role for dietary nitrate as an affordable, readily-available, adjunctive treatment in the management of patients with hypertension (funded by The British Heart Foundation). Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifier: NCT01405898.Abstract— Single dose administration of dietary inorganic nitrate acutely reduces blood pressure (BP) in normotensive healthy volunteers, via bioconversion to the vasodilator nitric oxide. We assessed whether dietary nitrate might provide sustained BP lowering in patients with hypertension. We randomly assigned 68 patients with hypertension in a double-blind, placebo-controlled clinical trial to receive daily dietary supplementation for 4 weeks with either dietary nitrate (250 mL daily, as beetroot juice) or a placebo (250 mL daily, as nitrate-free beetroot juice) after a 2-week run-in period and followed by a 2-week washout. We performed stratified randomization of drug-naive (n=34) and treated (n=34) patients with hypertension aged 18 to 85 years. The primary end point was change in clinic, ambulatory, and home BP compared with placebo. Daily supplementation with dietary nitrate was associated with reduction in BP measured by 3 different methods. Mean (95% confidence interval) reduction in clinic BP was 7.7/2.4 mm Hg (3.6–11.8/0.0–4.9, P <0.001 and P =0.050). Twenty-four-hour ambulatory BP was reduced by 7.7/5.2 mm Hg (4.1–11.2/2.7–7.7, P <0.001 for both). Home BP was reduced by 8.1/3.8 mm Hg (3.8–12.4/0.7–6.9, P <0.001 and P <0.01) with no evidence of tachyphylaxis over the 4-week intervention period. Endothelial function improved by ≈20% ( P <0.001), and arterial stiffness was reduced by 0.59 m/s (0.24–0.93; P <0.01) after dietary nitrate consumption with no change after placebo. The intervention was well tolerated. This is the first evidence of durable BP reduction with dietary nitrate supplementation in a relevant patient group. These findings suggest a role for dietary nitrate as an affordable, readily-available, adjunctive treatment in the management of patients with hypertension (funded by The British Heart Foundation). Clinical Trial Registration— URL: . Unique identifier: [NCT01405898][1]. # Novelty and Significance {#article-title-47} [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01405898&atom=%2Fhypertensionaha%2F65%2F2%2F320.atom

Physiological role for nitrate-reducing oral bacteria in blood pressure control.

Circulating nitrate (NO3−), derived from dietary sources or endogenous nitric oxide production, is extracted from blood by the salivary glands, accumulates in saliva, and is then reduced to nitrite (NO2−) by the oral microflora. This process has historically been viewed as harmful, because nitrite can promote formation of potentially carcinogenic N-nitrosamines. More recent research, however, suggests that nitrite can also serve as a precursor for systemic generation of vasodilatory nitric oxide, and exogenous administration of nitrate reduces blood pressure in humans. However, whether oral nitrate-reducing bacteria participate in “setting” blood pressure is unknown. We investigated whether suppression of the oral microflora affects systemic nitrite levels and hence blood pressure in healthy individuals. We measured blood pressure (clinic, home, and 24-h ambulatory) in 19 healthy volunteers during an initial 7-day control period followed by a 7-day treatment period with a chlorhexidine-based antiseptic mouthwash. Oral nitrate-reducing capacity and nitrite levels were measured after each study period. Antiseptic mouthwash treatment reduced oral nitrite production by 90% (p < 0.001) and plasma nitrite levels by 25% (p = 0.001) compared to the control period. Systolic and diastolic blood pressure increased by 2–3 .5 mm Hg, increases correlated to a decrease in circulating nitrite concentrations (r2 = 0.56, p = 0.002). The blood pressure effect appeared within 1 day of disruption of the oral microflora and was sustained during the 7-day mouthwash intervention. These results suggest that the recycling of endogenous nitrate by oral bacteria plays an important role in determination of plasma nitrite levels and thereby in the physiological control of blood pressure.

Enhanced Vasodilator Activity of Nitrite in Hypertension Critical Role for Erythrocytic Xanthine Oxidoreductase and Translational Potential

Elevation of circulating nitrite (NO2 −) levels causes vasodilatation and lowers blood pressure in healthy volunteers. Whether these effects and the underpinning mechanisms persist in hypertension is unknown. Therefore, we investigated the consequences of systemic nitrite elevation in spontaneously hypertensive rats and conducted proof-of-principle studies in patients. Nitrite caused dose-dependent blood pressure–lowering that was profoundly enhanced in spontaneously hypertensive rats versus normotensive Wistar Kyoto controls. This effect was virtually abolished by the xanthine oxidoreductase (XOR) inhibitor, allopurinol, and associated with hypertension-specific XOR-dependent nitrite reductase activity localized to the erythrocyte but not the blood vessel wall. To determine whether these pathways translate to human hypertension, we investigated the effects of elevation of circulating nitrite levels in 15 drug naïve grade 1 hypertensives. To elevate nitrite, we used a dose of dietary nitrate (≈3.5 mmol) that elevated nitrite levels ≈1.5-fold (P<0.01); a rise shown previously to exert no significant blood pressure–lowering effects in normotensives. This dose caused substantial reductions in systolic (≈12 mm Hg) and diastolic blood pressures (P<0.001) and pulse wave velocity (P<0.05); effects associated with elevations in erythrocytic XOR expression and XOR-dependent nitrite reductase activity. Our observations demonstrate the improved efficacy of inorganic nitrate and nitrite in hypertension as a consequence of increased erythrocytic XOR nitrite reductase activity and support the concept of dietary nitrate supplementation as an effective, but simple and inexpensive, antihypertensive strategy.Elevation of circulating nitrite (NO2−) levels causes vasodilatation and lowers blood pressure in healthy volunteers. Whether these effects and the underpinning mechanisms persist in hypertension is unknown. Therefore, we investigated the consequences of systemic nitrite elevation in spontaneously hypertensive rats and conducted proof-of-principle studies in patients. Nitrite caused dose-dependent blood pressure–lowering that was profoundly enhanced in spontaneously hypertensive rats versus normotensive Wistar Kyoto controls. This effect was virtually abolished by the xanthine oxidoreductase (XOR) inhibitor, allopurinol, and associated with hypertension-specific XOR-dependent nitrite reductase activity localized to the erythrocyte but not the blood vessel wall. To determine whether these pathways translate to human hypertension, we investigated the effects of elevation of circulating nitrite levels in 15 drug naive grade 1 hypertensives. To elevate nitrite, we used a dose of dietary nitrate (≈3.5 mmol) that elevated nitrite levels ≈1.5-fold ( P <0.01); a rise shown previously to exert no significant blood pressure–lowering effects in normotensives. This dose caused substantial reductions in systolic (≈12 mm Hg) and diastolic blood pressures ( P <0.001) and pulse wave velocity ( P <0.05); effects associated with elevations in erythrocytic XOR expression and XOR-dependent nitrite reductase activity. Our observations demonstrate the improved efficacy of inorganic nitrate and nitrite in hypertension as a consequence of increased erythrocytic XOR nitrite reductase activity and support the concept of dietary nitrate supplementation as an effective, but simple and inexpensive, antihypertensive strategy. # Novelty and Significance {#article-title-61}

Inorganic nitrate ingestion improves vascular compliance but does not alter flow-mediated dilatation in healthy volunteers

Highlights ► Inorganic nitrate supplementation does not alter endothelial function in healthy volunteers. ► Despite this, there was a reduction in blood pressure and improvement in arterial compliance. ► Improvements in vascular function are likely to contribute to the beneficial effects of inorganic nitrate on blood pressure.

Inorganic nitrate and the cardiovascular system

Fruit and vegetable-rich diets reduce blood pressure and risk of ischaemic stroke and ischaemic heart disease. While the cardioprotective effects of a fruit and vegetable-rich diet are unequivocal, the exact mechanisms of this effect remain uncertain. Recent evidence has highlighted the possibility that dietary nitrate, an inorganic anion found in large quantities in vegetables (particularly green leafy vegetables), may have a part to play. This beneficial activity lies in the processing in vivo of nitrate to nitrite and thence to the pleiotropic molecule nitric oxide. In this review, recent preclinical and clinical evidence identifying the mechanisms involved in nitrate bioactivity, and the evidence supporting the potential utility of exploitation of this pathway for the prevention and/or treatment of cardiovascular diseases are discussed.

Antiplatelet effects of dietary nitrate in healthy volunteers: involvement of cGMP and influence of sex.

Ingestion of vegetables rich in inorganic nitrate has emerged as an effective method, via the formation of a nitrite intermediate, for acutely elevating vascular NO levels. As such a number of beneficial effects of dietary nitrate ingestion have been demonstrated including the suggestion that platelet reactivity is reduced. In this study we investigated whether inorganic nitrate supplementation might also reduce platelet reactivity in healthy volunteers and have determined the mechanisms involved in the effects seen. We conducted two randomised crossover studies each in 24 (12 of each sex) healthy subjects assessing the acute effects of dietary nitrate (250 ml beetroot juice) or potassium nitrate capsules (KNO3, 8 mmol) vs placebo control on platelet reactivity. Inorganic nitrate ingested either from a dietary source or via supplementation raised circulating nitrate and nitrite levels in both sexes and attenuated ex vivo platelet aggregation responses to ADP and, albeit to a lesser extent, collagen but not epinephrine in male but not female volunteers. These inhibitory effects were associated with a reduced platelet P-selectin expression and elevated platelet cGMP levels. In addition, we show that nitrite reduction to NO occurs at the level of the erythrocyte and not the platelet. In summary, our results demonstrate that inorganic nitrate ingestion, whether via the diet or through supplementation, causes a modest decrease in platelet reactivity in healthy males but not females. Our studies provide strong support for further clinical trials investigating the potential of dietary nitrate as an adjunct to current antiplatelet therapies to prevent atherothrombotic complications. Moreover, our observations highlight a previously unknown sexual dimorphism in platelet reactivity to NO and intimate a greater dependence of males on the NO-soluble guanylate cyclase pathway in limiting thrombotic potential.

Clinical evidence demonstrating the utility of inorganic nitrate in cardiovascular health

The discovery of nitric oxide and its role in almost every facet of human biology opened a new avenue for treatment through manipulation of its canonical signaling and by attempts to augment endogenous nitric oxide generation through provision of substrate and co-factors to the endothelial nitric oxide synthase complex. This has been particularly so in the cardiovascular system and it is well recognized that there is reduced bioavailable nitric oxide in patients with both cardiovascular risk factors and manifest vascular disease. However, these attempts have failed to deliver the expected benefits of such an approach. Recently, an alternative pathway for nitric oxide synthesis has been elucidated that can produce authentic nitric oxide from the 1 electron reduction of inorganic nitrite. Furthermore, it has long been known that symbiotic, facultative, oral microflora can facilitate the reduction of inorganic nitrate, that is ingested in the average diet in millimolar amounts, to inorganic nitrite itself. Thus, there exists an alternative reductive pathway from nitrate, via nitrite as an intermediate, to nitric oxide that provides a novel pathway that may be amenable to therapeutic manipulation. As such, various research groups have explored the utility of manipulation of this nitrate-nitrite-nitric oxide pathway in situations in which nitric oxide is known to have a prominent role. Animal and early-phase human studies of both inorganic nitrite and nitrate supplementation have shown beneficial effects in blood pressure control, platelet function, vascular health and exercise capacity. This review considers in detail the pathways of inorganic nitrate bioactivation and the evidence of clinical utility to date on the cardiovascular system.

Joint UK societies' 2014 consensus statement on renal denervation for resistant hypertension.

Resistant hypertension continues to pose a major challenge to clinicians worldwide and has serious implications for patients who are at increased risk of cardiovascular morbidity and mortality with this diagnosis. Pharmacological therapy for resistant hypertension follows guidelines-based regimens although there is surprisingly scant evidence for beneficial outcomes using additional drug treatment after three antihypertensives have failed to achieve target blood pressure. Recently there has been considerable interest in the use of endoluminal renal denervation as an interventional technique to achieve renal nerve ablation and lower blood pressure. Although initial clinical trials of renal denervation in patients with resistant hypertension demonstrated encouraging office blood pressure reduction, a large randomised control trial (Symplicity HTN-3) with a sham-control limb, failed to meet its primary efficacy end point. The trial however was subject to a number of flaws which must be taken into consideration in interpreting the final results. Moreover a substantial body of evidence from non-randomised smaller trials does suggest that renal denervation may have an important role in the management of hypertension and other disease states characterised by overactivation of the sympathetic nervous system. The Joint UK Societies does not recommend the use of renal denervation for treatment of resistant hypertension in routine clinical practice but remains committed to supporting research activity in this field. A number of research strategies are identified and much that can be improved upon to ensure better design and conduct of future randomised studies.

Accelerated resolution of inflammation underlies sex differences in inflammatory responses in humans

BACKGROUND. Cardiovascular disease occurs at lower incidence in premenopausal females compared with age-matched males. This variation may be linked to sex differences in inflammation. We prospectively investigated whether inflammation and components of the inflammatory response are altered in females compared with males. METHODS. We performed 2 clinical studies in healthy volunteers. In 12 men and 12 women, we assessed systemic inflammatory markers and vascular function using brachial artery flow-mediated dilation (FMD). In a further 8 volunteers of each sex, we assessed FMD response to glyceryl trinitrate (GTN) at baseline and at 8 hours and 32 hours after typhoid vaccine. In a separate study in 16 men and 16 women, we measured inflammatory exudate mediators and cellular recruitment in cantharidin-induced skin blisters at 24 and 72 hours. RESULTS. Typhoid vaccine induced mild systemic inflammation at 8 hours, reflected by increased white cell count in both sexes. Although neutrophil numbers at baseline and 8 hours were greater in females, the neutrophils were less activated. Systemic inflammation caused a decrease in FMD in males, but an increase in females, at 8 hours. In contrast, GTN response was not altered in either sex after vaccine. At 24 hours, cantharidin formed blisters of similar volume in both sexes; however, at 72 hours, blisters had only resolved in females. Monocyte and leukocyte counts were reduced, and the activation state of all major leukocytes was lower, in blisters of females. This was associated with enhanced levels of the resolving lipids, particularly D-resolvin. CONCLUSIONS. Our findings suggest that female sex protects against systemic inflammation-induced endothelial dysfunction. This effect is likely due to accelerated resolution of inflammation compared with males, specifically via neutrophils, mediated by an elevation of the D-resolvin pathway. TRIAL REGISTRATION. ClinicalTrials.gov NCT01582321 and NRES: City Road and Hampstead Ethics Committee: 11/LO/2038. FUNDING. The authors were funded by multiple sources, including the National Institute for Health Research, the British Heart Foundation, and the European Research Council.