Nigel Benjamin

Acute Blood Pressure Lowering, Vasoprotective, and Antiplatelet Properties of Dietary Nitrate via Bioconversion to Nitrite

Diets rich in fruits and vegetables reduce blood pressure (BP) and the risk of adverse cardiovascular events. However, the mechanisms of this effect have not been elucidated. Certain vegetables possess a high nitrate content, and we hypothesized that this might represent a source of vasoprotective nitric oxide via bioactivation. In healthy volunteers, approximately 3 hours after ingestion of a dietary nitrate load (beetroot juice 500 mL), BP was substantially reduced (&Dgr;max −10.4/8 mm Hg); an effect that correlated with peak increases in plasma nitrite concentration. The dietary nitrate load also prevented endothelial dysfunction induced by an acute ischemic insult in the human forearm and significantly attenuated ex vivo platelet aggregation in response to collagen and ADP. Interruption of the enterosalivary conversion of nitrate to nitrite (facilitated by bacterial anaerobes situated on the surface of the tongue) prevented the rise in plasma nitrite, blocked the decrease in BP, and abolished the inhibitory effects on platelet aggregation, confirming that these vasoprotective effects were attributable to the activity of nitrite converted from the ingested nitrate. These findings suggest that dietary nitrate underlies the beneficial effects of a vegetable-rich diet and highlights the potential of a “natural” low cost approach for the treatment of cardiovascular disease.

Chemical generation of nitric oxide in the mouth from the enterosalivary circulation of dietary nitrate.

High concentrations of nitrite present in saliva (derived from dietary nitrate) may, upon acidification, generate nitrogen oxides in the stomach in sufficient amounts to provide protection from swallowed pathogens. We now show that, in the rat, reduction of nitrate to nitrite is confined to a specialized area on the posterior surface of the tongue, which is heavily colonized by bacteria, and that nitrate reduction is absent in germ-free rats. We also show that in humans increased salivary nitrite production resulting from nitrate intake enhances oral nitric oxide production. We propose that the salivary generation of nitrite is accomplished by a symbiotic relationship involving nitrate-reducing bacteria on the tongue surface, which is designed to provide host defence against microbial pathogens in the mouth and lower gut. These results provide further evidence for beneficial effects of dietary nitrate.

Nitrate, bacteria and human health

Nitrate is generally considered a water pollutant and an undesirable fertilizer residue in the food chain. Research in the 1970s indicated that, by reducing nitrate to nitrite, commensal bacteria might be involved in the pathogenesis of gastric cancers and other malignancies, as nitrite can enhance the generation of carcinogenic N-nitrosamines. More recent studies indicate that the bacterial metabolism of nitrate to nitrite and the subsequent formation of biologically active nitrogen oxides could be beneficial. Here, we will consider the evidence that nitrate-reducing commensals have a true symbiotic role in mammals and facilitate a previously unrecognized but potentially important aspect of the nitrogen cycle.

Xanthine oxidoreductase catalyses the reduction of nitrates and nitrite to nitric oxide under hypoxic conditions

Xanthine oxidoreductase (XOR) catalyses the reduction of the therapeutic organic nitrate, nitroglycerin (glyceryl trinitrate, GTN), as well as inorganic nitrate and nitrite, to nitric oxide (NO) under hypoxic conditions in the presence of NADH. Generation of nitric oxide is not detectable under normoxic conditions and is inhibited by the molybdenum site‐specific inhibitors, oxypurinol and (−)BOF 4272. These enzymic reactions provide a mechanism for generation of NO under hypoxic conditions where nitric oxide synthase does not function, suggesting a vasodilatory role in ischaemia.

Measuring Forearm Blood Flow and Interpreting the Responses to Drugs and Mediators

Venous occlusion plethysmography has been widely used to study forearm blood flow. The principle of the technique is straightforward: the rate of swelling of the forearm during occlusion of venous return is used to assess the rate of arterial inflow. Provided that perfusion pressure (arterial blood pressure) remains constant, changes in flow reflect changes in smooth muscle tone in small arteries and arterioles. Local infusion into the brachial artery allows assessment of the direct effect of drugs on vascular tone and has been used to probe the roles of endogenous mediators. The technique is at its most powerful when dose-response relationships to different drugs or mediators within a single study are being compared but can also be used for comparison of responses to drugs between healthy control subjects and patient populations. However, when responses between groups are being compared, it is important to take into account the starting conditions of baseline blood flow and pressure. This article describes venous occlusion plethysmography, discusses the presentation and analysis of data (dose of drug or concentration? forearm blood flow or resistance?), and highlights certain potential problems and limitations of the technique as a means of studying disease states.

Nitrate and nitrite in biology, nutrition and therapeutics

Inorganic nitrate and nitrite from endogenous or dietary sources are metabolized in vivo to nitric oxide (NO) and other bioactive nitrogen oxides. The nitrate-nitrite-NO pathway is emerging as an important mediator of blood flow regulation, cell signaling, energetics and tissue responses to hypoxia. The latest advances in our understanding of the biochemistry, physiology and therapeutics of nitrate, nitrite and NO were discussed during a recent 2-day meeting at the Nobel Forum, Karolinska Institutet in Stockholm.

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.

Basal nitric oxide synthesis in essential hypertension

BACKGROUND There is indirect evidence that nitric oxide (NO) synthesis in vascular endothelium of patients with hypertension is altered. The aim of this study was to estimate more directly NO production in patients with untreated essential hypertension by measurement of synthesis of inorganic nitrate, which is the end product of NO oxidation in humans. Two separate studies were undertaken in patients with hypertension and appropriate healthy controls. METHODS In the first study, ten patients and 13 controls were given a diet containing 82 mumoles nitrate per day for 2 days, with urinary and plasma nitrate measurement and 24 h ambulatory blood pressure monitoring on the 2nd day. In the second study, 11 patients and 11 controls were studied in the postabsorptive state; a bolus of 200 mg L[15N]2 arginine was administered intravenously over 10 min. 24 h ambulatory blood pressure monitoring was done and complete urine collections were made for the next 36 h. FINDINGS In the first study, 24 h urinary nitrate excretion was lower in the hypertensive patients than in the control group (mean 450 [SEM 37] vs 760 mumoles [77] per 24 h; p < 0.001). There was an inverse correlation between average mean daytime ambulatory blood pressure and nitrate excretion (p = 0.007; r2 = -0.73). In the second study, mean 36 h urinary 15N nitrate excretion was significantly lower in the hypertensive than in the control group (1313 [50] vs 2133 [142] pmoles; p < 0.001). There was an inverse correlation also between average mean daytime ambulatory blood pressure and 24 h urinary 15N nitrate excretion expressed per mmole of creatinine (p = 0.002, r2 = -0.59). In addition, total urinary 15N nitrate excretion in the hypertensive group was significantly higher in women than in men (285 [16] vs 198 [14] micrograms 15N nitrate per kg; p = 0.026). INTERPRETATION These data suggest that whole-body NO production in patients with essential hypertension is diminished under basal conditions. The origin of the NO we measured is not known, and we cannot tell whether the impaired synthesis is primary or secondary to a rise in blood pressure.

Preserved Endothelium-Dependent Vasodilatation in Patients with Essential Hypertension

BACKGROUND Previous studies suggest that vascular endothelial function may be impaired in essential hypertension. Although muscarinic agonists dilate blood vessels by releasing an endothelium-derived relaxing factor closely related to nitric oxide, nitroprusside dilates vessels by a mechanism that is independent of the endothelium. The finding of an impaired response to muscarinic agonists but a normal response to nitroprusside in patients with hypertension has suggested that endothelial function is abnormal in hypertension. METHODS We reassessed this issue by measuring forearm blood flow by plethysmography during the infusion of vasodilators into the brachial arteries of 95 subjects: 37 normotensive controls (mean [+/- SE] arterial blood pressure, 92 +/- 1 mm Hg) and 58 patients with essential hypertension (mean arterial blood pressure, 121 +/- 1 mm Hg). RESULTS In an initial study, vascular responses to the vasodilators carbachol and nitroprusside were similar in normotensive controls (n = 19) and hypertensive patients (n = 17). We wondered whether this might be attributable to the use of previously untreated patients or to the choice of carbachol as the muscarinic agonist. However, we found that the vasodilator responses to nitroprusside, acetylcholine, carbachol, and isoproterenol were also similar in separate groups of normotensive controls (n = 18) and hypertensive subjects, whether the subjects had never been treated for hypertension (n = 24) or had had therapy withheld for two weeks (n = 17). The 95 percent confidence intervals for the difference between the controls and hypertensive patients in the ratio of endothelium-dependent vasodilatation induced by acetylcholine or carbachol to endothelium-independent vasodilatation induced by nitroprusside were -14 to +23 percent for acetylcholine and -13 to +12 percent for carbachol. CONCLUSIONS In contrast to previous studies, our findings suggest that selective impairment of the responsiveness of the forearm vasculature to muscarinic agonists is not universal in patients with essential hypertension.

Dietary nitrate supplementation reduces the O2 cost of walking and running: a placebo-controlled study

Dietary supplementation with beetroot juice (BR) has been shown to reduce resting blood pressure and the O(2) cost of submaximal exercise and to increase tolerance to high-intensity cycling. We tested the hypothesis that the physiological effects of BR were consequent to its high NO(3)(-) content per se, and not the presence of other potentially bioactive compounds. We investigated changes in blood pressure, mitochondrial oxidative capacity (Q(max)), and physiological responses to walking and moderate- and severe-intensity running following dietary supplementation with BR and NO(3)(-)-depleted BR [placebo (PL)]. After control (nonsupplemented) tests, nine healthy, physically active male subjects were assigned in a randomized, double-blind, crossover design to receive BR (0.5 l/day, containing ∼6.2 mmol of NO(3)(-)) and PL (0.5 l/day, containing ∼0.003 mmol of NO(3)(-)) for 6 days. Subjects completed treadmill exercise tests on days 4 and 5 and knee-extension exercise tests for estimation of Q(max) (using (31)P-magnetic resonance spectroscopy) on day 6 of the supplementation periods. Relative to PL, BR elevated plasma NO(2)(-) concentration (183 ± 119 vs. 373 ± 211 nM, P < 0.05) and reduced systolic blood pressure (129 ± 9 vs. 124 ± 10 mmHg, P < 0.01). Q(max) was not different between PL and BR (0.93 ± 0.05 and 1.05 ± 0.22 mM/s, respectively). The O(2) cost of walking (0.87 ± 0.12 and 0.70 ± 0.10 l/min in PL and BR, respectively, P < 0.01), moderate-intensity running (2.26 ± 0.27 and 2.10 ± 0.28 l/min in PL and BR, respectively, P < 0.01), and severe-intensity running (end-exercise O(2) uptake = 3.77 ± 0.57 and 3.50 ± 0.62 l/min in PL and BL, respectively, P < 0.01) was reduced by BR, and time to exhaustion during severe-intensity running was increased by 15% (7.6 ± 1.5 and 8.7 ± 1.8 min in PL and BR, respectively, P < 0.01). In contrast, relative to control, PL supplementation did not alter plasma NO(2)(-) concentration, blood pressure, or the physiological responses to exercise. These results indicate that the positive effects of 6 days of BR supplementation on the physiological responses to exercise can be ascribed to the high NO(3)(-) content per se.