The effect of heparins on plasma concentration of heparin-binding protein: a pilot study.

Background: Neutrophil-derived heparin-binding protein (HBP) plays a role in the pathophysiology of impaired endothelial dysfunction during inflammation. HBP has been suggested as a predictor of organ dysfunction and disease progression in sepsis. We investigated the effects of heparins on plasma concentrations of HBP in patients undergoing surgery. Methods: We studied three groups of patients receiving heparins during or after surgery. The vascular surgery group received 3000-7500 U, whereas the cardiac surgery group received 27 500-40 000 U. After major general surgery, the third group received 5000 U of low-molecular-weight heparin (LMWH) subcutaneously. Serial plasma HBP concentrations were measured after these treatments with two different methods: Axis-Shield ELISA and Joinstar FIC-Q100. In addition, plasma myeloperoxidase and syndecan-1 were measured in the cardiac surgery group. Results: During vascular surgery, heparin induced a six-fold increase in HBP within 2 min, from 3.6 (2.4-5.4) to 21.4 (9.0-35.4) ng ml-1 (P<0.001). During cardiac surgery, the higher dose of heparin elevated HBP concentrations from 5.3 (2.7-6.1) to 48.7 (38.4-70.1) ng ml-1 (P<0.0001) within 3 min. Patients receiving LMWH showed an increase from a baseline of 5.7 (3.7-12.1) ng ml-1 to a peak HBP concentration of 14.8 (9.5-18.1) ng ml-1 (P<0.0001) after 3 h. Plasma concentrations of myeloperoxidase, but not syndecan-1, also responded with a rapid increase after heparin. There was a strong correlation between the two methods for HBP analysis (r=0.94). Conclusions: Plasma concentrations of HBP increased rapidly and dose-dependently after heparin administration. Subcutaneous administration of LMWH increases plasma HBP, but to a lesser degree. Clinical trial registration: ClinicalTrials.gov identifier: NCT04146493.

Nitric oxide signaling in health and disease.

The surprising discovery that the diatomic gas nitric oxide (NO) is generated by mammalian cells and serves to regulate a multitude of physiological processes has continued to fascinate biologists for almost four decades. The biochemistry of NO is complex, and novel insights into the control of NO biosynthesis and mechanisms of signal transduction are continuously emerging. NO is a key regulator of cardiovascular function, metabolism, neurotransmission, immunity, and more, and aberrant NO signaling is a central feature of many major disorders including cardiovascular disease, diabetes, and cancer. Here, we discuss the basics of NO biology emphasizing recent advances in the field including novel means of increasing NO bioactivity with therapeutic and nutritional implications.

Inorganic nitrate and nitrite ameliorate kidney fibrosis by restoring lipid metabolism via dual regulation of AMP-activated protein kinase and the AKT-PGC1α pathway.

BACKGROUND: Renal fibrosis, associated with oxidative stress and nitric oxide (NO) deficiency, contributes to the development of chronic kidney disease and renal failure. As major energy source in maintaining renal physiological functions, tubular epithelial cells with decreased fatty acid oxidation play a key role in renal fibrosis development. Inorganic nitrate, found in high levels in certain vegetables, can increase the formation and signaling by bioactive nitrogen species, including NO, and dampen oxidative stress. In this study, we evaluated the therapeutic value of inorganic nitrate treatment on development of kidney fibrosis and investigated underlying mechanisms including regulation of lipid metabolism in tubular epithelial cells. METHODS: Inorganic nitrate was supplemented in a mouse model of complete unilateral ureteral obstruction (UUO)-induced fibrosis. Inorganic nitrite was applied in transforming growth factor ß-induced pro-fibrotic cells in vitro. Metformin was administrated as a positive control. Fibrosis, oxidative stress and lipid metabolism were evaluated. RESULTS: Nitrate treatment boosted the nitrate-nitrite-NO pathway, which ameliorated UUO-induced renal dysfunction and fibrosis in mice, represented by improved glomerular filtration and morphological structure and decreased renal collagen deposition, pro-fibrotic marker expression, and inflammation. In human proximal tubule epithelial cells (HK-2), inorganic nitrite treatment prevented transforming growth factor ß-induced pro-fibrotic changes. Mechanistically, boosting the nitrate-nitrite-NO pathway promoted AMP-activated protein kinase (AMPK) phosphorylation, improved AKT-mediated peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α) activity and restored mitochondrial function. Accordingly, treatment with nitrate (in vivo) or nitrite (in vitro) decreased lipid accumulation, which was associated with dampened NADPH oxidase activity and mitochondria-derived oxidative stress. CONCLUSIONS: Our findings indicate that inorganic nitrate and nitrite treatment attenuates the development of kidney fibrosis by targeting oxidative stress and lipid metabolism. Underlying mechanisms include modulation of AMPK and AKT-PGC1α pathways.

Nitric Oxide Is Locally Produced in the Human Middle Ear and Is Reduced by Acquired Cholesteatoma.

OBJECTIVE: To find out if nitric oxide (NO) can be locally produced in the middle ear and if chronic otitis media (COM) and acquired cholesteatoma affect the production. DESIGN: Case-control study. SETTING: Two tertiary-referral hospitals. PATIENTS: Gaseous NO from 11 patients with unilateral perforations or grommet openings was measured with chemiluminescence. Middle ear mucosa from 48 patients with COM and 26 patients with cholesteatoma was investigated. MAIN OUTCOME MEASURES: Detection of NO. Expression of nitric oxide synthase (NOS) mRNA, in mucosa from COM, cholesteatoma and healthy controls, quantified using polymerase chain reaction. RESULTS: The gaseous NO from ears with a unilateral tympanic membrane perforation or a grommet was higher (9 ±â€Š3 ppb, n = 11) than among the controls (4 ±â€Š1 ppb, n = 11, p = 0.04). Lower levels of eNOS (2.64 ±â€Š0.86 mol/100,000 mol ACTB) were detected in the pooled samples from the COM group (n = 48), compared with the control group (140.48 ±â€Š92 mol/100,000 mol ACTB, n = 45, p = 0.010). In the cholesteatoma group (n = 26), a lower expression of nNOS (5.78 × 10-6 ±â€Š1.13 × 10-6 ΔCt) was found in comparison with the controls (1.23 × 10-4 ±â€Š3.18 × 10-5 ΔCt, n = 15, p = 0.011). CONCLUSIONS: NO is likely a natural and permanent part of the gas mixture in the human middle ear. The presence of NOS enzymes in the middle ear mucosa indicates an ongoing NO production and the reduction of NOS in ears with cholesteatoma, and pooled samples from ears with COM, suggest a role for locally produced NO in middle ear disease.

Effects of chronic dietary nitrate supplementation on longevity, vascular function and cancer incidence in rats.

RATIONALE: Dietary nitrate and nitrite have a notoriously bad reputation because of their proposed association with disease, in particular cancer. However, more recent lines of research have challenged this dogma suggesting that intake of these anions also possess beneficial effects after in vivo conversion to the vital signaling molecule nitric oxide. Such effects include improvement in cardiovascular, renal and metabolic function, which is partly mediated via reduction of oxidative stress. A recent study even indicates that low dose of dietary nitrite extends life span in fruit flies. METHODS: In this study, 200 middle-aged Wistar rats of both sexes were supplemented with nitrate or placebo in the drinking water throughout their remaining life and we studied longevity, biochemical markers of disease, vascular reactivity along with careful determination of the cause of death. RESULTS: Dietary nitrate did not affect life span or the age-dependent changes in markers of oxidative stress, kidney and liver function, or lipid profile. Ex vivo examination of vascular function, however, showed improvements in endothelial function in rats treated with nitrate. Neoplasms were not more common in the nitrate group. CONCLUSION: We conclude that chronic treatment with dietary nitrate does not affect life span in rats nor does it increase the incidence of cancer. In contrast, vascular function was improved by nitrate, possibly suggesting an increase in health span.

Effects of inorganic nitrate on ischaemia-reperfusion injury after coronary artery bypass surgery: a randomised controlled trial.

BACKGROUND: Nitric oxide (NO) is an important signalling molecule in the cardiovascular system with protective properties in ischaemia-reperfusion injury. Inorganic nitrate, an oxidation product of endogenous NO production and a constituent in our diet, can be recycled back to bioactive NO. We investigated if preoperative administration of inorganic nitrate could reduce troponin T release and other plasma markers of injury to the heart, liver, kidney, and brain in patients undergoing cardiac surgery. METHODS: This single-centre, randomised, double-blind, placebo-controlled trial included 82 patients undergoing coronary artery bypass surgery with cardiopulmonary bypass. Oral sodium nitrate (700 mg×2) or placebo (NaCl) were administered before surgery. Biomarkers of ischaemia-reperfusion injury and plasma nitrate and nitrite were collected before and up to 72 h after surgery. Troponin T release was our predefined primary endpoint and biomarkers of renal, liver, and brain injury were secondary endpoints. RESULTS: Plasma concentrations of nitrate and nitrite were elevated in nitrate-treated patients compared with placebo. The 72-h release of troponin T did not differ between groups. Other plasma biomarkers of organ injury were also similar between groups. Blood loss was not a predefined outcome parameter, but perioperative bleeding was 18% less in nitrate-treated patients compared with controls. CONCLUSION: Preoperative administration of inorganic nitrate did not influence troponin T release or other plasma biomarkers of organ injury in cardiac surgery. CLINICAL TRIAL REGISTRATION: NCT01348971.

Dietary nitrite extends lifespan and prevents age-related locomotor decline in the fruit fly.

Aging is associated with decreased nitric oxide (NO) bioavailability and signalling. Boosting of a dietary nitrate-nitrite-NO pathway e.g. by ingestion of leafy green vegetables, improves cardiometabolic function, mitochondrial efficiency and reduces oxidative stress in humans and rodents, making dietary nitrate and nitrite an appealing intervention to address age-related disorders. On the other hand, these anions have long been implicated in detrimental health effects of our diet, particularly in formation of carcinogenic nitrosamines. The aim of this study was to assess whether inorganic nitrite affects lifespan in Drosophila melanogaster and investigate possible mechanisms underlying any such effect. In a survival assay, female flies fed a nitrite supplemented diet showed lifespan extension by 9 and 15% with 0.1 and 1 µM nitrite respectively, with no impact of nitrite on reproductive output. Interestingly, nitrite could also protect female flies from age-dependent locomotor decline, indicating a protective effect on healthspan. NO generation from nitrite involved Drosophila commensal bacteria and was indicated by a fluorescent probe as well as direct measurements of NO gas formation with chemiluminescence. Nutrient sensing pathways such as TOR and sirtuins, have been strongly implicated in lifespan extension. In aged flies, nitrite supplementation significantly downregulated dTOR and upregulated dSir2 gene expression. Total triglycerides and glucose were decreased, a described downstream effect of both TOR and sirtuin pathways. In conclusion, we demonstrate that very low doses of dietary nitrite extend lifespan and favour healthspan in female flies. We propose modulation of nutrient sensing pathways as driving mechanisms for such effects.

AMP-activated protein kinase activation and NADPH oxidase inhibition by inorganic nitrate and nitrite prevent liver steatosis.

Advanced age and unhealthy dietary habits contribute to the increasing incidence of obesity and type 2 diabetes. These metabolic disorders, which are often accompanied by oxidative stress and compromised nitric oxide (NO) signaling, increase the risk of adverse cardiovascular complications and development of fatty liver disease. Here, we investigated the therapeutic effects of dietary nitrate, which is found in high levels in green leafy vegetables, on liver steatosis associated with metabolic syndrome. Dietary nitrate fuels a nitrate-nitrite-NO signaling pathway, which prevented many features of metabolic syndrome and liver steatosis that developed in mice fed a high-fat diet, with or without combination with an inhibitor of NOS (l-NAME). These favorable effects of nitrate were absent in germ-free mice, demonstrating the central importance of host microbiota in bioactivation of nitrate. In a human liver cell line (HepG2) and in a validated hepatic 3D model with primary human hepatocyte spheroids, nitrite treatment reduced the degree of metabolically induced steatosis (i.e., high glucose, insulin, and free fatty acids), as well as drug-induced steatosis (i.e., amiodarone). Mechanistically, the salutary metabolic effects of nitrate and nitrite can be ascribed to nitrite-derived formation of NO species and activation of soluble guanylyl cyclase, where xanthine oxidoreductase is proposed to mediate the reduction of nitrite. Boosting this nitrate-nitrite-NO pathway results in attenuation of NADPH oxidase-derived oxidative stress and stimulation of AMP-activated protein kinase and downstream signaling pathways regulating lipogenesis, fatty acid oxidation, and glucose homeostasis. These findings may have implications for novel nutrition-based preventive and therapeutic strategies against liver steatosis associated with metabolic dysfunction.

Dietary nitrate attenuates renal ischemia-reperfusion injuries by modulation of immune responses and reduction of oxidative stress.

Ischemia-reperfusion (IR) injury involves complex pathological processes in which reduction of nitric oxide (NO) bioavailability is suggested as a key factor. Inorganic nitrate can form NO in vivo via NO synthase-independent pathways and may thus provide beneficial effects during IR. Herein we evaluated the effects of dietary nitrate supplementation in a renal IR model. Male mice (C57BL/6J) were fed nitrate-supplemented chow (1.0mmol/kg/day) or standard chow for two weeks prior to 30min ischemia and during the reperfusion period. Unilateral renal IR caused profound tubular and glomerular damage in the ischemic kidney. Renal function, assessed by plasma creatinine levels, glomerular filtration rate and renal plasma flow, was also impaired after IR. All these pathologies were significantly improved by nitrate. Mechanistically, nitrate treatment reduced renal superoxide generation, pro-inflammatory cytokines (IL-1ß, IL-6 and IL-12 p70) and macrophage infiltration in the kidney. Moreover, nitrate reduced mRNA expression of pro-inflammatory cytokines and chemo attractors, while increasing anti-inflammatory cytokines in the injured kidney. In another cohort of mice, two weeks of nitrate supplementation lowered superoxide generation and IL-6 expression in bone marrow-derived macrophages. Our study demonstrates protective effect of dietary nitrate in renal IR injury that may be mediated via modulation of oxidative stress and inflammatory responses. These novel findings suggest that nitrate supplementation deserve further exploration as a potential treatment in patients at high risk of renal IR injury.

Dietary nitrate markedly improves voluntary running in mice.

Nitrate supplementation is shown to increase submaximal force in human and mouse skeletal muscles. In this study, we test the hypothesis that the increased submaximal force induced by nitrate supplementation reduces the effort of submaximal voluntary running, resulting in increased running speed and distance. C57Bl/6N male mice were fed nitrate in the drinking water and housed with or without access to an in-cage running wheel. Nitrate supplementation in sedentary mice had no effect on endurance in a treadmill test, nor did it enhance mitochondrial function. However, after three weeks with in-cage running wheel, mice fed nitrate ran on average 20% faster and 30% further than controls (p<0.01). Compared to running controls, this resulted in ~13% improved endurance on a subsequent treadmill test (p<0.05) and increased mitochondrial oxidative capacity, as judged from a mean increase in citrate synthase activity of 14% (p<0.05). After six weeks with nitrate, the mice were running 58% longer distances per night. When nitrate supplementation was removed from the diet, the running distance and speed decreased to the control level, despite the improved endurance achieved during nitrate supplementation. In conclusion, low-frequency force improvement due to nitrate supplementation facilitates submaximal exercise such as voluntary running.

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.

In adenosine A2B knockouts acute treatment with inorganic nitrate improves glucose disposal, oxidative stress, and AMPK signaling in the liver.

RATIONALE: Accumulating studies suggest that nitric oxide (NO) deficiency and oxidative stress are central pathological mechanisms in type 2 diabetes (T2D). Recent findings demonstrate therapeutic effects by boosting the nitrate-nitrite-NO pathway, which is an alternative pathway for NO formation. This study aimed at investigating the acute effects of inorganic nitrate on glucose and insulin signaling in adenosine A2B receptor knockout mice (A(-/-) 2B), a genetic mouse model of impaired metabolic regulation. METHODS: Acute effects of nitrate treatment were investigated in aged wild-type (WT) and A(-/-) 2B mice. One hour after injection with nitrate (0.1 mmol/kg, i.p.) or placebo, metabolic regulation was evaluated by intraperitoneal glucose and insulin tolerance tests. NADPH oxidase-mediated superoxide production and AMPK phosphorylation were measured in livers obtained from non-treated or glucose-treated mice, with or without prior nitrate injection. Plasma was used to determine insulin resistance (HOMA-IR) and NO signaling. RESULTS: A(-/-) 2B displayed increased body weight, reduced glucose clearance, and attenuated overall insulin responses compared with age-matched WT mice. Nitrate treatment increased circulating levels of nitrate, nitrite and cGMP in the A(-/-) 2B, and improved glucose clearance. In WT mice, however, nitrate treatment did not influence glucose clearance. HOMA-IR increased following glucose injection in the A(-/-) 2B, but remained at basal levels in mice pretreated with nitrate. NADPH oxidase activity in livers from A(-/-) 2B, but not WT mice, was reduced by nitrate treatment. Livers from A(-/-) 2B displayed reduced AMPK phosphorylation compared with WT mice, and this was increased by nitrate treatment. Finally, injection with the anti-diabetic agent metformin induced similar therapeutic effects in the A(-/-) 2B as observed with nitrate. CONCLUSION: The A(-/-) 2B mouse is a genetic mouse model of metabolic syndrome. Acute treatment with nitrate improved the metabolic profile in it, at least partly via reduction in oxidative stress and improved AMPK signaling in the liver.

Effects of long-term dietary nitrate supplementation in mice.

BACKGROUND: Inorganic nitrate (NO3(-)) is a precursor of nitric oxide (NO) in the body and a large number of short-term studies with dietary nitrate supplementation in animals and humans show beneficial effects on cardiovascular health, exercise efficiency, host defense and ischemia reperfusion injury. In contrast, there is a long withstanding concern regarding the putative adverse effects of chronic nitrate exposure related to cancer and adverse hormonal effects. To address these concerns we performed in mice, a physiological and biochemical multi-analysis on the effects of long-term dietary nitrate supplementation. DESIGN: 7 week-old C57BL/6 mice were put on a low-nitrate chow and at 20 weeks-old were treated with NaNO3 (1 mmol/L) or NaCl (1 mmol/L, control) in the drinking water. The groups were monitored for weight gain, food and water consumption, blood pressure, glucose metabolism, body composition and oxygen consumption until one group was reduced to eight animals due to death or illness. At that point remaining animals were sacrificed and blood and tissues were analyzed with respect to metabolism, cardiovascular function, inflammation, and oxidative stress. RESULTS: Animals were supplemented for 17 months before final sacrifice. Body composition, oxygen consumption, blood pressure, glucose tolerance were measured during the experiment, and vascular reactivity and muscle mitochondrial efficiency measured at the end of the experiment with no differences identified between groups. Nitrate supplementation was associated with improved insulin response, decreased plasma IL-10 and a trend towards improved survival. CONCLUSIONS: Long term dietary nitrate in mice, at levels similar to the upper intake range in the western society, is not detrimental.

Ultrasound contrast agent loaded with nitric oxide as a theranostic microdevice.

The current study describes novel multifunctional polymer-shelled microbubbles (MBs) loaded with nitric oxide (NO) for integrated therapeutic and diagnostic applications (ie, theranostics) of myocardial ischemia. We used gas-filled MBs with an average diameter of 4 µm stabilized by a biocompatible shell of polyvinyl alcohol. In vitro acoustic tests showed sufficient enhancement of the backscattered power (20 dB) acquired from the MBs' suspension. The values of attenuation coefficient (0.8 dB/cm MHz) and phase velocities (1,517 m/s) were comparable with those reported for the soft tissue. Moreover, polymer MBs demonstrate increased stability compared with clinically approved contrast agents with a fracture threshold of about 900 kPa. In vitro chemiluminescence measurements demonstrated that dry powder of NO-loaded MBs releases its gas content in about 2 hours following an exponential decay profile with an exponential time constant equal to 36 minutes. The application of high-power ultrasound pulse (mechanical index =1.2) on the MBs resuspended in saline decreases the exponential time constant from 55 to 4 minutes in air-saturated solution and from 17 to 10 minutes in degassed solution. Thus, ultrasound-triggered release of NO is achieved. Cytotoxicity tests indicate that phagocytosis of the MBs by macrophages starts within 6-8 hours. This is a suitable time for initial diagnostics, treatment, and monitoring of the therapeutic effect using a single injection of the proposed multifunctional MBs.

Control of pathogen growth and biofilm formation using a urinary catheter that releases antimicrobial nitrogen oxides.

Antibacterial nitrogen oxides including nitric oxide are formed from nitrite under acidic conditions. In a continuous-flow model of the urinary bladder we used the retention cuff of an all-silicone Foley catheter as a depot for export of nitrogen oxides. The cuff was filled with sodium nitrite (50mM) and an acidic buffer solution (pH 3.6) and the growth of nine common uropathogens in the surrounding artificial urine was measured along with biofilm formation on the catheter surface. In experiments with control catheters (NaCl) bacteria grew readily and biofilm developed within hours in five of nine strains. In contrast, with test catheters bacterial counts were markedly reduced and biofilm formation by Pseudomonas aeruginosa, Klebsiella pneumoniae, and Enterobacter cloace was prevented, whereas Escherichia coli and Staphylococcus aureus were unaffected. We conclude that antibacterial nitrogen oxides generated in the retention cuff of a urinary catheter diffuse into urine and prevent the growth of urinary pathogens and biofilm formation. Although promising, future studies will reveal if this novel approach can be clinically useful for the prevention of catheter-associated urinary tract infections.

Novel aspects of dietary nitrate and human health.

The circulation of nitrogen in nature is a prerequisite for life on earth. In the nitrogen cycle atmospheric nitrogen is fixated by bacteria into forms that can be utilized by plants and mammals. Nitrate and nitrite are obligate intermediates in this cycle, and for more than half a century these anions have interested nutritional scientists, mostly in relation to cancer, because of their ability to form nitrosamines. However, after the discovery of mammalian endogenous nitric oxide (NO) generation and later that its oxidation products nitrate and nitrite can be recycled back to bioactive NO, a novel field of research has emerged that explores a potentially beneficial role of these anions in physiology, nutrition, and therapeutics. In our diet, vegetables are the major source of nitrate that can fuel a nitrate-nitrite-NO pathway. Herein we discuss the nutritional aspects of this pathway and what is presently known about the implications for human health.

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

Circulating nitrate (NO(3)(-)), 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 (NO(2)(-)) 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 .5mmHg, increases correlated to a decrease in circulating nitrite concentrations (r(2) = 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.

Biology of nitrogen oxides in the gastrointestinal tract.

Throughout the human gastrointestinal tract a variety of reactive nitrogen oxides are continuously formed as a result of a complex interplay between the host, commensal bacteria and dietary factors. These compounds include nitric oxide, nitrite, nitrate, peroxynitrite, S-nitrosothiols, nitrated fatty acids and N-nitrosamines, all of which are bioactive with the potential to affect physiological and pathological processes locally in the gut as well as systemically after absorption. Historically, the literature has been dominated by studies on the formation of potentially carcinogenic nitrosamines, but the focus was shifted in the 1980s with the seminal discovery of the L-arginine-nitric oxide pathway and its profound impact on normal physiological functions. More recently, a nitrate-nitrite-nitric oxide pathway has been discovered, with implications for local host defence and gut mucosal integrity and, intriguingly, also for systemic regulation of cardiovascular and metabolic function. This review discusses recent advances in the understanding of the formation, biochemistry, physiology and pathophysiology of reactive nitrogen oxides in the gastrointestinal tract. In addition, opportunities for nitric oxide-based pharmacological or dietary interventions are highlighted.

Dietary nitrate supplementation improves revascularization in chronic ischemia.

BACKGROUND: Revascularization is an adaptive repair mechanism that restores blood flow to undersupplied ischemic tissue. Nitric oxide plays an important role in this process. Whether dietary nitrate, serially reduced to nitrite by commensal bacteria in the oral cavity and subsequently to nitric oxide and other nitrogen oxides, enhances ischemia-induced remodeling of the vascular network is not known. METHODS AND RESULTS: Mice were treated with either nitrate (1 g/L sodium nitrate in drinking water) or sodium chloride (control) for 14 days. At day 7, unilateral hind-limb surgery with excision of the left femoral artery was conducted. Blood flow was determined by laser Doppler. Capillary density, myoblast apoptosis, mobilization of CD34(+)/Flk-1(+), migration of bone marrow-derived CD31(+)/CD45(-), plasma S-nitrosothiols, nitrite, and skeletal tissue cGMP levels were assessed. Enhanced green fluorescence protein transgenic mice were used for bone marrow transplantation. Dietary nitrate increased plasma S-nitrosothiols and nitrite, enhanced revascularization, increased mobilization of CD34(+)/Flk-1(+) and migration of bone marrow-derived CD31(+)/CD45(-) cells to the site of ischemia, and attenuated apoptosis of potentially regenerative myoblasts in chronically ischemic tissue. The regenerative effects of nitrate treatment were abolished by eradication of the nitrate-reducing bacteria in the oral cavity through the use of an antiseptic mouthwash. CONCLUSIONS: Long-term dietary nitrate supplementation may represent a novel nutrition-based strategy to enhance ischemia-induced revascularization.

Effects of fluid resuscitation with hypertonic saline dextrane or Ringer's acetate after nonhemorrhagic shock caused by pulmonary contusion.

BACKGROUND: Injured lungs are sensitive to fluid resuscitation after trauma. Such treatment can increase lung water content and lead to desaturation. Hypertonic saline with dextran (HSD) has hyperosmotic properties that promote plasma volume expansion, thus potentially reducing these side effects. The aim of this study was to (1) evaluate whether fluid treatment counteracts hypotension and improves survival after nonhemorrhagic shock caused by lung contusion and (2) analyze whether resuscitation with HSD is more efficient than treatment with Ringer's acetate (RA) in terms of blood oxygenation, the amount of lung water, circulatory effects, and inflammatory response. METHODS: Twenty-nine pigs, all wearing body armor, were shot with a 7.62-mm assault rifle to produce a standardized pulmonary contusion. These animals were allocated into three groups: HSD, RA, and an untreated shot control group. Exposed animals were compared with animals not treated with fluid and shot with blank ammunition. For 2 hours after the shot, the inflammatory response and physiologic parameters were monitored. RESULTS: The impact induced pulmonary contusion, desaturation, hypotension, increased heart rate, and led to an inflammatory response. No change in blood pressure was observed after fluid treatment. HSD treatment resulted in significantly less lung water (p < 0.05) and tended to give better Pao2 (p = 0.09) than RA treatment. Tumor necrosis factor-α release and heart rate were significantly lower in animals given fluids. CONCLUSION: Fluid treatment does not affect blood pressure or mortality in this model of nonhemorrhagic shock caused by lung contusion. However, our data indicate that HSD, when compared with RA, has advantages for the injured lung.