ArticleLiterature Review

Nitrate-Nitrite-Nitric Oxide Pathway Implications for Anesthesiology and Intensive Care

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Abstract

The gaseous radical nitric oxide is involved in numerous physiologic and pathophysiological events important in anesthesiology and intensive care. Nitric oxide is endogenously generated from the amino acid l-arginine and molecular oxygen in reactions catalyzed by complex nitric oxide synthases. Recently, an alternative pathway for nitric oxide generation was discovered, wherein the inorganic anions nitrate (NO3) and nitrite (NO2), most often considered inert end products from nitric oxide generation, can be reduced back to nitric oxide and other bioactive nitrogen oxide species. This nitrate-nitrite-nitric oxide pathway is regulated differently than the classic l-arginine-nitric oxide synthase nitric oxide pathway, and it is greatly enhanced during hypoxia and acidosis. Several lines of research now indicate that the nitrate-nitrite-nitric oxide pathway is involved in regulation of blood flow, cell metabolism, and signaling, as well as in tissue protection during hypoxia. The fact that nitrate is abundant in our diet gives rise to interesting nutritional aspects in health and disease. In this article, we present an overview of this field of research with emphasis on relevance in anesthesiology and intensive care.

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... -RNS "In fact, a significant decrease in NOx was observed in healthy volunteers after breathing at FiO2=1.0 for 30 minutes (15) and in patients with primary episodes of septic shock (16)." -----Does not ref. ...
... -----Does not ref. 16 Thank you for noticing this issue. We apologize for the miscitation. ...
... The value of NOx was also upregulated in patients with primary episodes of septic shock, despite a potential increased production of RNS 15 ." (as we deleted reference 5, ref 16 is now listed as ref 15). ...
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Oxygen administration is particularly relevant in patients undergoing surgery under general anesthesia and in those who suffer from acute or critical illness. Nevertheless, excess O2, or hyperoxia, is also known to be harmful. Toxicity arises from the enhanced formation of Reactive Oxygen Species (ROS) that, exceeding the antioxidant defense, may generate oxidative stress. Oxidative stress markers are used to quantify ROS toxicity in clinical and non-clinical settings and represent a promising tool to assess the optimal FiO2 in anesthesia and critical care setting. Despite controversial, the guidelines for the regulation of FiO2 in such settings suggest the adoption of high perioperative oxygen levels. However, hyperoxia has also been shown to be an independent mortality risk factor in critically ill patients. In this literature review, we discuss the biochemical mechanisms behind oxidative stress and the available biomarkers for assessing the pro-oxidant vs antioxidant status. Then, we summarize recent knowledge on the hyperoxia-related consequences in the most common anesthesia and critical care settings, such as traumatic brain injury or cardiac arrest. To this purpose, we searched the Pubmed database according to the following combination of key words: ("hyperoxia" OR "FiO2" OR "oxygen therapy") AND ("oxidative stress" OR "ROS" OR "RNS" OR "lipid peroxidation") AND ("anesthesia" OR "surgery" OR "intensive care"). We focused in the results from the past 20 years. Available evidence points toward a conservative monitoring and use of oxygen, unless there is solid proof of its efficacy.
... Nitrate, the inorganic anion NO 3 − , is found in many foods and mostly in vegetables [1]. Nitrate is an endogenous mammalian metabolite, which is supplied mostly through the diet [2][3][4]. In the body, nitrate is reduced to nitrite [5,6], mainly by the bacterial nitratereductase enzymes present in saliva [2,[5][6][7][8][9][10]. ...
... Nitrate is an endogenous mammalian metabolite, which is supplied mostly through the diet [2][3][4]. In the body, nitrate is reduced to nitrite [5,6], mainly by the bacterial nitratereductase enzymes present in saliva [2,[5][6][7][8][9][10]. In mammalian tissues, the rate of this reduction is low [9]. ...
... In mammalian tissues, the rate of this reduction is low [9]. The produced nitrite can then be reduced to the vasodilator nitric oxide (NO) [2,3,5]. ...
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Nitrate, the inorganic anion NO3−, is found in many foods and is an endogenous mammalian metabolite, which is supplied mostly through the diet. Although much is known about the safety of sodium nitrate when given per os, methodological safety data on intravenous bolus injection of sodium nitrate to rodents are lacking. Recently, we have proposed a new use for nitrate, as a contrast agent for magnetic resonance imaging that will be metal free and leave no traces in the body and the environment further to the imaging examination. It was shown that a stable isotope-labelled analog of this ion (15NO3−), in a sodium nitrate solution form and hyperpolarized state, produces a high magnetic resonance signal with prolonged visibility. Therefore, sodium nitrate was targeted for further preclinical development in this context. In the absence of methodological safety data on the potential effects of a high concentration sodium nitrate bolus intravenous injection into rodents, we carried out such an investigation in mice and rats (n = 12 of each, 6 males and 6 females in each group, altogether 24 animals). We show here that an intravenous bolus administration of sodium nitrate at a concentration of 150 mM and a dose of 51 mg/Kg does not lead to adverse effects in mice and rats. This is the first investigation of the tolerance of rodents to an intravenous injection of sodium nitrate.
... Nitrite is used as a food additive for cured meats 21 and approximately 7% of our ingested nitrite comes this source, while the remainder comes from the enterosalivary pathway. 3,22 While nitrites are noncarcinogenic, their ability to form nitrosamines can lead to toxicity 23 as examined by the research community and mainstream media. 24,25 The action of methmyogolbin production by nitrite is, however, beneficial in the treatment of cyanide poisoning and sodium nitrite remains as one of the primary antidotes for acute intoxication. ...
... The ratio 5 D :6 D was now 24:1 in all three samples. At higher loadings of D, the formation of [Ir(H) 2 (IMes)(D) 3 ]Cl is observed, as a single hydride resonance at δ H −23.00. Clearly, this catalyst does not transfer hyperpolarization to Na 15 NO 2 and hinders the overall 15 N signal gain because of consumption of p-H 2 . ...
... The data in Figure 5 details these results which confirm both separation and the fact that the associated signal strengths are sufficient to allow for high-sensitivity 15 (0.18% 15 N polarization) is observed (Figure 5b). No direct evidence for an NO 3 − containing complex could be found, and therefore, polarization transfer must occur through a very low concentration species. ...
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Here, we show how signal amplification by reversible exchange hyperpolarization of a range of 15N-containing synthons can be used to enable studies of their reactivity by 15N nuclear magnetic resonance (NO2- (28% polarization), ND3 (3%), PhCH2NH2 (5%), NaN3 (3%), and NO3- (0.1%)). A range of iridium-based spin-polarization transfer catalysts are used, which for NO2- work optimally as an amino-derived carbene-containing complex with a DMAP-d2 coligand. We harness long 15N spin-order lifetimes to probe in situ reactivity out to 3 × T1. In the case of NO2- (T1 17.7 s at 9.4 T), we monitor PhNH2 diazotization in acidic solution. The resulting diazonium salt (15N-T1 38 s) forms within 30 s, and its subsequent reaction with NaN3 leads to the detection of hyperpolarized PhN3 (T1 192 s) in a second step via the formation of an identified cyclic pentazole intermediate. The role of PhN3 and NaN3 in copper-free click chemistry is exemplified for hyperpolarized triazole (T1 < 10 s) formation when they react with a strained alkyne. We also demonstrate simple routes to hyperpolarized N2 in addition to showing how utilization of 15N-polarized PhCH2NH2 enables the probing of amidation, sulfonamidation, and imine formation. Hyperpolarized ND3 is used to probe imine and ND4+ (T1 33.6 s) formation. Furthermore, for NO2-, we also demonstrate how the 15N-magnetic resonance imaging monitoring of biphasic catalysis confirms the successful preparation of an aqueous bolus of hyperpolarized 15NO2- in seconds with 8% polarization. Hence, we create a versatile tool to probe organic transformations that has significant relevance for the synthesis of future hyperpolarized pharmaceuticals.
... However, following 2 h post-ROSC there is a substantial increase in the total concentration of nitrates and nitrites as compared to ROSC and control levels (P < 0.01 and P < 0.001, respectively; Fig. 3e). Increased nitrate/nitrite concentration suggests an increase in reactive nitrogen species (RNS) 2 h after CA and resuscitation, which is a subset of oxidative stress mediators that work in a similar fashion as ROS to cause damage (Dedon and Tannenbaum 2004;Weitzberg et al. 2010). As a major catalyst of oxidative stress, we measured the plasma concentrations of free iron that would participate in the Fenton reaction to produce damaging hydroxyl species. ...
... In fact, the decrease of DHE rate post-CA using febuxostat highly supports the role of XO in generation of a prooxidant environment in the plasma. Although previously considered as simple, less active end products of nitric oxide generation, nitrates and nitrites have been implicated in more complex physiology that can result in the protective modulation of cellular metabolism, vascular regulation, and cell signaling; however, in the setting of ischemia-reperfusion injury, the nitrate/ nitrite increase can facilitate the conversion to nitric oxide and peroxynitrite and begin an oxidative cycle contributing to tissue damage (Dedon and Tannenbaum 2004;Weitzberg et al. 2010;Warner et al. 2004). The substantial increase observed 2 h post-ROSC suggests that the deleterious effects of nitrate/nitrite may outweigh any beneficial vasodilatory effects during the initial ischemic phase. ...
Article
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Background Cardiac arrest (CA) results in loss of blood circulation to all tissues leading to oxygen and metabolite dysfunction. Return of blood flow and oxygen during resuscitative efforts is the beginning of reperfusion injury and is marked by the generation of reactive oxygen species (ROS) that can directly damage tissues. The plasma serves as a reservoir and transportation medium for oxygen and metabolites critical for survival as well as ROS that are generated. However, the complicated interplay among various ROS species and antioxidant counterparts, particularly after CA, in the plasma have not been evaluated. In this study, we assessed the equilibrium between pro- and anti-oxidants within the plasma to assess the oxidative status of plasma post-CA. Methods In male Sprague–Dawley rats, 10 min asphyxial-CA was induced followed by cardiopulmonary resuscitation (CPR). Plasma was drawn immediately after achieving return of spontaneous circulation (ROSC) and after 2 h post-ROSC. Plasma was isolated and analyzed for prooxidant capacity (Amplex Red and dihydroethidium oxidation, total nitrate and nitrite concentration, xanthine oxidase activity, and iron concentration) and antioxidant capacity (catalase and superoxide dismutase activities, Total Antioxidant Capacity, and Iron Reducing Antioxidant Power Assay). The consequent oxidative products, such as 4-Hydroxyl-2-noneal, malondialdehyde, protein carbonyl, and nitrotyrosine were evaluated to determine the degree of oxidative damage. Results After CA and resuscitation, two trends were observed: (1) plasma prooxidant capacity was lower during ischemia, but rapidly increased post-ROSC as compared to control, and (2) plasma antioxidant capacity was increased during ischemia, but either decreased or did not increase substantially post-ROSC as compared to control. Consequently, oxidation products were increased post-ROSC. Conclusion Our study evaluated the disbalance of pro- and anti-oxidants after CA in the plasma during the early phase after resuscitation. This disequilibrium favors the prooxidants and is associated with increased levels of downstream oxidative stress-induced end-products, which the body’s antioxidant capacity is unable to directly mitigate. Here, we suggest that circulating plasma is a major contributor to oxidative stress post-CA and its management requires substantial early intervention for favorable outcomes.
... the potential of targeting S-nitrosylated GAPDH for proteolysis as a therapeutic approach for alleviating tauopathy memory loss, AD, or other tau-related NDDs. [53][54][55]. In the mitochondria, oxygen is reduced to H + and H2O by cytochrome c oxidase to promote the production of ATP in the electron transport chain [53]. ...
... Ubiquitylation involves the sequential transfer of ubiquitin through a three-enzyme cascade-an ubiquitin-activating enzyme (E1), an ubiquitin-conjugating enzyme (E2), and are produced by various enzymes and reactions. Nitric oxide is produced from nitrate and nitrite (catalyzed by xanthine oxidoreductase) and L-arginine (catalyzed by the nitric oxide synthases) [53][54][55]. In the mitochondria, oxygen is reduced to H + and H 2 O by cytochrome c oxidase to promote the production of ATP in the electron transport chain [53]. ...
Article
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Neurodegeneration has been predominantly recognized as neuronal breakdown induced by the accumulation of aggregated and/or misfolded proteins and remains a preliminary factor in age-dependent disease. Recently, critical regulating molecular mechanisms and cellular pathways have been shown to induce neurodegeneration long before aggregate accumulation could occur. Although this opens the possibility of identifying biomarkers for early onset diagnosis, many of these pathways vary in their modes of dysfunction while presenting similar clinical phenotypes. With selectivity remaining difficult, it is promising that these neuroprotective pathways are regulated through the ubiquitin-proteasome system (UPS). This essential post-translational modification (PTM) involves the specific attachment of ubiquitin onto a substrate, specifically marking the ubiquitin-tagged protein for its intracellular fate based upon the site of attachment, the ubiquitin chain type built, and isopeptide linkages between different ubiquitin moieties. This review highlights both the direct and indirect impact ubiquitylation has in oxidative stress response and neuroprotection, and how irregularities in these intricate processes lead towards the onset of neurodegenerative disease (NDD).
... NO is rapidly (milliseconds) oxidized to the stable-end metabolites, nitrite and nitrate, in biologic systems [14]. The half-lives of nitrate and nitrite in the circulation are about 5-8 h and 20-45 min, respectively [15]. ...
... The half-lives of nitrate and nitrite in the circulation are about 5-8 h and 20-45 min, respectively [15]. As the final products of NO oxidation pathways and reversible NO metabolites [14], nitrite and nitrate may have the potential to distract or overlap hydroxyurea's efficacy as a NO releasing agent. ...
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In several systems, hydroxyurea has been shown to trigger nitric oxide (NO) release or activation of NO synthase (NOS). To elucidate this duality in its pharmacological effects, during myelosuppression, we individually examined hydroxyurea’s (NO releasing agent) and NO metabolites’ (stable NO degradation products) effects on erythroid colony growth and NOS/NO levels in mice using NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). Hydroxyurea and nitrite/nitrate decreased the bone marrow cellularity that was blocked by PTIO only for the NO metabolites. Hydroxyurea inhibition of colony-forming unit-erythroid (CFU-E) formation and reticulocytes was reversed by PTIO. Moreover, hydroxyurea, through a negative feedback mechanism, reduced inducible NOS (iNOS) expressing cells in CFU-E, also prevented by PTIO. Nitrate inhibition of burst-forming units-erythroid (BFU-E) colony growth was blocked by PTIO, but not in mature CFU-E. The presented results reveal that NO release and/or production mediates the hydroxyurea inhibition of mature erythroid colony growth and the frequency of iNOS immunoreactive CFU-E.
... NO is a colorless gas with a solubility of 2-3 mm in water (1 mm at body temperature), is converted to NO 2 − and NO 3 − through metabolic pathways, and is excreted via the urinary pathway (Feelish and Stamler 2005). Although increased concentration of nitrite and nitrate in the urine of patients with chronic inflammation, sepsis or acute microbial infection was noted in the earlier part of 20th century, their connection with elevated level of iNOS activity in the body has only been recently realized (Weitzberg et al. 2010). The reactions of NO with superoxide anion (O 2 − ) and other ROS produce peroxynitrite (ONOO − ), an oxidative species that causes rapid nitration of aromatic amino acid residues of proteins (such as superoxide dismutase). ...
... The reactions of NO with superoxide anion (O 2 − ) and other ROS produce peroxynitrite (ONOO − ), an oxidative species that causes rapid nitration of aromatic amino acid residues of proteins (such as superoxide dismutase). These nitrated proteins serve as hallmarks of inflammation (Weitzberg et al. 2010). Peroxynitrite (ONOO − ), an oxidative species, is responsible for certain types of NOmediated toxicity in vivo (Radi et al. 1991). ...
Article
Nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H 2 S) are gaseous molecules of major impact in biology. Despite their toxicity, these molecules have profound effects on mammalian physiology and major implications in therapeutics. At tiny concentrations in human biology, they play key signaling and regulatory functions and hence are now labeled as “gasotransmitters.” In this literature survey, an introduction to gasotransmitters in relevance with NO, CO and H 2 S has been primarily focused. A special attention has been given to the conjoint physiological, pathophysiological and therapeutic aspects of NO in this work. In addition to the aforementioned elements of the investigation being reported, this report gives a detailed account of some of the recent advancements covering the NO release from both the nitro as well as nitroso compounds. The importance of the metallic center on the eve of producing the reduction center on NO and to develop photolabile properties have been elaborated within the effect of a few examples of metallic centers. Also, theoretical investigations that have been reported in the recent past and some other current theories pertaining to NO chemistry have been enlightened in this review. From the overall study, it is eminent that a number of facts are yet to be explored in context with NO for deeper mechanistic insights, model design for these molecules, other key roles and the search to find the best fit formalism in theoretical chemistry.
... Therefore, potential immunomodulatory treatment should reduce inflammatory organ injury while preserving the viability and competence of the immune cells. Inhaled nitric oxide (iNO) has been proposed as an organ-protective treatment that also has vasoactive action [8]. iNO has also been shown to modulate systemic inflammation by reducing the production of inflammatory mediators and adhesion molecule expression at the tissue level [8]. ...
... Inhaled nitric oxide (iNO) has been proposed as an organ-protective treatment that also has vasoactive action [8]. iNO has also been shown to modulate systemic inflammation by reducing the production of inflammatory mediators and adhesion molecule expression at the tissue level [8]. iNO was shown to downregulate the lung cells NF-κB activation and the production of IL-6 and MCP-1 leading to decreased influx of neutrophils and monocytes [9,10]. ...
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Inhaled nitric oxide (iNO) remains one of the treatment modalities in shock, and in addition to its vasoactive properties, iNO exerts immunomodulatory effects. We used a porcine model of endotoxemia with shock resuscitation (control) and additional treatment with iNO and a steroid (treatment group). After 20 h, bone marrow (BM), peripheral blood (PB), and bronchoalveolar lavage fluid (BALF) were collected to analyze the immunophenotype and mitochondrial membrane potential (Δφ) in three subsets of monocytes. In both groups, SLA-DR expression decreased twofold on the circulating CD14+CD163+ and CD14−CD163+ monocytes, while it did not change on the CD14+CD163+. Δφ increased only in the CD14−CD163+ subpopulation (0.8 vs. 2.0, p < 0.001). The analysis of compartment-specific alterations showed that nearly 100% of BALF CD14+CD163+ and CD14−CD163+ monocytes expressed SLA-DR, and it was higher compared to PB (32% and 20%, p < 0.0001) and BM (93% and 67%, p < 0.001, respectively) counterparts. BALF CD14+CD163+ had a threefold higher Δφ than PB and BM monocytes, while the Δφ of the other subsets was highest in PB monocytes. We confirmed the compartmentalization of the monocyte response during endotoxemic shock, which highlights the importance of studying tissue-resident cells in addition to their circulating counterparts. The iNO/steroid treatment did not further impair monocyte fitness.
... This reaction decreases the size of the NO • pool. Thus, the observed decrease in NOx at t = 2 h after surgery in the FiO2 = 0.80 group (Figure 3e) may be considered a cardiovascular risk factor [22] because it decreases vasodilatation and may impair myocardial tissue perfusion and oxygenation. ...
Article
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Oxygen (O2) is commonly used in clinical practice to prevent or treat hypoxia, but if used in excess (hyperoxia), it may act as toxic. O2 toxicity arises from the enhanced formation of Reactive Oxygen Species (ROS) that exceed the antioxidant defenses and generate oxidative stress. In this study, we aimed at assessing whether an elevated fraction of inspired oxygen (FiO2) during and after general anesthesia may contribute to the unbalancing of the pro-oxidant/antioxidant equilibrium. We measured five oxidative stress biomarkers in blood samples from patients undergoing elective abdominal surgery, randomly assigned to FiO2 = 0.40 vs. 0.80: hydroperoxides, antioxidants, nitrates and nitrites (NOx), malondialdehyde (MDA), and glutathionyl hemoglobin (HbSSG). The MDA concentration was significantly higher 24 h after surgery, and the body antioxidant defense lower, in the FiO2 = 0.80 group with respect to both the FiO2 = 0.40 group and the baseline values (p ≤ 0.05, Student’s t-test). HbSSG in red blood cells was also higher in the FiO2 = 0.80 group at the end of the surgery. NOx was higher in the FiO2 = 0.80 group than the FiO2 = 0.40 group at t = 2 h after surgery. MDA, the main end product of the peroxidation of polyunsaturated fatty acids directly influenced by FiO2, may represent the best marker to assess the pro-oxidant/antioxidant equilibrium after surgery.
... Three different isoforms of the NO synthase enzyme (NOS) are responsible for NO production in mammalian biology; these enzymes are neuronal NOS (NOS I or nNOS), inducible NOS (NOS II or iNOS) and endothelial NOS (NOS III or eNOS) [396]. NO generation can also occur in a NOS-independent manner in the nitrate-nitrite-NO pathway, which involves the reduction of nitrate or nitrite to NO [397]. The nitratenitrite-NO pathway activity increases under low oxygen conditions (hypoxia) when the classical oxygen-dependent mechanism of NO synthesis is inhibited. ...
... Endogenously produced NO has a half life measured in seconds, and is rapidly oxidized to nitrite (NO 2 -) and nitrate (NO 3 --) end products, the latter of which is biologically inert (15). In the presence of microcirculatory ischemia and endothelial cell dysfunction, however, endogenous NO production by eNOS is much more limited. ...
Article
Background: Sodium nitrite has been reported to be effective in reducing chronic peripheral pain. Objectives: To evaluate the safety and efficacy of 40 and 80 mg, BID, of an oral sustained release formulation of sodium nitrite (SR-nitrite) in patients suffering from diabetic neuropathy, and to determine whether SR-nitrite would reduce the frequency of headaches reported previously by subjects receiving the same doses of an immediate release formulation. Study design: Phase II, single-center, randomized, double-blind, placebo controlled clinical trial. Setting: The Ohio Pain Clinic and Kettering Medical Center. Methods: Twenty-four patients were randomized to 40 mg or 80 mg SR-nitrite or placebo twice daily for 12 weeks. The primary objective was to determine whether headaches would be reduced using SR-nitrite. The primary efficacy endpoint was the mean difference in the change of the Neuropathic Pain Symptom Inventory (NPSI) pain score from baseline to that reported after 12 weeks of treatment. Secondary endpoints included changes from baseline for the Brief Pain Inventory (BPI) Scale, the RAND 36 questionnaire, Short Form McGill Questionnaire, daily patient reported score for neuropathic pain, changes in HbA1c, PulseOx and quantitative sensory testing. Results: The number of subjects reporting adverse events and the number of adverse events did not change with dose. There were no reports of treatment-related headaches. Although no significant differences were identified in patient responses to the questionnaires, a trend was observed. In the NPSI assessment, patients in the 40 mg and 80 mg dose group reported a 12.7% and 22.0% reduction in pain, respectively, compared to an 8.4% reduction by patients in the placebo group. A trend was also observed with the BPI total severity score. However, the 40 mg dosing group reported the greatest reduction in pain using the McGill Pain index and via patient logs of daily pain scores, where the mean of pain scores reported by subjects in the 40 mg group dropped by day 41 and generally stayed lower than the mean of scores reported by subjects in either of the other two groups. Patients in the 80 mg SR-nitrite group had an improvement in both Nerve Sensory Conductance and Nerve Sensory Velocity. No changes were observed in HbA1c levels or PulseOx. Limitations: Small sample size. Conclusion: Sustained release sodium nitrite prevents the prevalent reports of headaches by patients treated with an immediate release formulation of sodium nitrite. In a previous study of patients with peripheral arterial disease (PAD), 40 mg BID treatment led to a statistically significant reduction in reported pain, similar trends were observed at the end of the trial period for most of the pain questionnaires used in the study. The 80 mg BID treatment had the more pronounced affect on bioactivity (quantitative sensory testing), which was similar to the PAD study, where this dose group had the greatest improvement in FMD {AU: spell out FMD}. The ability to alleviate pain with BID treatment of SR-nitrite offers promise for a new non-addictive, non-sedating treatment of chronic pain and warrants further study. Key words: Diabetes, diabetic neuropathy, neuropathic pain, peripheral neuropathy, sodium nitrite.
... In the present study, the salivary levels of nitrate were well above this range both at 4 and 12 months of age indicating an uptake of nitrate in the salivary glands and concentration in saliva. However, in the 4 months old infants salivary nitrate levels were lower than what has been repeatedly shown in adults by our group, indicating that the active uptake of nitrate by the salivary glands at that age is probably less than in adults [10,[31][32][33]. At 12 months of age the salivary nitrate levels were more or less in parity with what is found in adults. ...
Article
The inorganic anions nitrate and nitrite are oxidation products from endogenous nitric oxide (NO) generation and constituents in our diet. A nitrate-nitrite-NO pathway exists in which nitrate can be serially reduced to bioactive NO. The first step of this pathway occurs in the oral cavity where oral bacteria convert salivary nitrate to nitrite, whereafter nitrite is reduced to NO systemically by several enzymatic and non-enzymatic pathways. Data are scarce regarding salivary levels and oral conversion capacity of these anions in infants. We measured salivary nitrate and nitrate in infants at 4 and 12 months of age and related values to age, sex, dietary pattern and oral microbiome. Saliva was collected from a total of 188 infants at 4 and 12 months of age. Salivary nitrate, nitrite and nitrite/nitrate ratio as a measure of oral nitrate-reducing capacity were analyzed by HPLC and related to age, sex, type of diet (breast milk or formula) and oral microbiome. There was no difference in salivary nitrate, nitrite or nitrite/nitrate ratio between boys and girls at any age. At 4 months levels of these parameters were lower than what has been described in adults but they had all increased significantly at 12 months of age. At 4 months of age salivary nitrite/nitrate ratio was lower in breast-fed compared to formula-fed infants, but these differences disappeared at 12 months. Several bacterial species were associated with oral nitrate reducing capacity including Prevotella, Veillonella, Alloprevotella and Leptotrichia. We conclude that in infants there is an increase in salivary nitrate and nitrite as well as in oral nitrate-reductase capacity during the first year of life. Differences observed at 4 months of age between breast-fed and formula-fed infants disappear at one year of age.
... These genes are crucial to the L-Arginine-NO pathway, and have been shown to alter ocular blood flow (Schmetterer and Polak, 2001). If this pathway is inhibited due to a deleterious change in the aforementioned genes -as in glaucoma -the body can utilize an alternative method whereby nitrates from the diet are converted to nitrites by commensal bacteria in the saliva, and then converted to NO in the stomach (the Nitrate-nitrite-NO pathway) (Lundberg et al., 2008;Weitzberg et al., 2010). Interestingly, it has already been shown that high levels of nitrates in the diet can reduce the risk of glaucoma by 20e30% (Kang et al., 2016). ...
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The purpose of this study was to determine if there is an association between tinnitus and glaucoma. We tested this by first completing a clinic-based cross-sectional questionnaire study in which we sent a series of tinnitus-related questions to glaucoma patients and healthy subjects, and then followed up with a large population-based cross-sectional study in which glaucoma and tinnitus were also assessed by questionnaire. For the clinical study, we received 209 responses from glaucoma patients and 109 responses from healthy subjects (primarily the spouses of the patients). For the population-based study, we evaluated 79,866 participants. Logistic regression models were used to test the relationship between glaucoma and tinnitus; the clinical study analysis was adjusted for age, gender, BMI, hypertension, and diabetes and the population-based study was adjusted for these same variables with the addition of socioeconomic status and subjective hearing loss. For the clinical study, glaucoma patients had an 85% increase in odds for tinnitus (adjusted OR 1.85, 95% CI 1.10 to 3.05). The effect did not depend on pretreatment intraocular pressure, and the associated symptoms were not pulsatile in nature. For the population-based study, glaucoma patients had a 19% increase in odds for tinnitus (adjusted OR 1.19, 95% CI 1.02 to 1.40). Overall, our results suggest that those with glaucoma are more likely to have tinnitus than those without glaucoma. These results provide hypotheses for a mechanism involved in both tinnitus and glaucoma. One possible mechanism could be vascular dysregulation due to impairment of nitric oxide production.
... In the latter part of the 20 th century, many studies revealed important biological roles for NO, which is produced by a small number of physiological processes. These include pathways that were once thought to be inconsequential, such as nitrite reduction, that are emerging as important facets of nitroso group metabolism [4][5][6]. The best studied and potentially most relevant biological source of NO is the nitric oxide synthase (NOS) family. ...
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Nitric oxide synthases are the major sources of nitric oxide, a critical signaling molecule involved in a wide range of cellular and physiological processes. These enzymes comprise a family of genes that are highly conserved across all eukaryotes. The three family members found in mammals are important for inter- and intra-cellular signaling in tissues that include the nervous system, the vasculature, the gut, skeletal muscle, and the immune system, among others. We summarize major advances in the understanding of biochemical and tissue-specific roles of nitric oxide synthases, with a focus on how these mechanisms enable tissue adaptation and health or dysfunction and disease. We highlight the unique mechanisms and processes of neuronal nitric oxide synthase, or NOS1. This was the first of these enzymes discovered in mammals, and yet much remains to be understood about this highly conserved and complex gene. We provide examples of two areas that will likely be of increasing importance in nitric oxide biology. These include the mechanisms by which these critical enzymes promote adaptation or disease by 1) coordinating communication by diverse cell types within a tissue and 2) directing cellular differentiation/activation decisions processes.
... Humans can obtain nitrate and nitrite by two main sources, the first involves the classic endogenous L-arginine-NOS (nitric oxide synthase) pathway, where the NOS enzymes utilize the L arginine and molecular oxygen to produce the NO, which then is oxidized in the blood vessels and tissues to form nitrate and nitrite as a result of the reaction in which NO with oxyhaemoglobin produces nitrate and methaemoglobin, while oxidation of NO forms nitrite is given by a process that is catalyzed in plasma and in the presence of the multi-copper oxidase and the NO oxidase ceruloplasmin, it is important to mention that there are two more isoforms of NOS, neuronal NOS (nNOS) and inducible NOS (iNOS), the latter is induced by the presence of inflammation or systemic infection (Lundberg et al.2004;Lundberg et al. 2008;Weitzberg et al. 2010); the second source is an exogenous route in which the nitrate and nitrite are acquired through the diet, where the most prominent consumption is that of vegetables mostly green leafy (for nitrate), followed by cured meats, sausages, food preservatives (for nitrite), and water, where the latter on surface and normal conditions contains low levels of these compounds (Nujić and Habuda-Stanić 2017;Lundberg and Weitzberg 2017). This exogenous route contemplates the nitrate-nitrite-NO reductive pathway and requires the presence and action of commensal facultative anaerobic bacteria and the enzyme nitrate reductase to promote the reduction of inorganic nitrate to nitrite in the mouth, after that, nitrite is swallowed and reach the acidic conditions of the stomach, once is absorbed it passes into the systemic circulation reaching the blood and tissues where is then metabolized to nitric oxide and other bioactive nitrogen oxides (as S-nitrosothiols and nitrated species) through enzymatic (catalyzed by deoxygenated hemoglobin, myoglobin, and xanthine oxidoreductase) and non-enzymatic mechanisms, these reactions are generally carried out during hypoxic/ischemich and acidic conditions (Lundberg et al. 2008(Lundberg et al. , 2018Bailey et al. 2012). ...
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Nowadays, the nitrates have been established as carcinogenic components due to the endogenous formation of N-nitroso compounds, however, the consumption of water contaminated with nitrates has only been strongly related to the presence of methemoglobinemia in infants, as an acute effect, leaving out other side effects that demand attention. The thyroid gland takes relevance because it can be altered by many pollutants known as endocrine disruptors, which are agents capable of interfering with the synthesis of hormones, thus far, it is known that nitrates may disrupt the amount of iodine uptake causing most of the time hypothyroidism and affecting the metabolic functions of the organism in all development stages, resulting in an important health burden for the exposed population. Here, this review and update highlighted the impact of consumption of water contaminated with nitrates and effects on the thyroid gland in humans, concluding that nitrates could act as true endocrine disruptor.
... In addition to the regular pathway, nitric oxide can also be synthesized via the enterosalivary pathway. It is also known as the nitrate-nitrite-nitric oxide pathway [26]. In this pathway, the endogenous nitric oxide can be recycled to nitrate via oxidation [27]. ...
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Proton pump inhibitors (PPIs) are amongst the most commonly prescribed medications in the clinical setting. They are commonly used for the treatment of gastroesophageal reflux disease and are readily available over the counter. However, their irrational long term use has been repeatedly discouraged. PPIs are highly lipophilic and may adversely affect multiple physiological pathways of various organs. Over the past few decades, multiple clinical studies have linked PPI use with an increased risk of cardiovascular disease. In this review, we discuss the PPI-induced pathophysiological mechanisms that contribute to cardiovascular disease. Furthermore, we highlight clinical evidence that associates PPI use with an increased risk for various cardiovascular manifestations.
... It derives either from enzymatic reactions catalyzed by neuronal and endothelial nitric oxide synthase (nNOS and eNOS, respectively) or from the inorganic nitrate (NO 3 − ) and nitrite (NO 2 − ) anions. 24 In human skeletal muscles nNOS is highly expressed and is mainly active during contraction, being activated by cytosolic free Ca 2+ concentration as low as 200 nM. 25 Interestingly, nNOS co-localizes with RyR1 in human skeletal muscles and markedly increases in pathological conditions characterized by prominent muscle weakness and impaired work capacity, i.e., nNOS enhances in an animal model of arthritis-induced skeletal muscle weakness. ...
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INTRODUCTIONː Calcium release through ryanodine receptor type 1 (RyR1) channel triggers skeletal muscle contraction, which is finely regulated during physical activity allowing muscles to adapt to stress. However, both prolonged physical activity and inactivity decrease contractile function. The adaptive mechanisms are related to changes in structure, function, and expression of RyR1 channel. EVIDENCE ACQUISITIONː The research on the effects of physical activities on RyR1 structure and function covered PubMed and Scopus databases from 2008 onward. The search term used was RyR1 combined with exercise, fatigue, exertional rhabdomyolysis, or aging. EVIDENCE SYNTHESISː Physical activity induces RyR1 dynamic post-translational modifications, driven essentially by increased reactive oxygen and nitrogen species. RyR1 redox-dependent modifications increase channel activity, but impaired Ca2+ handling may lead to muscle fatigue. Furthermore, imbalance of redox equilibrium may induce RyR1 fragmentation. During exercise RyR1 is also hyperphosphorylated and dissociated from its stabilizing subunit calastabin1, resulting in ‘‘leaky’’ channels and decreased exercise tolerance. RyR1 modifications also occur during aging. Conversely, reduced RyR1 protein expression occurs in some physiopathological conditions characterized by low-exercise capacity, i.e., heart failure and prolonged bed rest. Impaired muscle performance is also linked to RyR1 mutations, with altered channel structure and/or function. Moreover, in apparently healthy subjects with RyR1 mutations, physical exercise can trigger skeletal muscle stiffness at low temperatures, or exertional rhabdomyolysis. CONCLUSIONSː We provide an overview of the molecular aspects linking skeletal muscle activity to RyR1 modifications, with particular attention to the effects produced by different type of exercises, i.e. aerobic and anaerobic exercise, by some pathophysiological conditions, by age-related loss of muscle function and by RyR1 mutations. Key words: Physical activity - RyR1 - ROS/RNS - Post-translation modifications - Expression
... This in turn propagates endothelial and tubular epithelial cell injuries [7], which contribute to the development of reduced kidney function. In addition to hypoxia during the ischemic period, excessive production of reactive oxygen species (ROS) generated in excess by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondria during the reperfusion phase scavenges NO [8]. New approaches that reduce oxidative stress and maintain NO bioactivity may have therapeutic value in the battle against IR-induced kidney failure. ...
Article
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Background: Renal ischemia-reperfusion (IR) injury is a common cause of acute kidney injury (AKI), which is associated with oxidative stress and reduced nitric oxide (NO) bioactivity and increased risk of developing chronic kidney disease (CKD) and cardiovascular disease (CVD). New strategies that restore redox balance may have therapeutic implications during AKI and associated complications. Aim: To investigate the therapeutic value of boosting the nitrate-nitrite-NO pathway during development of IR-induced renal and cardiovascular dysfunction. Methods: Male C57BL/6 J mice were given sodium nitrate (10 mg/kg, i. p) or vehicle 2 h prior to warm ischemia of the left kidney (45 min) followed by sodium nitrate supplementation in the drinking water (1 mmol/kg/day) for the following 2 weeks. Blood pressure and glomerular filtration rate were measured and blood and kidneys were collected and used for biochemical and histological analyses as well as renal vessel reactivity studies. Glomerular endothelial cells exposed to hypoxia-reoxygenation, with or without angiotensin II, were used for mechanistic studies. Results: IR was associated with reduced renal function and slightly elevated blood pressure, in combination with renal injuries, inflammation, endothelial dysfunction, increased Ang II levels and Ang II-mediated vasoreactivity, which were all ameliorated by nitrate. Moreover, treatment with nitrate (in vivo) and nitrite (in vitro) restored NO bioactivity and reduced mitochondrial oxidative stress and injuries. Conclusions: Acute treatment with inorganic nitrate prior to renal ischemia may serve as a novel therapeutic approach to prevent AKI and CKD and associated risk of developing cardiovascular dysfunction.
... Although a direct hit of excessive nitric oxide to neurons appear to be detrimental to its mitochondrial function, the role of systemic nitric oxide is dependent on its source and cellular context. Nitric oxide generated from inorganic nitrite has been shown to play a protective role in variety of pathologies including cardiovascular diseases (Weitzberg et al. 2010, Rocha et al. 2016. Nitrite administration in zebrafish protected dopaminergic neurodegeneration induced by 1-methyl-4-phenylpyridinium (MPP+), in rat protected dopaminergic neurodegeneration induced by rotenone or 6-hydroxydopamine, and in dermal fibroblasts from human PD patients with LRRK2 mutations (Chiara Milanese 2017). ...
Article
Parkinson's disease (PD) is a movement disorder with widespread neurodegeneration in the brain. Significant oxidative, reductive, metabolic, and proteotoxic alterations have been observed in PD postmortem brains. The alterations of mitochondrial function resulting in decreased bioenergetic health is important and needs to be further examined to help develop biomarkers for PD severity and prognosis. It is now becoming clear that multiple hits on metabolic and signaling pathways are likely to exacerbate PD pathogenesis. Indeed, data obtained from genetic and genome association studies have implicated interactive contributions of genes controlling protein quality control and metabolism. For example, loss of key proteins that are responsible for clearance of dysfunctional mitochondria through a process called mitophagy has been found to cause PD, and a significant proportion of genes associated with PD encode proteins involved in the autophagy-lysosomal pathway. In this review, we highlight the evidence for the targeting of mitochondria by proteotoxic, redox and metabolic stress and the role autophagic surveillance in maintenance of mitochondrial quality. Furthermore, we summarize the role of α-synuclein, LRRK2, and tau in modulating mitochondrial function and autophagy. Among the stressors that can overwhelm the mitochondrial quality control mechanisms, we will discuss 4-hydroxynonenal (HNE) and nitric oxide. The impact of autophagy is context depend and as such can have both beneficial and detrimental effects. Furthermore, we highlight the potential of targeting mitochondria and autophagic function as an integrated therapeutic strategy and the emerging contribution of the microbiome to PD susceptibility.
... The majority of information about NOx physiology comes from studies of nitric oxide, metabolized from nitrite, nitrate via nitrite, Larginine, or pharmaceutical NO donors such as sodium nitroprusside (SNP). NO is a highly diffusible gas with a half-life ranging from microseconds to more than a minute (Herold et al., 2001; reviewed by Kelm, 1999;Weitzberg et al., 2010). The half-life of NO is shortest when NO concentration is high, oxygen is available, and oxygen-based free radicals, NO-metabolizing enzymes, or metal containing compounds such as hemoglobin, myoglobin, or neuroglobin are present. ...
... The predominant NO oxidation product in plasma is nitrate with micromolar concentrations at least two orders of magnitude higher than nitrite. In circulation, the half-lives of nitrate and nitrite are approximately 6 hours and 20 min respectively, nitrate and nitrite are excreted in urine and can be used as a measure of NO production 231,232 . However nitrate and nitrite can also be reduced and recycled back to NO by several pathways including by haemoglobin acting as a nitrite reductase as wells as ...
Thesis
Oxidative stress has recently become understood as a disruption of redox signalling and control, and is strongly associated with a wide number of physiological conditions and diseases. Redox metabolism plays a key role in the defence of cells against oxidative damage. Attempts to produce better outcomes in these conditions by correcting the redox imbalance with antioxidants have produced mixed results at best. Therefore, there is an increasing need to better understand redox metabolism and how the many redox pathways involved are connected. In particular, hydrogen sulfide (H2S) has emerged as a key signalling molecule in redox regulation, along with its associated metabolites and redox pathways. Whilst there are existing analytical methods available for the analysis of H2S and its metabolites, they are often limited in scope, and neglect detailed investigation of important steps in the analysis including sample processing. The ascorbate metabolome comprises a separate redox pathway and has close links to the sulfide metabolome. Some aspects of the ascorbate metabolome, such as ascorbic acid-2-sulfate,however have not been widely analysed in humans. In order to better understand the sulfide redox metabolome, an ultra-high pressure liquid chromatography mass spectrometry (UHPLC-MS) based method was developed to quantify sulfide and other key metabolites. Important aspects of sample processing including collection, preparation and storage, were investigated to determine their impact on the measured metabolites. N-Ethylmaleimide was used to trap the sulfide and prevent oxidation and degradation of the other metabolites. The same method was then utilised to analyse persulfides and polysulfides, which are additional redox relevant sulfide related metabolites. The kinetics of the trapping reactions were also investigated in more detail. Additionally, a UHPLC-MS method for the simultaneous analysis of the two key components of ascorbate metabolism, ascorbic acid and dehydroascorbic acid along with ascorbic acid-2sulfate was developed, and specific aspects of sample preparation for this analysis investigated. The developed method for the analysis of the sulfide metabolome was successfully applied to the analysis of plasma from a cohort of ten healthy volunteers as a proof of concept study. This robust and versatile method could be easily applied to both existing sample cohorts and new studies, and provide new insights into sulfur redox metabolism. It could also be used in conjunction with stable isotope tracing and established methodologies for the analysis of other redox metabolomes. The analysis of polysulfides provided new insights into previously unknown sulfur related metabolic activities of the antioxidant enzyme superoxide dismutase. The method for the analysis of the ascorbate metabolome requires further work due to factors involving sample processing.
... shock. An example is therapeutic nitrite infusion to drive nitric oxide production under hypoxic conditions to reopen collapsed capillaries [17,18]. Nitric oxide also downregulates mitochondrial respiration during hypoxia which may reduce ROS generation during reperfusion [15]. ...
... Here we observed that sepsis increased the levels of nitrite in the lung and the brain of sedentary rats, while it was significantly decreased in the hippocampus along with a tendency toward diminishing in the prefrontal cortex of trained septic rats. Nitrite is generated due to the oxidation of the RNS nitric oxide (NO) by different nitric oxide synthases (NOS), and it can be further reduced to NO and other bioactive nitrogen oxides [61]. Particularly, different cells produce NO, including hematopoietic, immune, and parenchymal derived from the lung, liver, kidney, and gut [62,63]. ...
Article
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Sepsis is life-threatening organ dysfunction caused by a dysregulated host response to infection. The crosstalk occurs between the primary focus of infection and lung and other organ systems including the central nervous system via soluble and cellular inflammatory mediators and that this involves both the innate and adaptive immune systems. These interactions are reflected by genomic changes and abnormal rates of cellular apoptosis. The lungs and the brain are rapidly affected due to an inflammatory response and oxidative stress in sepsis. Physical exercise promotes positive responses in the inflammatory cascade and oxidative/antioxidant system. In this sense, we aimed at determining the possible protectant effects of a physical exercise program against inflammation and oxidative stress on the lungs and the brain of rats subjected to sepsis. Adult male Wistar rats were randomly assigned to the sham + sedentary (S), sham + trained (T), and cecal ligation and perforation (CLP) + S and CLP + T and subjected to a physical exercise program using a treadmill for 21 days. Forty-eight hours after the last training session, sepsis was induced by the CLP model. Twenty-four hours later, the animals were euthanized and the lungs, the hippocampus, and the prefrontal cortex were harvested to determine the levels of cytokines by enzyme-linked immunosorbent assay (ELISA) and nitrite and reactive oxygen species production, oxidative damage to proteins, and antioxidant enzymes by spectrophotometric method. Sepsis increased the lung and brain levels of TNF-α, IL-1β, and IL-6, while diminished IL-10 levels, elevated nitrite levels and reactive oxygen species production, augmented the levels of protein carbonyls and diminished the sulfhydryl content, and decreased SOD activity and GSH levels. The exercise program diminished the levels of TNF-α, IL-1β, IL-6, nitrite, and reactive oxygen species production, as well as the levels of protein carbonyls but augmented the sulfhydryl content, and elevated SOD activity. In conclusion, the exercise program protected the lungs and the brain of septic rats against inflammation and oxidative stress.
... This rapid oxidation partly regulates NO bioactivity, where nitrate is the predominant oxidation product in the circulation and diet is the main contributor of exogenous nitrate. In blood and tissues, nitrite can be reduced to NO and other bioactive nitrogen oxides [72]. Taken together, the measurement of NOx is an indirect but helpful assessment of NO. ...
Article
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Coronary microvascular dysfunction is prevalent among people with diabetes and is correlated with cardiac mortality. Compromised endothelial-dependent dilation (EDD) is an early event in the progression of diabetes, but its mechanisms remain incompletely understood. Nitric oxide (NO) is the major endothelium-dependent vasodilatory metabolite in the healthy coronary circulation, but this switches to hydrogen peroxide (H2O2) in coronary artery disease (CAD) patients. Because diabetes is a significant risk factor for CAD, we hypothesized that a similar NO-to-H2O2 switch would occur in diabetes. Vasodilation was measured ex vivo in isolated coronary arteries from wild type (WT) and microRNA-21 (miR-21) null mice on a chow or high-fat/high-sugar diet, and B6.BKS(D)-Leprdb/J (db/db) mice using myography. Myocardial blood flow (MBF), blood pressure, and heart rate were measured in vivo using contrast echocardiography and a solid-state pressure sensor catheter. RNA from coronary arteries, endothelial cells, and cardiac tissues was analyzed via quantitative real-time PCR for gene expression, and cardiac protein expression was assessed via western blot analyses. Superoxide was detected via electron paramagnetic resonance. (1) Ex vivo coronary EDD and in vivo MBF were impaired in diabetic mice. (2) Nω-Nitro-L-arginine methyl ester, an NO synthase inhibitor (L-NAME), inhibited ex vivo coronary EDD and in vivo MBF in WT. In contrast, polyethylene glycol-catalase, an H2O2 scavenger (Peg-Cat), inhibited diabetic mouse EDD ex vivo and MBF in vivo. (3) miR-21 was upregulated in diabetic mouse endothelial cells, and the deficiency of miR-21 prevented the NO-to-H2O2 switch and ameliorated diabetic mouse vasodilation impairments. (4) Diabetic mice displayed increased serum NO and H2O2, upregulated mRNA expression of Sod1, Sod2, iNos, and Cav1, and downregulated Pgc-1α in coronary arteries, but the deficiency of miR-21 reversed these changes. (5) miR-21-deficient mice exhibited increased cardiac PGC-1α, PPARα and eNOS protein and reduced endothelial superoxide. (6) Inhibition of PGC-1α changed the mRNA expression of genes regulated by miR-21, and overexpression of PGC-1α decreased the expression of miR-21 in high (25.5 mM) glucose treated coronary endothelial cells. Diabetic mice exhibit a NO-to-H2O2 switch in the mediator of coronary EDD, which contributes to microvascular dysfunction and is mediated by miR-21. This study represents the first mouse model recapitulating the NO-to-H2O2 switch seen in CAD patients in diabetes.
... This concept of NO ⇄ NO 2 − /NO 3 − interconversion is utilized to modulate blood flow to and from certain organs in humans (e.g. brain, heart etc.) [56][57], thereby efficiently regulating dioxygen distribution [58][59] and formation/concentrations of ROS (reactive oxygen species) and RNOS species, while also maintaining an anti-inflammatory/antiapoptotic environment [60]. In light of the notable significance of physiological nitrogen oxide (NO x ) concentrations and their interconversion equilibria, how these are modulated by the presence of metalloproteins/their activities has drawn considerable chemical and biological research interests. ...
Article
Dioxygen activating heme enzymes have long predicted to be powerhouses for nitrogen oxide interconversion, especially for nitric oxide (NO) oxidation which has far-reaching biological and/or environmental impacts. Lending credence, reactivity of NO with high-valent heme‑oxygen intermediates of globin proteins has recently been implicated in the regulation of a variety of pivotal physiological events such as modulating catalytic activities of various heme enzymes, enhancing antioxidant activity to inhibit oxidative damage, controlling inflammatory and infectious properties within the local heme environments, and NO scavenging. To reveal insights into such crucial biological processes, we have investigated low temperature NO reactivities of two classes of synthetic high-valent heme intermediates, Compound-II and Compound-I. In that, Compound-II rapidly reacts with NO yielding the six-coordinate (NO bound) heme ferric nitrite complex, which upon warming to room temperature converts into the five-coordinate heme ferric nitrite species. These ferric nitrite complexes mediate efficient substrate oxidation reactions liberating NO; i.e., shuttling NO2⁻ back to NO. In contrast, Compound-I and NO proceed through an oxygen-atom transfer process generating the strong nitrating agent NO2, along with the corresponding ferric nitrosyl species that converts to the naked heme ferric parent complex upon warmup. All reaction components have been fully characterized by UV–vis, ²H NMR and EPR spectroscopic methods, mass spectrometry, elemental analyses, and semi-quantitative determination of NO2⁻ anions. The clean, efficient, potentially catalytic NOx interconversions driven by high-valent heme species presented herein illustrate the strong prospects of a heme enzyme/O2/NOx dependent unexplored territory that is central to human physiology, pathology, and therapeutics.
... New evidence has indicated that NO inhalation leads to the formation of new compounds which may be carried as thiol groups attached to proteins in the blood or which may act indirectly through nitrite metabolites. 12,13 NO in tissue has been shown to exert anti-inflammatory effects and inhibit the expression of cytokines, adhesion molecules, interleukins, and other inflammatory mediators. 14 Taken together, these data support the concept that inhaled NO could be a novel treatment option for a disease characterized by systemic endothelial dysfunction. ...
Article
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This study evaluated the effects of inhaled nitric oxide (iNO) therapy combined with intravenous (IV) corticosteroids on hemodynamics, selected cytokines, and kidney messenger RNA toll-like receptor 4 (mRNA TLR4) expression in ischemia–reperfusion injury animal model. The primary endpoint was the evaluation of circulatory, respiratory, and renal function over time. We also investigated the profile of selected cytokines and high-mobility group box 1 (HMGB1) protein, as well as renal mRNA TLR4 activation determined by quantitative real-time polymerase chain reaction analysis. Pigs (n = 19) under sevoflurane AnaConDa anesthesia/sedation were randomized and subjected to abdominal laparotomy and alternatively suprarenal aortic cross-clamping (SRACC) for 90 min or sham surgery: Group 1 (n = 8) iNO (80 ppm) + IV corticosteroids (25 mg ×3) started 30 min before SRACC and continued 2 h after SRACC release, followed with decreased iNO (30 ppm) until the end of observation, Group 2 (n = 8) 90 min SRACC, Group 3 (n = 3)—sham surgery. Renal biopsies were sampled 1 hr before SRACC and at 3 and 20 h after SRACC release. Aortic clamping increased TLR4 mRNA expression in ischemic kidneys, but significant changes were recorded only in the control group (P = 0.016). Treatment with iNO and hydrocortisone reduced TLR4 mRNA expression to pre-ischemic conditions, and the difference observed in mRNA expression was significant between control and treatment group after 3 h (P = 0.042). Moreover, animals subjected to treatment with iNO and hydrocortisone displayed an attenuated systemic inflammatory response and lowered pulmonary vascular resistance plus increased oxygen delivery. The results indicated that iNO therapy combined with IV corticosteroids improved central and systemic hemodynamics, oxygen delivery, and diminished the systemic inflammatory response and renal mRNA TLR4 expression.
... Although our knowledge on the effects of nitrate and nitrite is constantly increasing less is known about the metabolic fate of these anions in various organs. The plasma half-life of nitrate and nitrite has been estimated to 5-6 h and 40 min, respectively [16]. However, after intake of nitrate the enterosalivary circulation of nitrate will generate a prolonged increase in plasma nitrite. ...
Article
Numerous studies have shown beneficial cardiovascular and metabolic effects of dietary nitrate but the release or uptake of these anions on an organ level is still poorly elucidated. Here we administered sodium nitrate in the pig and measured acute changes in release/uptake of nitrate and nitrite across several organs as well as cardiovascular and metabolic functions. In 17 anesthetized pigs multiple venous catheters and arterial ultrasonic blood flow probes were positioned. After pretreatment with the NO synthase (NOS) inhibitor l-NAME to minimize involvement of NOS-dependent nitrate/nitrite generation, the animals received bolus injections of either sodium nitrate or sodium chloride. Organ blood flows and release/uptake of nitrate and nitrite were measured in the pulmonary, splanchnic, hepatic and renal circulations for up to two hours. In addition, small intestinal luminal NO, gut secretion of nitrate, as well as hepatic and renal NADPH oxidase activity were measured. At baseline there was a significant uptake of nitrite in the liver and kidneys together with a release of nitrite from the lungs. In the control pigs, arterial plasma nitrite progressively declined during the observation period (−54%) but was stable in the nitrate group, indicating conversion of nitrate to nitrite. Sodium nitrate led to a marked accumulation of nitrate in the small intestinal lumen with a parallel increase in luminal nitrite. This was coupled with release of nitrite in the portal vein and a concomitant uptake of this anion in the liver. There was a trend towards reduced NADPH oxidase-dependent superoxide generation in the liver but an increase in the kidney. Nitrate had no acute effects on cardiovascular parameters or regional and systemic oxygen consumption. In conclusion, we found a notable difference in release and uptake of nitrate and nitrite between the organs investigated. Our findings indicate an acute conversion of nitrate to nitrite, most likely independent of oral bacteria but by a mammalian nitrate reductase and/or gut bacteria.
... The half-life of NO is only several seconds; thus NO is rapidly oxidised to both nitrite and nitrate. Although inorganic nitrite and nitrate have been considered as stable end metabolites of NO, recent studies have highlighted the possibility of using the nitrate-nitrite-NO conversion pathway as a means of increasing circulating NO [11]. ...
Article
Non-alcoholic fatty liver disease (NAFLD) is considered the hepatic representation of the metabolic disorders. Inorganic nitrate/nitrite can be converted to nitric oxide, regulate glucose metabolism, lower lipid levels, and reduce inflammation, thus raising the hypothesis that inorganic nitrate/nitrite could be beneficial for improving NAFLD. This study assessed the therapeutic effects of chronic dietary nitrate on NAFLD in a mouse model. 60 ApoE−/- mice were fed a high-fat diet (HFD) for 12 weeks to allow for the development of atherosclerosis with associated NAFLD. The mice were then randomly assigned to different groups (20/group) for a further 12 weeks: (i) HFD + NaCl (1 mmol/kg/day), (ii) HFD + NaNO3 (1 mmol/kg/day), and (iii) HFD + NaNO3 (10 mmol/kg/day). A fourth group of ApoE−/- mice consumed a normal chow diet for the duration of the study. At the end of the treatment, caecum contents, serum, and liver were collected. Consumption of the HFD resulted in significantly greater lipid accumulation in the liver compared to mice on the normal chow diet. Mice whose HFD was supplemented with dietary nitrate for the second half of the study, showed an attenuation in hepatic lipid accumulation. This was also associated with an increase in hepatic AMPK activity compared to mice on the HFD. In addition, a significant difference in bile acid profile was detected between mice on the HFD and those receiving the high dose nitrate supplemented HFD. In conclusion, dietary nitrate attenuates the progression of liver steatosis in ApoE−/- mice fed a HFD.
... To date, it is known that nitric oxide is endogenously generated from the amino acid l-arginine following a process that is regulated by complex nitric oxide synthases. In 2010 Weitzberg et al proposed an alternative pathway of nitric oxide generation that is based in back reduction of nitrate and nitrite (which are generally considered to be inert end products) 17 . Since then, however, there has been no study to support the potential interaction of nitrate consumption with infertility; hence, this remains to be investigated in forthcoming projects. ...
Article
Nitrate is a nitrogen oxoanion formed by loss of a proton from nitric acid. It is an important source of nitrogen which is essential for protein formation. Regular levels of nitrate in the environment do not exceed 2-5 mg/L. Fertilizers, herbicides and pesticides increase, however, the amount of nitrate in drinking water. In 2011 the World Health Organization (WHO) has issued a revised document on nitrate and nitrite levels in drinking water to help establish a common guideline that will help countries. To date, the majority of guidelines on drinking water quality that is directly related to nitrate levels are based in its toxic effect in infants and neonates (primarily the occurrence of methemoglobinemia) and suggest as an arbitrary cut-off the existence of less than 10 mg/L as a measure of safety. Exposure to nitrate levels has been linked to adverse neurodevelopmental effects, fetal malformations and several forms of cancer. However, the associations remain scarce in several fields as the majority of available data is retrieved from articles that investigate the impact of nitrosatable drugs, rather than environmental exposure. In Greece, seven vulnerable regions have been recently identified, following the country`s referral to the Court of Justice by Commission. The aim of the present article is to summarize current evidence and provide recommendations for clinical practice and future research in the field.
... Ultimately some nitrite is absorbed into the circulation where it acts as a storage pool for subsequent NO production. The conversion of nitrite to NO is expedited in conditions of acidosis or hypoxemia which likely occurs in regions of the pulmonary vasculature in COPD patients, especially during exercise [37][38][39]. ...
Article
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Background: During exercise as pulmonary blood flow rises, pulmonary capillary blood volume increases and gas exchange surface area expands through distention and recruitment. We have previously demonstrated that pulmonary capillary recruitment is limited in COPD patients with poorer exercise tolerance. Hypoxia and endothelial dysfunction lead to pulmonary vascular dysregulation possibly in part related to nitric oxide related pathways. Purpose: To determine if increasing dietary nitrate might influence lung surface area for gas exchange and subsequently impact exercise performance. Methods: Subjects had stable, medically treated COPD (n = 25), gave informed consent, filled out the St George Respiratory Questionnaire (SGRQ), had a baseline blood draw for Hgb, performed spirometry, and had exhaled nitric oxide (exNO) measured. Then they performed the intra-breath (IB) technique for lung diffusing capacity for carbon monoxide (DLCO) as well as pulmonary blood flow (Qc). Subsequently they completed a progressive semi-recumbent cycle ergometry test to exhaustion with measures of oxygen saturation (SpO2) and expired gases along with DLCO and Qc measured during the 1st work load only. Subjects were randomized to nitrate supplement group (beetroot juice) or placebo group (black currant juice). Results: Exhaled nitric oxide levels rose >200% in the nitrate group (p < 0.05) with minimal change in placebo group. The SGRQ suggested a small fall in perceived symptom limitation in the nitrate group, but no measure of resting pulmonary function differed post nitrate supplementation. With exercise, there was no influence of nitrate supplementation on peak VO2or other measures of respiratory gas exchange. There was a tendency for the exercise DLCO to increase slightly in the nitrate group with a trend towards a rise in the DLCO/Qc relationship (p = 0.08) but not in the placebo group. The only other significant finding was a fall in the exercise blood pressure in the nitrate group, but not placebo group (p < 0.05). Conclusion: Despite evidence of a rise in exhaled nitric oxide levels with nitrate supplementation, there was minimal evidence for improvement in exercise performance or pulmonary gas exchange surface area in a stable medically treated COPD population.
... It is regulated by its rapid oxidation to NO2 and then, in the presence of oxyhemoglobin, to NO3. In the presence of carbonic anhydrase, vitamin C, or polyphenols, NO2 is reduced to NO, thus systemizing the nitrogen cycle in the body 12 . ...
Article
Background: Oxidative stress is thought to be closely related to epileptogenesis. We have previously reported that nitric oxide (NO) levels are higher in epilepsy-prone EL mice between the ages of 3 and 8 weeks than in control mice. However, NO is divided into two fractions, nitrite (NO2) and nitrate (NO3), which appear to play different roles in epileptogenesis. Methods: NO2 and NO3 levels were measured, in EL mice and the control mice, in the parietal cortex, which is thought to be the primary epileptogenetic center in EL mice, and measured in the hippocampus, which is thought to be the secondary center. Results: NO3 levels in the hippocampus and parietal cortex of the immature EL mice (3 to 8 weeks of age) were significantly higher than those in the control mice; NO2 levels were significantly higher in the EL mice throughout the study period. The NO3 levels were significantly higher than the NO2 levels in the immature EL mice, but after the onset of ictogenesis at 10 weeks of age, the relative levels of the two fractions reversed. Conclusion: The reversal of the NO fraction distribution at the onset of seizures that we observed may be related to the developmental process of seizure susceptibility in the neural network of EL mice.
... Low tissue oxygen tension significantly decreased the oxygen-dependent NO synthesis of endothelial NO synthase during ischemia (Webb et al., 2004). Furthermore, overproduction of malondialdehyde (MDA) in the reperfusion phase further consumes endogenous NO (Weitzberg et al., 2010). Reduced bioavailability of NO can lead to endothelial and microvascular functional imbalance, bringing about the "noreflow phenomenon" after ischemic tissue reperfusion is initiated (Eltzschig and Eckle, 2011). ...
Article
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Objectives: Hepatic ischemia-reperfusion injury (HIRI) is of common occurrence during liver surgery and liver transplantation and may cause hepatic impairment, resulting in acute liver dysfunction. Nitrate plays an important physiological regulatory role in the human body. Whether dietary nitrate could prevent HIRI is, however, unknown. Methods: A HIRI mouse model was established in that the blood supply to the median lobe and left lateral lobe was blocked for 60 min through the portal vein and related structures using an atraumatic clip. Sodium nitrate (4 mM) was administrated in advance through drinking water to compare the influence of sodium nitrate and normal water on HIRI. Results: Liver necrosis and injury aggravated after HIRI. The group treated with sodium nitrate showed the lowest activities of plasma aminotransferase and lactate dehydrogenase and improved outcomes in histological investigation and TUNEL assay. Mechanistically, sodium nitrate intake increased plasma and liver nitric oxide levels, upregulated nuclear factor erythroid 2-related factor 2 (NRF2)–related molecules to reduce malondialdehyde level, and increased the activities of antioxidant enzymes to modulate hepatic oxidative stress. Conclusions: Dietary inorganic nitrate could prevent HIRI, possibly by activating the NRF2 pathway and modulating oxidative stress. Our study provides a novel therapeutic compound that could potentially prevent HIRI during liver transplantation or hepatic surgery.
... ; https://doi.org/10.1101/2021.05.18.444667 doi: bioRxiv preprint product in the circulation, but diet is the main contributor of exogenous nitrate. In blood and tissues, nitrite can be further reduced to nitric oxide and other bioactive nitrogen oxides [63]. Taken together, the measurement of NOx is an indirect but useful measurement NO. ...
Preprint
Coronary microvascular dysfunction is prevalent among diabetics and is correlated with cardiac mortality. Compromised endothelial-dependent dilation (EDD) is an early event in the progression of diabetes, but the mechanisms remain incompletely understood. Nitric oxide (NO) is the major endothelium-dependent vasodilatory metabolite in the healthy coronary circulation, but switches to hydrogen peroxide (H2O2) in coronary artery disease (CAD) patients. Because diabetes is a major risk factor for CAD we hypothesized that a similar switch from NO-to-H2O2 occurs in diabetes. Methods: Vasodilation was measured ex vivo in isolated coronary arteries from wild type (WT) and microRNA-21 (miR-21) null mice fed chow or high fat and sugar diet, and LepR null (db/db) mice using myography. Myocardial blood flow (MBF), blood pressure, and heart rate were measured in vivo using contrast echocardiography and a solid-state pressure sensor catheter. RNA from coronary arteries, endothelial cells and hearts were analyzed via qPCR for gene expression and protein expression was assessed via Western-Blot analyses. Superoxide was detected via electron paramagnetic resonance (EPR). Results: 1) Ex vivo coronary EDD and in vivo MBF was impaired in diabetes. 2) L-NAME (NO-synthase inhibitor) inhibited ex vivo coronary EDD and in vivo MBF in WT, while PEG-catalase (H2O2 scavenger) inhibited diabetic EDD ex vivo and MBF in vivo. 5) miR-21 deficiency blocked the NO-to-H2O2 switch and prevented diabetic vasodilation impairments. 6) Diabetic mice displayed increased serum NO and H2O2, upregulated mRNA expression of Sod1, Sod2, iNos, and Cav-1, and downregulated Pgc-1α. Deficiency of miR-21 reversed these changes. 7) miR-21 deficiency increased PGC1α, PPARα and eNOS protein and reduced detection of endothelial superoxide. Conclusions: Diabetics exhibit an NO-to-H2O2 switch in the mediator of EDD coronary dilation, which contributed to microvascular dysfunction and is mediated by miR-21. This study represents the first mouse model recapitulating the NO-to-H2O2 switch seen in CAD patients.
... Diminished oxygen supply requires metabolic adaptation to cope with the resulting energetic stress and to maintain the supply of ATP [1]. Diminished oxygen levels decrease utilization of the electron transport chain in the mitochondria and will shift mitochondrial oxygen reduction to water in the direction of nitrite reduction to nitric oxide to induce vasodilatation and increase blood supply [2]. Additional adaptations to hypoxic stress could be due to changes in gene expression under the control of hypoxia-inducible factor 1-α (HIF-1α) [3], a master regulator that mediates most transcriptional changes during hypoxia. ...
Article
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Blood cholesterol levels have been connected to high-altitude adaptation. In the present study, we treated mice with high-cholesterol diets following exposure to acute hypoxic stress and evaluated the effects of the diets on whole-body, liver glucose, and liver fat metabolism. For rapid cholesterol liver uptake, 6-week-old male C57BL/J6 mice were fed with high-cholesterol/cholic acid (CH) diet for 6 weeks and then were exposed to gradual oxygen level reduction for 1 h and hypoxia at 7% oxygen for additional 1 hour using a hypoxic chamber. Animals were than sacrificed, and metabolic markers were evaluated. Hypoxic treatment had a strong hypoglycemic effect that was completely blunted by CH treatment. Decreases in gluconeogenesis and glycogenolysis as well as an increase in ketone body formation were observed. Such changes indicate a metabolic shift from glucose to fat utilization due to activation of the inducible nitric oxide synthase/AMPK axis in the CH-treated animals. Increased ketogenesis was also observed in vitro in hepatocytes after cholesterol treatment. In conclusion, our results show for the first time that cholesterol contributes to metabolic shift and adaptation to hypoxia in vivo and in vitro through induction of HIF-1 α and iNOS expression.
Article
Reports on gadolinium deposits in the body and brains of adults and children who underwent contrast-enhanced MRI examinations warrant development of new, metal free, contrast agents for MRI. Nitrate is an abundant ion in mammalian biochemistry and sodium nitrate can be safely injected intravenously. We show that hyperpolarized [¹⁵N]nitrate can potentially be used as an MR tracer. The ¹⁵N site of hyperpolarized [¹⁵N]nitrate showed a T1 of more than 100 s in aqueous solutions, which was prolonged to more than 170 s below 20 °C. Capitalizing on this effect for polarization storage we obtained a visibility window of 9 min in blood. Conversion to [¹⁵N]nitrite, the bioactive reduced form of nitrate, was not observed in human blood and human saliva in this time frame. Thus, [¹⁵N]nitrate may serve as a long-lived hyperpolarized tracer for MR. Due to its ionic nature, the immediate applications appear to be perfusion and tissue retention imaging.
Article
The role of the oral microbiome in maintaining health is becoming increasingly apparent (1). Oral nitrate-reducing commensal bacteria reduce nitrate to nitrite, which is then reduced to nitric oxide in the acidic environment of the stomach via the nitrate-nitrite-nitric oxide pathway. Nitric oxide has a key role in maintaining gastric mucosal blood flow and thickness, thereby protecting against bacterial infection. Gastric nitric oxide levels are nearly abolished in intubated and ventilated intensive care patients, which might be linked to the prevalence of ulcers and bacterial overgrowth (2). In addition, it has been reported that the nitrate content of both enteral and parenteral nutrition is minimal (3) thereby further reducing the activity of the nitrate-nitrite-nitric oxide pathway. Patients on intensive care may receive salivary suction, antibacterial mouthwash, low nitrate feeds and broad-spectrum antibiotics, all of which could affect the oral microbiome. Poor oral health and periodontal disease is associated with ventilator-associated pneumonia (4). As part of a wider study we investigated the effects of antibacterial mouthwash on oral nitrate-reducing capacity and salivary pH, glucose and lactate as well as salivary and plasma nitrate and nitrite concentrations.
Article
Objectives: Sepsis is a severe organic dysfunction caused by an infection that affects the normal regulation of several organ systems, including the central nervous system. Inflammation and oxidative stress play crucial roles in the development of brain dysfunction in sepsis. The aim of this study was to determine the effect of a fish oil (FO)-55-enriched lipid emulsion as an important anti-inflammatory compound on brain dysfunction in septic rats. Methods: Wistar rats were subjected to sepsis by cecal ligation and perforation (CLP) or sham (control) and treated orally with FO (600 µL/kg after CLP) or vehicle (saline; sal). Animals were divided into sham+sal, sham+FO, CLP+sal and CLP+FO groups. At 24 h and 10 d after surgery, the hippocampus, prefrontal cortex, and total cortex were obtained and assayed for levels of interleukin (IL)-1β and IL-10, blood-brain barrier permeability, nitrite/nitrate concentration, myeloperoxidase activity, thiobarbituric acid reactive species formation, protein carbonyls, superoxide dismutase and catalase activity, and brain-derived neurotrophic factor levels. Behavioral tasks were performed 10 d after surgery. Results: FO reduced BBB permeability in the prefrontal cortex and total cortex of septic rats, decreased IL-1β levels and protein carbonylation in all brain structures, and diminished myeloperoxidase activity in the hippocampus and prefrontal cortex. FO enhanced brain-derived neurotrophic factor levels in the hippocampus and prefrontal cortex and prevented cognitive impairment. Conclusions: FO diminishes the negative effect of polymicrobial sepsis in the rat brain by reducing inflammatory and oxidative stress markers.
Chapter
Due to different biotic and abiotic factors, agriculture is unable to produce enough quantity of food, fodder, feed, biofuel and industrial raw materials from available land resources. Currently food security depends upon a limited number of staple food crops, fruits and vegetables having different environmental, ecological, biological (insect, pests), nutritional and economic issues due to which handful amount of productivity of these crops is impossible, resulting in uncertainty in food security. Therefore, to fulfil the food demands of ever-growing population of the world, use of underutilized vegetables and other crops is the better option. The underutilized crops have better adaptation for adverse biotic and abiotic stresses. There are many underutilized vegetables available, which are the good source of protein, carbohydrates, micronutrients and vitamins. Additionally they are being used as medicinal plants and fodder of animals, and their vegetative portions are used as industrial raw materials; these valuable components are necessary to attain food security. Generally, vegetables have commercial value, so they are the good source of income for farmers owing small lands. As very limited research has been done on different aspects related to their adaptations, genetics, agronomy, economics, etc., the existing wild types and available varieties having wide adaptation and commercial potential could be recommended for cultivations. In this chapter, nutritional and medicinal values of underutilized vegetables and their role in poverty alleviation and food security are briefly discussed.
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Nitrites can be formed from and away by the nitrate-nitrite-nitric oxide pathway. The properties of nitrite oxidation and metHb formation are carefully studied in mechanism, forming either N-binding or O-binding structures. Apart from nitrate and nitric oxide, nitrites can also form carcinogenic nitrosamines in acidic environments. MetHb can cause hypoxia and vasodilation, while symptoms are revealed in different degrees under recalled or present hypoxic conditions. The study thoroughly studied nitrite’s metabolic properties, chemical pathways, and dosage effects on health. The cancer risks of consuming dietary nitrite need more statistical support, while its metabolite N-nitrosodimethylamine and NDMA concentration are well considered with increasing cancer risks. ED50 of human vasodilation is identified, and lethal doses on juvenile pike-perch can be further utilized to predict related doses for humans. More studies should be done to investigate relative nitrite doses to boost utilization and studies about this chemical.
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Incidence of glaucoma, a severe disease leading to irreversible loss of vision, is increasing with global aging populations. Lowering intraocular pressure (IOP) is the only proven treatment method for glaucoma. Nitric oxide (NO) is an emerging material targeting the conventional outflow pathway by relaxing the trabecular meshwork (TM). However, there is little understanding on NO level effective to IOP lowering without toxicity. Here we report a novel long-term NO-releasing polydiazeniumdiolate (NOP) that enables lowering IOP via the conventional outflow pathway. NOP is composed of carbon-bound polydiazeniumdiolate, stable NO donor moiety. NO release was monitored with accurate parameters by detection of real-time gas and accumulated form. Based on the NO release information, the selected safe level of NOP exhibited effective TM relaxation and potential an IOP lowering effect in vivo without side effects. This work provides new insights to nitric oxide release behavior that should be considered for glaucoma treatment.
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The vascular endothelium provides a direct interface between circulating blood cells and parenchymal cells. Thus, it has a key role in vasomotor tone regulation, primary hemostasis, vascular barrier, and immunity. In the case of systemic inflammation, endothelial cell (EC) activation initiates a powerful innate immune response to eliminate the pathogen. In some specific conditions, ECs may also contribute to the activation of adaptive immunity and the recruitment of antigen-specific lymphocytes. However, the loss of EC functions or an exaggerated activation of ECs during sepsis can lead to multiorgan failure.
Article
Background Nitric oxide (NO) is a relevant molecule for vascular homeostasis. The level of serum NO metabolites (NOx), which consist of nitrite and nitrate, has been investigated as an alternative biomarker of NO production, but its clinical value has not yet been determined. Methods and results 143 patients (66 ± 12 years old) were followed up after coronary catheterization. During a median (inter-quartile range) observation period of 6.13 (3.32–9.21) years, there were 20 (14 %) all-cause deaths, including 11 (8 %) cardiovascular deaths, 17 (12 %) major adverse cardiovascular events, and 17 (12 %) hospital admissions for heart failure. Median NOx level was 34.5 μmol/L (23.9–54.3). NOx was a risk factor for all-cause death [hazard ratio (HR) by unit increase, 1.010, 95 % confidence interval (CI) 1.001–1.018; p = 0.021] and heart failure (HR 1.010, CI 1.001–1.019; p = 0.029). Even after adjustment for age, sex, coronary risk factors, C-reactive protein, log-transformed brain natriuretic peptide, estimated glomerular filtration rate, and nitrate treatment, NOx was a risk factor for all-cause death (HR 1.015, CI 1.004–1.027; p = 0.008) and admission with heart failure (HR 1.018, CI 1.005–1.018, p = 0.007). Conclusions An increase in serum NOx level does not herald a benign clinical course but is an independent predictor of high risk of any-cause mortality and heart failure.
Article
Endothelial dysfunction, with impaired bioavailability and/or bioactivity of the vasoprotective molecule, nitric oxide, appears to be a vital step in the initiation of atherosclerosis. Several studies have shown that dietary nitrate/nitrite can have significant benefits on human cardiovascular homeostasis. Although serum nitrite concentrations can reach micromolar levels, the physiological significance of nitrate/nitrite in normal tissues has not been fully elucidated. We investigated in vitro the chronic effects of nitrate/nitrite on endothelial nitric oxide synthase (eNOS) to determine the potential vasoprotective effects of nitrate/nitrite and the underlying molecular mechanisms. Our results demonstrate the expression of phosphorylated eNOS at Ser1177 and phosphorylated adenosine monophosphate activated protein kinase (AMPK) at Thr172 in human aortic endothelial cells were increased after nitrite treatment. We suggest that nitrite stimulation may enhance eNOS activation, which is due, in part, to AMPK activation. The AMPK–eNOS activation by nitrite may be a possible molecular mechanism underlying the vascular protective effects of dietary nitrate.
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Pro-inflammatory cytokines and reactive oxygen species (ROS) are produced in acute spinal cord injury, leading to myelin breakdown, inflammation, mitochondrial dysfunction, and apoptosis of neurons and glial cells. The aim of the present study was to investigate possible protective effects of L-carnitine (carn) or atorvastatin (ator) on spinal cord ischemia-reperfusion injury (IRI). Rats were randomized into nine equal groups (n = 8): control and control taking carn (100 mg/kg BW), ator (2.5 mg/kg BW) or both, as well as sham-operation, IRI and IRI taking same doses of carn, ator or both. Neurological assessments were done 48 hours after IRI, and serum nitrite/nitrate was measured. Finally, lumbar segments of spinal cord were excised, and part was homogenized and prepared for measuring tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), malondialdehyde (MDA), advanced oxidation protein products (AOPP), reduced glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase. The other part was sectioned for evaluation of histopathological changes and for immunostaining by glial fibrillary acidic protein (GFAP), Bax and Bcl-2. The IRI increased ROS (nitrite/nitrate, MDA, AOPP) and pro-inflammatory cytokines (TNF-α, IL-1β), and decreased antioxidants (GSH, GPx, SOD, catalase) with impaired sensory and motor functions. Astrogliosis was detected by GFAP, and increased apoptosis was demonstrated by increasing Bax and decreasing Bcl-2. Treatment with carn or ator alone decreased TNF-α, IL-1β, nitrite/nitrate, MDA and AOPP, and increased GSH, GPx, SOD, and catalase with improvement of neurological functions and histological studies. Combination of carn and ator improved most of measured IRI-affected parameters better than isolated carn or ator administration.
Article
Background: Previous clinical studies have shown controversial results regarding the effect of inorganic nitrate supplementation on blood pressure (BP) in older individuals. We performed this systematic review and meta-analysis to assess the effect of inorganic nitrate on BP in older adults. Methods: Eligible studies were searched in Cochrane Library, PubMed, Scopus, Web of Science, and Embase. Randomized controlled trials which evaluated the effect of inorganic nitrate consumption on BP in older adults were recruited. The random-effect model was used to calculate the pooled effect sizes. Results: 22 studies were included in this meta-analysis. Overall, inorganic nitrate consumption significantly reduced systolic blood pressure (SBP) by -3.90 mmHg (95% confidence interval: -5.23 to -2.57; P<0.001) and diastolic blood pressure (DBP) by -2.62 mmHg (95% confidence interval: -3.86 to -1.37; P<0.005) comparing with the control group. Subgroup analysis showed that the BP was significantly reduced when participants aged≥65, BMI>30, or the baseline BP in prehypertension stage. And both SBP and DBP decreased significantly after acute nitrate supplementation of a single dose (<1 day) or more than 1-week. However, participants with hypertension at baseline were not associated with significant changes in both SBP and DBP. Subgroup analysis of measurement methods showed that only the resting BP group showed a significant reduction in SBP and DBP, compared with the 24-hour ambulatory BP monitoring (ABPM) group and daily home BP measurement group. Conclusion: These results demonstrated that consuming inorganic nitrate could significantly reduce SBP and DBP in older adults, especially in whose age ≥ 65, BMI>30, or baseline BP in prehypertension stage.
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Arsenic exposure causes immense health distress by increasing risk of cardiovascular abnormalities, diabetes mellitus, neurotoxicity, and nephrotoxicity. The present study explored the role of inducible nitric oxide synthase (iNOS) inhibitors against sodium arsenite-induced renal and hepatic dysfunction in rats. Female Sprague Dawley rats were subjected to arsenic toxicity by administering sodium arsenite (5 mg/kg/day, oral) for 4 weeks. The iNOS inhibitors, S-methylisothiourea (10 mg/kg, i.p.) and aminoguanidine (100 mg/kg, i.p.) were given one hour before sodium arsenite administration in rats for 4 weeks. Sodium arsenite led rise in serum creatinine, urea, uric acid, electrolytes (potassium, fractional excretion of sodium), microproteinuria, and decreased creatinine clearance (p < 0.001) indicated renal dysfunction in rats. Arsenic-intoxication resulted in significant oxidative stress in rat kidneys, which was measured in terms of increase in lipid peroxides, superoxide anion generation and decrease in reduced glutathione (p < 0.001) levels. A threefold increase in renal hydroxyproline level in arsenic intoxicated rats indicated fibrosis. Hematoxylin–eosin staining indicated tubular damage, whereas picrosirius red staining highlighted collagen deposition in rat kidneys. S-methylisothiourea and aminoguanidine improved renal function and attenuated arsenic led renal oxidative stress, fibrosis, and decreased the kidney injury score. Additionally, arsenite-intoxication resulted in significant rise in hepatic parameters (serum aspartate aminotransferase, alanine transferase, alkaline phosphatase, and bilirubin (p < 0.001) along with multi-fold increase in oxidative stress, fibrosis and liver injury score in rats, which was significantly (p < 0.001) attenuated by concurrent administration of iNOS inhibitors). Hence, it is concluded that iNOS inhibitors attenuate sodium arsenite-induced renal and hepatic dysfunction in rats.
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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.
Article
Nitrate and nitrite ions taken from food are the sources of bioavailable nitric oxide (NO) in the nitrogen cycle. Some beneficial effects of honey on health are attributed to the ability of honey to increase NO production. The variation of nitrate and nitrite levels of honey samples collected from different Anatolia regions were clarified using capillary electrophoresis technique. The sensitivities of both anions were improved with the application of the sample stacking method. Separation buffer consisted of 30 mmol L ⁻¹ formic acid and 30 mmol L ⁻¹ sodium sulfate at a pH of 4.0. The CE technique revealed that 18 honey samples contained nitrate anion ranged between 2.53 and 31.8 mg kg ⁻¹ . Nitrite amounts were found in lower amounts in the honey samples as between non-detected and 0.533 mg kg ⁻¹ . The observed differences in nitrate levels between honey varieties may be a way to determine honey's origin.
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Monitoring of reactive nitrogen species (RNS) released from cells under high glucose induction is of great importance due to their significant contributions in the occurrence and development of diabetes mellitus (DM). Here, we developed a novel ratiometric electrochemical sensor based on platinum/hollow tubular conjugated organic microporous polymer/chitosan (Pt/HTCMP/CTS) and used for the detection of peroxynitrite anion (ONOO⁻), which is the main member of RNS. A "signal on-off" ratiometric dual-signal strategy was designed by using of the redox characteristics of K3[Fe(CN)6] ([Fe(CN)6]⁴⁻) and the analyte (ONOO⁻) for the first time. Then, due to the significant enhancement for the ratio of oxidation current value (IONOO⁻/I[Fe(CN)6]4-), the sensor showed excellent analytical performance over a wide range from 3.09 × 10⁻¹⁰ to 1.20 × 10⁻⁴ M and a detection limit as low as 0.103 nM (S/N = 3). Importantly, in high glucose environment, ONOO⁻ and H2O2 released from cancer cells were monitored directly with the proposed sensor for the first time. Therefore, the study provides an effective method for realizing fast, reliable and ultra-sensitive determination of ONOO-, and exhibits significant application value in biomedical analysis and clinical diagnosis.
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Figure 3 (a and b) Correlation between MSI and AAG (a) or APE1 (b) activity. Bar graphs represent means ± SEM. There was a significant trend for MSI and AAG activity (robust regression analysis, P = 0.0012). Although this trend was not observed between MSI and APE1, there was a significant increase in APE1 activity in the MSI-High group (n = 5; one-way ANOVA with Scheffe multiple comparison test, P = 0.0004). *, AAG activity is significantly higher in the MSI-Low group (n = 10) than in the microsatellite stable group (n = 15). **, AAG activity is significantly higher in the MSI-High group (n = 5) than in the MSI-Low group (n = 10). ***, APE1 activity is significantly higher in the MSI-High group (n = 5) than in the MSI-Low (n = 10) and microsatellite stable (n = 15) groups. (c–e) Number of samples belonging to a specific AAG and APE1 activity category. AAG and APE1 activities were ranked in order, then placed into tertiles as samples with activity belonging to the Lower 1/3, Middle 1/3, or Top 1/3. (c) Of the 60 samples , 43 did not have a band shift and were characterized as microsatellite stable samples. (d) Of the 60 samples, 11 had a band shift in one of the markers examined (including TGFβRII and BLM) and were characterized as MSI-Low samples. (e) Of the 60 samples, six had a band shift in two or more of the markers examined (including TGFβRII and BLM) and were characterized as MSI-High samples. Shaded boxes represent activities where there is an imbalance of AAG and APE1 activities. The simple κ statistic indicates a trend for imbalance between AAG and APE1 as MSI levels increase. The simple κ statistic of 1.0 indicates no imbalance. A simple κ statistic moving toward zero indicates greater imbalance between the two enzymes.
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The present study explored the role of myoglobin (Mb) in cardiac NO homeostasis and its functional relevance by employing isolated hearts of wild-type (WT) and myoglobin knockout mice. ¹H NMR spectroscopy was used to measure directly the conversion of oxygenated Mb (MbO2) to metmyoglobin (metMb) by reaction with NO. NO was applied intracoronarily (5 nM to 25 μM), or its endogenous production was stimulated with bradykinin (Bk; 10 nM to 2 μM). We found that infusion of authentic NO solutions dose-dependently (≥ 2.5 μM NO) increased metMb formation in WT hearts that was rapidly reversible on cessation of NO infusion. Likewise, Bk-induced release of NO was associated with significant metMb formation in the WT (≥1 μM Bk). Hearts lacking Mb reacted more sensitively to infused NO in that vasodilatation and the cardiodepressant actions of NO were more pronounced. Similar results were obtained with Bk. The lower sensitivity of WT hearts to changes in NO concentration fits well with the hypothesis that in the presence of Mb, a continuous degradation of NO takes place by reaction of MbO2 + NO to metMb + NO3⁻, thereby effectively reducing cytosolic NO concentration. This breakdown protects myocytic cytochromes against transient rises in cytosolic NO. Regeneration of metMb by metMb reductase to Mb and subsequent association with O2 leads to reformation of MbO2 available for another NO degradation cycle. Our data indicate that this cycle is crucial in the breakdown of NO and substantially determines the dose–response curve of the NO effects on coronary blood flow and cardiac contractility.
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Background. The gas nitric oxide (NO) is an important endothelium-derived relaxing factor, inactivated by rapid combination with heme in hemoglobin. Methods and Results. Awake spontaneously breathing lambs inhaled 5-80 ppm NO with an acutely constricted pulmonary circulation due to either infusion of the stable thromboxane endoperoxide analogue U46619 or breathing a hypoxic gas mixture. Within 3 minutes after adding 40 ppm NO or more to inspired gas, pulmonary hypertension was reversed. Systemic vasodilation did not occur. Pulmonary hypertension resumed within 3-6 minutes of ceasing NO inhalation. During U46619 infusion pulmonary vasodilation was maintained up to 1 hour without tolerance. In the normal lamb, NO inhalation produced no hemodynamic changes. Breathing 80 ppm NO for 3 hours did not increase either methemoglobin or extravascular lung water levels nor modify lung histology compared with control lambs. Conclusions. Low dose inhaled NO (5-80 ppm) is a selective pulmonary vasodilator reversing both hypoxia- and thromboxane-induced pulmonary hypertension in the awake lamb [corrected].
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Cranberries have been used widely for several decades for the prevention and treatment of urinary tract infections (UTIs). This is the third update of our review first published in 1998 and updated in 2004 and 2008. To assess the effectiveness of cranberry products in preventing UTIs in susceptible populations. Search methods: We searched MEDLINE, EMBASE, the Cochrane Central Register of Controlled Trials (CENTRAL in The Cochrane Library) and the Internet. We contacted companies involved with the promotion and distribution of cranberry preparations and checked reference lists of review articles and relevant studies. Date of search: July 2012. Selection criteria: All randomised controlled trials (RCTs) or quasi-RCTs of cranberry products for the prevention of UTIs. Data collection and analysis: Two authors independently assessed and extracted data. Information was collected on methods, participants, interventions and outcomes (incidence of symptomatic UTIs, positive culture results, side effects, adherence to therapy). Risk ratios (RR) were calculated where appropriate, otherwise a narrative synthesis was undertaken. Quality was assessed using the Cochrane risk of bias assessment tool. This updated review includes a total of 24 studies (six cross-over studies, 11 parallel group studies with two arms; five with three arms, and two studies with a factorial design) with a total of 4473 participants. Ten studies were included in the 2008 update, and 14 studies have been added to this update. Thirteen studies (2380 participants) evaluated only cranberry juice/concentrate; nine studies (1032 participants) evaluated only cranberry tablets/capsules; one study compared cranberry juice and tablets; and one study compared cranberry capsules and tablets. The comparison/control arms were placebo, no treatment, water, methenamine hippurate, antibiotics, or lactobacillus. Eleven studies were not included in the meta-analyses because either the design was a cross-over study and data were not reported separately for the first phase, or there was a lack of relevant data. Data included in the meta-analyses showed that, compared with placebo, water or not treatment, cranberry products did not significantly reduce the occurrence of symptomatic UTI overall (RR 0.86, 95% CI 0.71 to 1.04) or for any the subgroups: women with recurrent UTIs (RR 0.74, 95% CI 0.42 to 1.31); older people (RR 0.75, 95% CI 0.39 to 1.44); pregnant women (RR 1.04, 95% CI 0.97 to 1.17); children with recurrent UTI (RR 0.48, 95% CI 0.19 to 1.22); cancer patients (RR 1.15 95% CI 0.75 to 1.77); or people with neuropathic bladder or spinal injury (RR 0.95, 95% CI: 0.75 to 1.20). Overall heterogeneity was moderate (I² = 55%). The effectiveness of cranberry was not significantly different to antibiotics for women (RR 1.31, 95% CI 0.85, 2.02) and children (RR 0.69 95% CI 0.32 to 1.51). There was no significant difference between gastrointestinal adverse effects from cranberry product compared to those of placebo/no treatment (RR 0.83, 95% CI 0.31 to 2.27). Many studies reported low compliance and high withdrawal/dropout problems which they attributed to palatability/acceptability of the products, primarily the cranberry juice. Most studies of other cranberry products (tablets and capsules) did not report how much of the 'active' ingredient the product contained, and therefore the products may not have had enough potency to be effective. Prior to the current update it appeared there was some evidence that cranberry juice may decrease the number of symptomatic UTIs over a 12 month period, particularly for women with recurrent UTIs. The addition of 14 further studies suggests that cranberry juice is less effective than previously indicated. Although some of small studies demonstrated a small benefit for women with recurrent UTIs, there were no statistically significant differences when the results of a much larger study were included. Cranberry products were not significantly different to antibiotics for preventing UTIs in three small studies. Given the large number of dropouts/withdrawals from studies (mainly attributed to the acceptability of consuming cranberry products particularly juice, over long periods), and the evidence that the benefit for preventing UTI is small, cranberry juice cannot currently be recommended for the prevention of UTIs. Other preparations (such as powders) need to be quantified using standardised methods to ensure the potency, and contain enough of the 'active' ingredient, before being evaluated in clinical studies or recommended for use.
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Few studies have evaluated the relationship between fruit and vegetable intake and cardiovascular disease. To examine the associations between fruit and vegetable intake and ischemic stroke. Prospective cohort studies, including 75 596 women aged 34 to 59 years in the Nurses' Health Study with 14 years of follow-up (1980-1994), and 38683 men aged 40 to 75 years in the Health Professionals' Follow-up Study with 8 years of follow-up (1986-1994). All individuals were free of cardiovascular disease, cancer, and diabetes at baseline. Incidence of ischemic stroke by quintile of fruit and vegetable intake. A total of 366 women and 204 men had an ischemic stroke. After controlling for standard cardiovascular risk factors, persons in the highest quintile of fruit and vegetable intake (median of 5.1 servings per day among men and 5.8 servings per day among women) had a relative risk (RR) of 0.69 (95% confidence interval [CI], 0.52-0.92) compared with those in the lowest quintile. An increment of 1 serving per day of fruits or vegetables was associated with a 6% lower risk of ischemic stroke (RR, 0.94; 95 % CI, 0.90-0.99; P =.01, test for trend). Cruciferous vegetables (RR, 0.68 for an increment of 1 serving per day; 95% CI, 0.49-0.94), green leafy vegetables (RR, 0.79; 95% CI, 0.62-0.99), citrus fruit including juice (RR, 0.81; 95% CI, 0.68-0.96), and citrus fruit juice (RR, 0.75; 95% CI, 0.61-0.93) contributed most to the apparent protective effect of total fruits and vegetables. Legumes or potatoes were not associated with lower ischemic stroke risk. The multivariate pooled RR for total stroke was 0.96 (95% CI, 0.93-1.00) for each increment of 2 servings per day. These data support a protective relationship between consumption of fruit and vegetables-particularly cruciferous and green leafy vegetables and citrus fruit and juice-and ischemic stroke risk.
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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.
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Changes in plasma nitrite concentration in the human forearm circulation have recently been shown to reflect acute changes in endothelial nitric oxide synthase (eNOS)-activity. Whether basal plasma nitrite is a general marker of constitutive NOS-activity in vivo is yet unclear. Due to the rapid metabolism of nitrite in blood and the difficulties in its analytical determination literature data on levels of nitrite in mammals are largely inconsistent. We hypothesized that constitutive NOS-activity in the circulatory system is relatively uniform throughout the mammalian kingdom. If true, this should result in comparable systemic plasma nitrite levels in different species. Using three different analytical approaches we determined plasma nitrite concentration to be in a nanomolar range in a variety of species: humans (305 +/- 23 nmol/l), monkeys (367 +/- 62 nmol/l), minipigs (319 +/- 24 nmol/l), dogs (305 +/- 50 nmol/l), rabbits (502 +/- 21 nmol/l), guinea pigs (412 +/- 44 nmol/l), rats (191 +/- 43 nmol/l), and mice (457 +/- 51 nmol/l). Application of different NOS-inhibitors in humans, minipigs, and dogs decreased NOS-activity and thereby increased vascular resistance. This was accompanied by a significant, up to 80%, decrease in plasma nitrite concentration. A comparison of plasma nitrite concentrations between eNOS(-/-) and NOS-inhibited wild-type mice revealed that 70 +/- 5% of plasma nitrite is derived from eNOS. These results provide evidence for a uniform constitutive vascular NOS-activity across mammalian species.
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The nitrite anion is reduced to nitric oxide (NO*) as oxygen tension decreases. Whereas this pathway modulates hypoxic NO* signaling and mitochondrial respiration and limits myocardial infarction in mammalian species, the pathways to nitrite bioactivation remain uncertain. Studies suggest that hemoglobin and myoglobin may subserve a fundamental physiological function as hypoxia dependent nitrite reductases. Using myoglobin wild-type ((+/+)) and knockout ((-/-)) mice, we here test the central role of myoglobin as a functional nitrite reductase that regulates hypoxic NO* generation, controls cellular respiration, and therefore confirms a cytoprotective response to cardiac ischemia-reperfusion (I/R) injury. We find that myoglobin is responsible for nitrite-dependent NO* generation and cardiomyocyte protein iron-nitrosylation. Nitrite reduction to NO* by myoglobin dynamically inhibits cellular respiration and limits reactive oxygen species generation and mitochondrial enzyme oxidative inactivation after I/R injury. In isolated myoglobin(+/+) but not in myoglobin(-/-) hearts, nitrite treatment resulted in an improved recovery of postischemic left ventricular developed pressure of 29%. In vivo administration of nitrite reduced myocardial infarction by 61% in myoglobin(+/+) mice, whereas in myoglobin(-/-) mice nitrite had no protective effects. These data support an emerging paradigm that myoglobin and the heme globin family subserve a critical function as an intrinsic nitrite reductase that regulates responses to cellular hypoxia and reoxygenation [corrected]
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Sodium nitrite has been shown to have vasodilator activity in experimental animals and in human subjects. However, the mechanism by which nitrite anion is converted to vasoactive nitric oxide (NO) is uncertain. It has been hypothesized that deoxyhemoglobin, xanthine oxidoreductase, mitochondrial aldehyde dehydrogenase, and other heme proteins can reduce nitrite to NO, but studies in the literature have not identified the mechanism in the intact rat, and several studies report no effect of inhibitors of xanthine oxidoreductase. In the present study, the effects of the xanthine oxidoreductase inhibitor allopurinol and the mitochondrial aldehyde dehydrogenase inhibitor cyanamide on decreases in mean systemic arterial pressure in response to i.v. sodium nitrite administration were investigated in the rat. The decreases in mean systemic arterial pressure in response to i.v. administration of sodium nitrite were inhibited in a selective manner after administration of allopurinol in a dose of 25 mg/kg i.v. A second 25 mg/kg i.v. dose had no additional inhibitory effect on the response to sodium nitrite. The decreases in mean systemic arterial pressure in response to sodium nitrite were attenuated by cyanamide and a second 25 mg/kg i.v. dose had no additional inhibitory effect. In L-NAME-treated animals, allopurinol attenuated responses to sodium nitrite and a subsequent administration of cyanamide had no additional effect. When the order of administration of the inhibitors was reversed, responses to sodium nitrite were attenuated by administration of cyanamide and a subsequent administration of allopurinol had no additional inhibitory effect. The results of these studies suggest that nitrite can be reduced to vasoactive NO in the systemic vascular bed of the rat by xanthine oxidoreductase and mitochondrial aldehyde dehydrogenase and that the 2 pathways of nitrite activation act in a parallel manner.
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Nitrite (NO(2)(-)), previously viewed as a physiologically inert metabolite and biomarker of the endogenous vasodilator NO, was recently identified as an important biological NO reservoir in vasculature and tissues, where it contributes to hypoxic signaling, vasodilation, and cytoprotection after ischemia-reperfusion injury. Reduction of nitrite to NO may occur enzymatically at low pH and oxygen tension by deoxyhemoglobin, deoxymyoglobin, xanthine oxidase, mitochondrial complexes, or NO synthase (NOS). We show that nitrite treatment, in sharp contrast with the worsening effect of NOS inhibition, significantly attenuates hypothermia, mitochondrial damage, oxidative stress and dysfunction, tissue infarction, and mortality in a mouse shock model induced by a lethal tumor necrosis factor challenge. Mechanistically, nitrite-dependent protection was not associated with inhibition of mitochondrial complex I activity, as previously demonstrated for ischemia-reperfusion, but was largely abolished in mice deficient for the soluble guanylate cyclase (sGC) alpha1 subunit, one of the principal intracellular NO receptors and signal transducers in the cardiovasculature. Nitrite could also provide protection against toxicity induced by Gram-negative lipopolysaccharide, although higher doses were required. In conclusion, we show that nitrite can protect against toxicity in shock via sGC-dependent signaling, which may include hypoxic vasodilation necessary to maintain microcirculation and organ function, and cardioprotection.
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Addition of nitrite achieves kill of micro-organisms where acid alone allows growth to continue. The synergism in antimicrobial action of acid and nitrite is evident against common gut pathogens such as the Enterobacteriaceae, including Escherichia coli 0157, but also against the stomach pathogen Helicobacter pylori, normally very resistant to acid alone. The antibacterial action of acidified nitrite becomes apparent at physiological concentrations of acid and nitrite after exposure times that are within the passage time of a food bolus through the stomach. The antimicrobial activity of acidified nitrite is enhanced by thiocyanate, also present in gastric juice. Ascorbic acid provides protection against the antibacterial action of acidified nitrite, suggesting that NO is not the antibacterial agent.
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The objective of this study was to determine whether nitric oxide (NO) is responsible for the vascular smooth muscle relaxation elicited by endothelium-derived relaxing factor (EDRF). EDRF is an unstable humoral substance released from artery and vein that mediates the action of endothelium-dependent vasodilators. NO is an unstable endothelium-independent vasodilator that is released from vasodilator drugs such as nitroprusside and glyceryl trinitrate. We have repeatedly observed that the actions of NO on vascular smooth muscle closely resemble those of EDRF. In the present study the vascular effects of EDRF released from perfused bovine intrapulmonary artery and vein were compared with the effects of NO delivered by superfusion over endothelium-denuded arterial and venous strips arranged in a cascade. EDRF was indistinguishable from NO in that both were labile (t1/2 = 3-5 sec), inactivated by pyrogallol or superoxide anion, stabilized by superoxide dismutase, and inhibited by oxyhemoglobin or potassium. Both EDRF and NO produced comparable increases in cyclic GMP accumulation in artery and vein, and this cyclic GMP accumulation was inhibited by pyrogallol, oxyhemoglobin, potassium, and methylene blue. EDRF was identified chemically as NO, or a labile nitroso species, by two procedures. First, like NO, EDRF released from freshly isolated aortic endothelial cells reacted with hemoglobin to yield nitrosylhemoglobin. Second, EDRF and NO each similarly promoted the diazotization of sulfanilic acid and yielded the same reaction product after coupling with N-(1-naphthyl)-ethylenediamine. Thus, EDRF released from artery and vein possesses identical biological and chemical properties as NO.
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Recent studies surprisingly show that dietary inorganic nitrate, abundant in vegetables, can be metabolized in vivo to form nitrite and then bioactive nitric oxide. A reduction in blood pressure was recently noted in healthy volunteers after dietary supplementation with nitrate; an effect consistent with formation of vasodilatory nitric oxide. Oral bacteria have been suggested to play a role in bioactivation of nitrate by first reducing it to the more reactive anion nitrite. In a cross-over designed study in seven healthy volunteers we examined the effects of a commercially available chlorhexidine-containing antibacterial mouthwash on salivary and plasma levels of nitrite measured after an oral intake of sodium nitrate (10mg/kg dissolved in water). In the control situation the salivary and plasma levels of nitrate and nitrite increased greatly after the nitrate load. Rinsing the mouth with the antibacterial mouthwash prior to the nitrate load had no effect on nitrate accumulation in saliva or plasma but abolished its conversion to nitrite in saliva and markedly attenuated the rise in plasma nitrite. We conclude that the acute increase in plasma nitrite seen after a nitrate load is critically dependent on nitrate reduction in the oral cavity by commensal bacteria. The removal of these bacteria with an antibacterial mouthwash will very likely attenuate the NO-dependent biological effects of dietary nitrate.
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Nitrite represents a bioactive reservoir of nitric oxide (NO) that may modulate vasodilation, respiration and cytoprotection after ischemia-reperfusion injury. Although nitrite formation is thought to occur via reaction of NO with oxygen, this third-order reaction cannot compete kinetically with the reaction of NO with hemoglobin to form nitrate. Indeed, the formation of nitrite from NO in the blood is limited when plasma is substituted with physiological buffers, which suggests that plasma contains metal-based enzymatic pathways for nitrite synthesis. We therefore hypothesized that the multicopper oxidase, ceruloplasmin, could oxidize NO to NO+, with subsequent hydration to nitrite. Accordingly, plasma NO oxidase activity was decreased after ceruloplasmin immunodepletion, in ceruloplasmin knockout mice and in people with congenital aceruloplasminemia. Compared to controls, plasma nitrite concentrations were substantially reduced in ceruloplasmin knockout mice, which were more susceptible to liver infarction after ischemia and reperfusion. The extent of hepatocellular infarction normalized after nitrite repletion. These data suggest new functions for the multicopper oxidases in endocrine NO homeostasis and nitrite synthesis, and they support the hypothesis that physiological concentrations of nitrite contribute to hypoxic signaling and cytoprotection.