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Objective: To determine whether nitrite can enhance exercise training (ET) effects in heart failure with preserved ejection fraction (HFpEF)., Methods: In this multicenter, double-blind, placebo-controlled, randomized trial conducted at 1 urban and 9 rural outreach centers between November 22, 2016, and December 9, 2021, patients with HFpEF underwent ET along with inorganic nitrite 40 mg or placebo 3 times daily. The primary end point was peak oxygen consumption (VO 2 ). Secondary end points included Kansas City Cardiomyopathy Questionnaire overall summary score (KCCQ-OSS, range 0 to 100; higher scores reflect better health status), 6-minute walk distance, and actigraphy., Results: Of 92 patients randomized, 73 completed the trial because of protocol modifications necessitated by loss of drug availability. Most patients were older than 65 years (80%), were obese (75%), and lived in rural settings (63%). At baseline, median peak VO 2 (14.1 mL·kg -1 ·min -1 ) and KCCQ-OSS (63.7) were severely reduced. Exercise training improved peak VO 2 (+0.8 mL·kg -1 ·min -1 ; 95% CI, 0.3 to 1.2; P<.001) and KCCQ-OSS (+5.5; 95% CI, 2.5 to 8.6; P<.001). Nitrite was well tolerated, but treatment with nitrite did not affect the change in peak VO 2 with ET (nitrite effect, -0.13; 95% CI, -1.03 to 0.76; P=.77) or KCCQ-OSS (-1.2; 95% CI, -7.2 to 4.9; P=.71). This pattern was consistent across other secondary outcomes., Conclusion: For patients with HFpEF, ET administered for 12 weeks in a predominantly rural setting improved exercise capacity and health status, but compared with placebo, treatment with inorganic nitrite did not enhance the benefit from ET., Trial Registration: ClinicalTrials.gov identifier: NCT02713126., (Copyright © 2023 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.)

Background: Interleukin (IL)-6 is a central inflammatory mediator and potential therapeutic target in heart failure (HF). Prior studies have shown that IL-6 concentrations are elevated in patients with HF, but much fewer data are available in heart failure with preserved ejection fraction (HFpEF)., Objectives: This study aims to determine how IL-6 relates to changes in cardiac function, congestion, body composition, and exercise tolerance in HFpEF., Methods: Clinical, laboratory, body composition, exercise capacity, physiologic and health status data across 4 National Heart, Lung, and Blood Institute-sponsored trials were analyzed according to the tertiles of IL-6., Results: IL-6 was measured in 374 patients with HFpEF. Patients with highest IL-6 levels had greater body mass index; higher N-terminal pro-B-type natriuretic peptide, C-reactive protein, and tumor necrosis factor-α levels; worse renal function; and lower hemoglobin levels, and were more likely to have diabetes. Although cardiac structure and function measured at rest were similar, patients with HFpEF and highest IL-6 concentrations had more severely impaired peak oxygen consumption (12.3 ± 3.3 mL/kg/min 13.1 ± 3.1 mL/kg/min 14.4 ± 3.9 mL/kg/min, P < 0.0001) as well as 6-minute walk distance (276 ± 107 m vs 332 ± 106 m vs 352 ± 116 m, P < 0.0001), even after accounting for increases in IL-6 related to excess body mass. IL-6 concentrations were associated with increases in total body fat and trunk fat, more severe symptoms during submaximal exercise, and poorer patient-reported health status., Conclusions: IL-6 levels are commonly elevated in HFpEF, and are associated with greater symptom severity, poorer exercise capacity, and more upper body fat accumulation. These findings support testing the hypothesis that therapies that inhibit IL-6 in patients with HFpEF may improve clinical status. (Clinical Trial Registrations: Phosphodiesterase-5 Inhibition to Improve Clinical Status and Exercise Capacity in Diastolic Heart Failure [RELAX], NCT00763867; Nitrate's Effect on Activity Tolerance in Heart Failure With Preserved Ejection Fraction, NCT02053493; Inorganic Nitrite Delivery to Improve Exercise Capacity in HFpEF, NCT02742129; Inorganic Nitrite to Enhance Benefits From Exercise Training in Heart Failure With Preserved Ejection Fraction [HFpEF], NCT02713126)., Competing Interests: Funding Support and Author Disclosures Dr Borlaug is supported by R01 HL128526, U01 HL160226, and R01 HL162828, from the National Institutes of Health (NIH), and W81XWH2210245, from the U.S. Department of Defense. Dr Alogna is supported by the Deutsche Forschungsgemeinschaft (DFG; CRC 1470, Z01). Drs Kirkland and Tchkonia are supported by R37 AG013925, P01 AG062413, and R33 AG061456 from NIH, the Connor Fund, Robert J. and Theresa W. Ryan, and the Noaber Foundation. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2023 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Introduction: Attention deficit hyperactivity disorder (ADHD) is a mental disorder that was once thought to occur only in children. Meanwhile, it is known that adults can also be affected. The first-line drug in children and adults to treat symptoms of inattention, impulsivity, lack of self-regulation, and hyperactivity is methylphenidate (MPH). Known adverse effects of MPH include cardiovascular problems, such as elevated blood pressure and heart rate. Therefore, biomarkers to monitor potential cardiovascular side effects of MPH are needed. The l-Arginine/Nitric oxide (Arg/NO) pathway is involved in noradrenaline and dopamine release as well as in normal cardiovascular functioning and is therefore a prime candidate for the search of biomarkers. The aim of the present study was to investigate the Arg/NO pathway as well as oxidative stress in adult ADHD patients in plasma and urine and the potential influence of MPH medication., Methods: In plasma and urine samples of 29 adults with ADHD (39.2 ± 10.9 years) and 32 healthy adults serving as controls (CO) (38.0 ± 11.6 years) the major NO metabolites nitrite and nitrate, Arg, the NO synthesis inhibitor asymmetric dimethylarginine (ADMA) and its major urinary metabolite dimethylamine (DMA) as well as malondialdehyde (MDA) were measured by gas chromatography-mass spectrometry., Results: Of the 29 patients with ADHD 14 were currently without MPH treatment (-MPH) and 15 were treated with MPH (+MPH). Plasma nitrate concentrations were significantly higher in patients not treated with MPH vs. CO (-MPH 60.3 μM [46.2-76.0] vs. CO 44.4 μM [35.0-52.7]; p = 0.002), while plasma nitrite tended to be higher in -MPH patients (2.77 μM [2.26-3.27]) vs. CO (2.13 μM [1.50-2.93]; p = 0.053). Additionally, plasma creatinine concentrations were significantly different, with -MPH showing significantly higher concentrations than the other two groups (-MPH 141 μM [128-159]; +MPH 96.2 μM [70.2-140]; Co 75.9 μM [62.0-94.7]; p < 0.001). Urinary creatinine excretion tended to be lowest in -MPH group vs. +MPH and CO (-MPH 11.4 ± 8.88 mM; +MPH 20.7 ± 9.82 mM; 16.6 ± 7.82 mM; p = 0.076). None of the other metabolites, including MDA, a marker of oxidative stress, showed a difference between the groups., Conclusion: Adult patients with ADHD, who are not treated with MPH (-MPH), showed varied Arg/NO pathway, but Arg bioavailability seemed to be consistent over the groups. Our findings imply that urinary reabsorption may be increase and/or excretion of nitrite and nitrate may be decreased in ADHD, resulting in an increase in the plasma concentration of nitrite. MPH seems to partially reverse these effects by not yet known mechanisms, and does not affect oxidative stress., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Hypoandrogenism is a cause of erectile dysfunction (ED). Vascular smooth muscle cell contraction and relaxation are regulated by TRPV1-4 channels. However, the influence of hypoandrogenism on TRPV1-4 and its relationship with erectile function remain unclear., Aim: To reveal whether hypoandrogenism affects erectile function by influencing TRPV1-4 expression in the corpus cavernosum of rats., Methods: Male Sprague-Dawley rats (N = 36) aged 8 weeks were assigned to 6 groups at random (n = 6): sham operation, castrated, castrated + testosterone replacement, sham operation + transfection, castrated + transfection, and castrated + empty transfection. Four weeks after castration, 20 μL of lentiviral vector (1 × 108 TU/mL) carrying the TRPV4 gene was injected into the penile cavernous tissue of the transfection groups. One week after transfection, the maximum intracavernous pressure (ICPmax)/mean arterial pressure (MAP) and the content of TRPV1-4, phosphorylated eNOS (p-eNOS)/eNOS, and nitric oxide (NO) in penile cavernous tissue of each group were measured., Outcomes: Under low androgen conditions, TRPV4 expression in endothelial cells in the rat penile cavernosum was sharply reduced, resulting in a decrease in p-eNOS/eNOS and NO content, which could inhibit erectile function., Results: In rat penile cavernous tissue, TRPV1-4 was expressed in the cell membranes of endothelial cells and smooth muscle cells. The ICPmax/MAP and the content of TRPV4, p-eNOS/eNOS, and NO end product nitrite level in rat penile cavernous tissue was markedly reduced in the castrated group as compared with the sham group (P < .05). The ICPmax/MAP and the content of TRPV4, p-eNOS/eNOS, and NO end product nitrite level in rat penile cavernous tissue were markedly improved in the castrated + transfection group vs the castrated group (P < .01)., Clinical Implications: Upregulation of TRPV4 expression in penile cavernosum tissue might be a viable therapeutic for ED caused by hypoandrogenism., Strengths and Limitations: The specific mechanism of TRPV4 in ED needs to be further verified by androgen receptor or TRPV4 gene knockout experiments., Conclusion: Hypoandrogenism may cause ED by reducing the expression of TRPV4 in rat penile cavernous tissue. Upregulation of TRPV4 expression in penile cavernous tissue can increase the ratio of p-eNOS/eNOS and NO levels and ameliorate the erectile function of castrated rats., (© The Author(s) 2023. Published by Oxford University Press on behalf of The International Society of Sexual Medicine. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: The efficacy of nitrite therapy for the treatment of heart failure remains controversial. We conducted a systematic review and meta-analysis to explore the impact of nitrite therapy on heart failure., Methods: We searched the PubMed, EMbase, Web of Science, EBSCO, and Cochrane Library databases through November 2019 for randomized controlled trials (RCTs) assessing the effect of nitrite therapy on heart failure. This meta-analysis was performed using the random-effect model., Results: Three RCTs are included in the meta-analysis. Overall, compared with the control group for heart failure, nitrite therapy is associated with significantly reduced PCWP (Std. MD=-1.22; 95% CI=-1.81 to -0.63; P < 0.0001) and improved PAC (Std. MD=0.71; 95% CI=0.16 to 1.27; P = 0.01), but reveals no substantial influence on peak VO2 (Std. MD=-0.19; 95% CI=-0.49 to 0.11; P = 0.21), systolic BP (Std. MD=-3.98; 95% CI=-8.24 to 0.28; P = 0.07), mean BP (Std. MD=-1.53; 95% CI=-3.37 to 0.31; P = 0.10), or heart rate (Std. MD=0.40; 95% CI=-0.14 to 0.94; P = 0.15)., Conclusions: Nitrite therapy may show some benefits to heart failure.

β-Carotene exhibits antioxidant and hepatoprotective activities via a multitude of biochemical mechanisms. However, the action mechanism involved in antioxidant and anti-inflammatory effects of this carotene in chronic liver diseases is not fully understood. In the present investigation, we have attempted to outline a plausible mechanism of β-carotene action against liver fibrosis in albino Wistar rats. To induce hepatic fibrosis, diethylnitrosamine (DEN) was administered in experimental rats for two weeks. DEN treated rats were divided into four groups, wherein each group comprised of five rats. β-Carotene supplement attenuated DEN-induced elevation in LFT markers (P < 0.05); averted depletion of glycogen (24%, P < 0.05) and, increased nitrite (P < 0.05), hydroxyproline (~67%, P < 0.05) and collagen levels (~65%, P < 0.05). Confocal microscopy of tissue sections stained with picrosirius red revealed accrued collagen in DEN-administered group, which was found to be reduced by β-carotene supplementation. Furthermore, β-carotene decreased the expression of iNOS/NOS-2 and NF-κB, as revealed by immunohistochemistry and Western immunoblotting. Collectively, these results demonstrate that β-carotene mitigates experimental liver fibrosis via inhibition of iNOS and NF-κB in-vivo . Thus, β-carotene may be suggested as a possible nutraceutical to curb experimental liver fibrosis.

The surprising discovery that the diatomic gas nitric oxide (NO) is generated by mammalian cells and serves to regulate a multitude of physiological processes has continued to fascinate biologists for almost four decades. The biochemistry of NO is complex, and novel insights into the control of NO biosynthesis and mechanisms of signal transduction are continuously emerging. NO is a key regulator of cardiovascular function, metabolism, neurotransmission, immunity, and more, and aberrant NO signaling is a central feature of many major disorders including cardiovascular disease, diabetes, and cancer. Here, we discuss the basics of NO biology emphasizing recent advances in the field including novel means of increasing NO bioactivity with therapeutic and nutritional implications., Competing Interests: Declaration of interests Lundberg and Weitzberg are named inventors on patents and patent applications relating to the medical uses of inorganic nitrate and nitrite and co-directors of HeartBeet., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Leishmaniasis is a parasitic disease caused by Leishmania protozoa, which presents a large spectrum of clinical manifestations. In the present study, a quinoline derivative salt named N-(2-((7-chloroquinolin-4-yl)amino)ethyl)-N-(prop-2-yn-1-yl)prop-2-yn-1-aminium chloride or QDS3 was in vitro and in vivo tested against L. infantum by means of its incorporation in Poloxamer 407-based polymeric micelles (QDS3/M). The in vitro antileishmanial activity of QDS3 and QDS3/M was investigated in L. infantum promastigotes, axenic amastigotes and infected macrophages. BALB/c mice were infected with L. infantum, and parasitological parameters were evaluated 1 and 15 days post-treatment by determining the parasite load by a limiting dilution assay, besides a quantitative PCR (qPCR) method. Immunological response was assessed based on production of cellular cytokines, as well as by quantification of nitrite levels and specific antibodies. In vitro results showed that QDS3 free or in micelles presented effective antileishmanial action against both parasite stages, being more effective in amastigotes. In vivo data showed that treatment using QDS3 or QDS3/M reduced the parasite load in the livers, spleens, draining lymph nodes (dLN) and bone marrows of the treated animals, 1 and 15 days after treatment, when compared to values found in the control groups. Additionally, treated mice developed a polarized Th1-type immune response, with higher levels of IL-12, IFN-γ, GM-CSF and nitrite, besides high production of specific IgG2a antibodies, when compared to the controls. Parasitological and immunological data obtained using the micellar composition were better than the others. In conclusion, QDS3, mainly when applied in a delivery adjuvant system, could be considered for future studies as therapeutic candidate against VL., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cutaneous thermal injuries from burns/explosives are a major cause of morbidity and mortality and represent a monumental burden on our current health care system. Injury severity is predominantly due to potentially lethal sepsis caused by multidrug-resistant (MDR) bacteria such as Pseudomonas aeruginosa (MDR-PA). Thus, there is a critical need to develop novel and effective antimicrobials for the (i) prevention, (ii) treatment, and (iii) healing of such wounds that are complicated by MDR-P. aeruginosa and other bacterial infections. AB569 is a novel bactericidal tandem consisting of acidified NaNO 2 (A-NO 2- ) and Na 2 -EDTA. Here, we first show that AB569 acts synergistically to kill all human burn wound strains of P. aeruginosa in vitro. This was found to be due, in part, to the generation of A-NO 2- -mediated nitric oxide (NO) formation coupled with the metal chelating properties of Na 2 -EDTA. Using a murine scald burn wound model of P. aeruginosa infection, an AB569-Solosite gel formulation eradicated all bacteria. Futher, we also demonstrate enhanced AB569-mediated wound healing by not only accelerating wound contraction, but also by reducing levels of the proinflammatory cytokines interleukin-6 (IL-6) and IL-1β while increasing the levels of anti-inflammatory cytokine, IL-10, and granulocyte-colony-stimulating factor (G-CSF). We also observed better epidermal restoration in AB569-treated wounds. Taken together, we conclude that this study provides solid foundational evidence that AB569 can be used topically to treat highly problematic dermal insults, including wound, burn, blast, and likely, diabetic infections in civilian and military populations, and help relieve the economical burden that MDR organisms have on the global health care system.

Testicular torsion is a urologic emergency induced by torsion of the spermatic cord, interrupting blood circulation to the testis. Therapeutic options for testicular torsion, except surgical restoration of testis, are rarely applied in clinical practice. This study, therefore, investigated whether topical application of nitrite (NO 2 - ) is beneficial in tissue damage due to testicular ischemia-reperfusion (I/R) injury in rats. Pubertal Sprague-Dawley rats were assigned to seven groups: group A, sham-operated control group; group B, I/R with no treatment; groups C, D, and E, I/R followed by treatment with three different doses of nitrite; group F, I/R followed by administration of nitrite and a NO scavenger, C-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt); and group G, I/R followed by administration of nitrate (NO 3 - ). Unilateral testicular ischemia was maintained for 5 h, followed by reperfusion for 24 h. Nitrite and nitrate were topically administered before reperfusion. Compared to group A, germ cell apoptosis, oxidative stress, antioxidant enzymatic function, and lipid peroxidation were significantly increased, along with abnormal morphology and impaired spermatogenesis in group B ( P < 0.05). In contrast, testicular damage was generally attenuated in the nitrite treatment groups due to a reduction in superoxide and peroxynitrite levels and the inhibition of caspase-3-dependent apoptosis ( P < 0.05 vs. group B). These therapeutic effects of nitrite-derived NO were suppressed after injection of C-PTIO, which showed in group F. Taken together, our results demonstrate that topical application of nitrite may be one of the therapeutic strategies for testicular ischemia-reperfusion injury., Competing Interests: The authors have no conflict of interest to disclose., (Copyright © 2021 Jae Won Lee et al.)

Cold atmospheric plasma (CAP) and plasma-treated liquids (PTLs) have recently become a promising option for cancer treatment, but the underlying mechanisms of the anti-cancer effect are still to a large extent unknown. Although hydrogen peroxide (H2O2) has been recognized as the major anti-cancer agent of PTL and may enable selectivity in a certain concentration regime, the co-existence of nitrite can create a synergistic effect. We develop a mathematical model to describe the key species and features of the cellular response toward PTL. From the numerical solutions, we define a number of dependent variables, which represent feasible measures to quantify cell susceptibility in terms of the H2O2 membrane diffusion rate constant and the intracellular catalase concentration. For each of these dependent variables, we investigate the regimes of selective versus non-selective, and of synergistic versus non-synergistic effect to evaluate their potential role as a measure of cell susceptibility. Our results suggest that the maximal intracellular H2O2 concentration, which in the selective regime is almost four times greater for the most susceptible cells compared to the most resistant cells, could be used to quantify the cell susceptibility toward exogenous H2O2. We believe our theoretical approach brings novelty to the field of plasma oncology, and more broadly, to the field of redox biology, by proposing new ways to quantify the selective and synergistic anti-cancer effect of PTL in terms of inherent cell features.

Hypertension is a highly prevalent disease marked by vascular and cardiac maladaptive remodelling induced mainly by renin-angiotensin system activation followed by oxidative stress. Here, we briefly describe these damages and review the current evidence supporting a potential role for nitrate and nitrite as antihypertensive molecules that act via nitric oxide (NO) formation-dependent and NO formation-independent mechanisms and how nitrate/nitrite inhibits cardiovascular remodelling in hypertension. The renin-angiotensin system activation and oxidative stress converge to activate proteases involved in cardiovascular remodelling in hypertension. Besides these proteases, several investigations have demonstrated that reduced endogenous NO bioavailability is a central pathological event in hypertension. In this regard, nitrate/nitrite, long considered inert products of NO, is now known as physiological molecules able to reduce blood pressure in hypertensive patients and in different experimental models of hypertension. These effects are associated with the formation of NO and other NO-related molecules, which could induce S-nitrosylation of target proteins. However, it remains unclear whether S-nitrosylation is an essential mechanism for the anti-remodelling effects of nitrate/nitrite in hypertension. Moreover, nitrate/nitrite produces antioxidant effects associated with the inhibition of signalling pathways involved in cardiovascular remodelling. Together, these findings may help to establish nitrate and nitrite as effective therapies in hypertension-induced cardiovascular remodelling., (© 2020 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Cardiovascular disease (CVD) is the main cause of death worldwide, and aging is its leading risk factor. Aging is much accelerated in Hutchinson-Gilford progeria syndrome (HGPS), an ultra-rare genetic disorder provoked by the ubiquitous expression of a mutant protein called progerin. HGPS patients die in their teens, primarily due to cardiovascular complications. The primary causes of age-associated CVD are endothelial dysfunction and dysregulated vascular tone; however, their contribution to progerin-induced CVD remains poorly characterized. In the present study, we found that progeroid Lmna G609G / G609G mice with ubiquitous progerin expression show both endothelial dysfunction and severe contractile impairment. To assess the relative contribution of specific vascular cell types to these anomalies, we examined Lmna LCS / LCS Tie2Cre tg /+ and Lmna LCS / LCS Sm22αCre tg /+ mice, which express progerin specifically in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), respectively. Whereas vessel contraction was impaired in mice with VSMC-specific progerin expression, we observed no endothelial dysfunction in mice with progerin expression restricted to VSMCs or ECs. Vascular tone regulation in progeroid mice was ameliorated by dietary sodium nitrite supplementation. Our results identify VSMCs as the main cell type causing contractile impairment in a mouse model of HGPS that is ameliorated by nitrite treatment.

Background: Poppers are nitrite-containing liquids, which are inhaled for their aphrodisiac and hallucinogenic effects. Despite some cases of severe poisonings, poppers are often perceived as harmless by consumers. Inhalation and ingestion of poppers are well known, but, according to our literature review, intravenous abuse has not been reported before., Case Presentation: A 34-year-old man injected poppers intravenously for recreational purposes. He then suffered from dyspnoea and general discomfort. Upon arrival of emergency medical services, the patient was dyspnoeic with blue-grey skin colour and oxygen saturation was 82% on ambient air. Non-invasive ventilation was necessary, and he was transferred to the intensive care unit. Toluidine blue was administered because of a methaemoglobinaemia of 40% and methaemoglobin levels dropped to 0.4%. He was discharged home after a 24-hour observation. We additionally analysed the contents of the poppers bottle: isopropyl nitrite, isopropanol and acetone were detected. Possible complications and the treatment regarding intravenous administration of poppers are discussed., Conclusion: We present the first published case of intravenous poppers abuse. Our patient suffered from methaemoglobinaemia and was rapidly discharged after treatment with toluidine blue. No specific treatment regarding the contents of the poppers bottle, apart from isopropyl nitrite, was necessary., (© Royal College of Physicians 2020. All rights reserved.)

Nitrite, an anion produced from the oxidative breakdown of nitric oxide (NO), has traditionally been viewed as an inert molecule. However, this dogma has been challenged with the findings that nitrite can be readily reduced to NO under pathological conditions, hence representing a physiologically relevant storage reservoir of NO either in the blood or tissues. Nitrite administration has been demonstrated to improve myocardial function in subjects with heart failure and to lower the blood pressure in hypertensive subjects. Thus, extensive amount of work has since been carried out to investigate the therapeutic potential of nitrite in treating cardiovascular diseases, especially hypertension. Studies done on several animal models of hypertension have demonstrated the efficacy of nitrite in preventing and ameliorating the pathological changes associated with the disease. This brief review of the current findings aims to re-evaluate the use of nitrite for the treatment of hypertension and in particular to highlight its role in improving endothelial function.

Background: Dietary nitrate improves exercise performance by reducing the oxygen cost of exercise, although the mechanisms responsible are not fully understood., Objectives: We tested the hypothesis that nitrate and nitrite treatment would lower the oxygen cost of exercise by improving mitochondrial function and stimulating changes in the availability of metabolic fuels for energy production., Methods: We treated 9-mo-old zebrafish with nitrate (sodium nitrate, 606.9 mg/L), nitrite (sodium nitrite, 19.5 mg/L), or control (no treatment) water for 21 d. We measured oxygen consumption during a 2-h, strenuous exercise test; assessed the respiration of skeletal muscle mitochondria; and performed untargeted metabolomics on treated fish, with and without exercise., Results: Nitrate and nitrite treatment increased blood nitrate and nitrite levels. Nitrate treatment significantly lowered the oxygen cost of exercise, as compared with pretreatment values. In contrast, nitrite treatment significantly increased oxygen consumption with exercise. Nitrate and nitrite treatments did not change mitochondrial function measured ex vivo, but significantly increased the abundances of ATP, ADP, lactate, glycolytic intermediates (e.g., fructose 1,6-bisphosphate), tricarboxylic acid (TCA) cycle intermediates (e.g., succinate), and ketone bodies (e.g., β-hydroxybutyrate) by 1.8- to 3.8-fold, relative to controls. Exercise significantly depleted glycolytic and TCA intermediates in nitrate- and nitrite-treated fish, as compared with their rested counterparts, while exercise did not change, or increased, these metabolites in control fish. There was a significant net depletion of fatty acids, acyl carnitines, and ketone bodies in exercised, nitrite-treated fish (2- to 4-fold), while exercise increased net fatty acids and acyl carnitines in nitrate-treated fish (1.5- to 12-fold), relative to their treated and rested counterparts., Conclusions: Nitrate and nitrite treatment increased the availability of metabolic fuels (ATP, glycolytic and TCA intermediates, lactate, and ketone bodies) in rested zebrafish. Nitrate treatment may improve exercise performance, in part, by stimulating the preferential use of fuels that require less oxygen for energy production., (Copyright © American Society for Nutrition 2019.)

Introduction: Besides therapeutic hypothermia or targeted temperature management no novel therapies have been developed to improve outcomes of patients after cardiac arrest (CA). Recent studies suggest that nitrite reduces neurological damage after asphyxial CA. Nitrite is also implicated as a new mediator of remote post conditioning produced by tourniquet inflation-deflation, which is under active investigation in CA. However, little is known about brain penetration or pharmacokinetics (PK). Therefore, to define the optimal use of this agent, studies on the PK of nitrite in experimental ventricular fibrillation (VF) are needed. We tested the hypothesis that nitrite administered after resuscitation from VF is detectable in cerebrospinal fluid (CSF), brain and other organ tissues, produces no adverse hemodynamic effects, and improves neurologic outcome in rats., Methods: After return of spontaneous circulation (ROSC) of 5 min untreated VF, adult male Sprague-Dawley rats were given intravenous nitrite (8 μM, 0.13 mg/kg) or placebo as a 5 min infusion beginning at 5 min after CA. Additionally, sham groups with and without nitrite treatment were also studied. Whole blood nitrite levels were serially measured. After 15 min, CSF, brain, heart and liver tissue were collected. In a second series, using a randomized and blinded treatment protocol, rats were treated with nitrite or placebo after arrest. Neurological deficit scoring (NDS) was performed daily and eight days after resuscitation, fear conditioning testing (FCT) and brain histology were assessed., Results: In an initial series of experiments, rats (n = 21) were randomized to 4 groups: VF-CPR and nitrite therapy (n = 6), VF-CPR and placebo therapy (n = 5), sham (n = 5), or sham plus nitrite therapy (n = 5). Whole blood nitrite levels increased during drug infusion to 57.14 ± 10.82 μM at 11 min post-resuscitation time (1 min after dose completion) in the VF nitrite group vs. 0.94 ± 0.58 μM in the VF placebo group (p < 0.001). There was a significant difference between the treatment and placebo groups in nitrite levels in blood between 7.5 and 15 min after CPR start and between groups with respect to nitrite levels in CSF, brain, heart and liver. In a second series (n = 25 including 5 shams), 19 out of 20 animals survived until day 8. However, NDS, FCT and brain histology did not show any statistically significant difference between groups., Conclusions: Nitrite, administered early after ROSC from VF, was shown to cross the blood brain barrier after a 5 min VF cardiac arrest. We characterized the PK of intravenous nitrite administration after VF and were able to demonstrate nitrite safety in this feasibility study., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Acute pulmonary embolism may cause right heart failure due to increased pulmonary vascular resistance and arterial hypoxemia. Effective vasodilator therapy of the pulmonary hypertension is highly needed. Therefore, we investigated the effects of a newly developed effective pulmonary vasodilator, the organic mononitrites of 1,2-propanediol (PDNO), in a rabbit model of acute pulmonary embolism. In anesthetized and ventilated rabbits, systemic and pulmonary hemodynamics, exhaled nitric oxide (NO), plasma nitrite concentration, and blood gases were monitored. First, dose-response experiments with intravenous and left heart ventricle infusions of PDNO and inorganic nitrite were done in naive animals and in pulmonary hypertension induced by a thromboxane A 2 analogue. Second, acute pulmonary embolism was induced and either PDNO or placebo were administered intravenously within 20 minutes and evaluated within 1 hour after pulmonary embolization. PDNO intravenously, in contrast to inorganic nitrite intravenously, increased exhaled NO and counteracted pulmonary hypertension and vasodilated the systemic circulation, dose-dependently, thereby showing efficient NO donation. Pulmonary embolization induced pulmonary hypertension and gas exchange disturbances. PDNO significantly decreased and normalized pulmonary vascular resistance and the right ventricle rate-pressure product, without causing tolerance, with no significant side effects on the systemic circulation, nor on blood-gas values or on methemoglobin formation. In conclusion, PDNO is a NO donor and an efficient vasodilator in the pulmonary circulation. Treatment with this or similar organic nitrites intravenously may be a future option to avoid right heart failure in life-threatening acute pulmonary embolism.

Hypertension is usually associated with deficient nitric oxide (NO) bioavailability, and therefore stimulating NO activity is an important antihypertensive strategy. Recently, many studies have shown that both nitrite and nitrate anions are not simple products of NO metabolism and indeed may be reduced back to NO. While enzymes with nitrite-reductase activity capable of generating NO from nitrite may contribute to antihypertensive effects of nitrite, another mechanism involving the generation of NO-related species in the stomach from nitrite has been validated. Under the acidic conditions of the stomach, nitrite generates NO-related species that form S-nitrosothiols. Conversely, drugs that increase gastric pH may impair the gastric formation of S-nitrosothiols, which may mediate antihypertensive effects of oral nitrite or nitrate. Therefore, it is now becoming clear that promoting gastric formation of S-nitrosothiols may result in effective antihypertensive responses, and this mechanism opens a window of opportunity in the therapy of hypertension. In this review, we discuss the recent studies supporting the gastric generation of S-nitrosothiols as a potential antihypertensive mechanism of oral nitrite. We also highlight some drugs that increase S-nitrosothiols bioavailability, which may also improve the responses to nitrite/nitrate therapy. This new approach may result in increased nitrosation of critical pharmacological receptors and enzymes involved in the pathogenesis of hypertension, which tend to respond less to their activators resulting in lower blood pressure., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Importance: There are few effective treatments for heart failure with preserved ejection fraction (HFpEF). Short-term administration of inorganic nitrite or nitrate preparations has been shown to enhance nitric oxide signaling, which may improve aerobic capacity in HFpEF., Objective: To determine the effect of 4 weeks' administration of inhaled, nebulized inorganic nitrite on exercise capacity in HFpEF., Design, Setting, and Participants: Multicenter, double-blind, placebo-controlled, 2-treatment, crossover trial of 105 patients with HFpEF. Participants were enrolled from July 22, 2016, to September 12, 2017, at 17 US sites, with final date of follow-up of January 2, 2018., Interventions: Inorganic nitrite or placebo administered via micronebulizer device. During each 6-week phase of the crossover study, participants received no study drug for 2 weeks (baseline/washout) followed by study drug (nitrite or placebo) at 46 mg 3 times a day for 1 week followed by 80 mg 3 times a day for 3 weeks., Main Outcomes and Measures: The primary end point was peak oxygen consumption (mL/kg/min). Secondary end points included daily activity levels assessed by accelerometry, health status as assessed by the Kansas City Cardiomyopathy Questionnaire (score range, 0-100, with higher scores reflecting better quality of life), functional class, cardiac filling pressures assessed by echocardiography, N-terminal fragment of the prohormone brain natriuretic peptide levels, other exercise indices, adverse events, and tolerability. Outcomes were assessed after treatment for 4 weeks., Results: Among 105 patients who were randomized (median age, 68 years; 56% women), 98 (93%) completed the trial. During the nitrite phase, there was no significant difference in mean peak oxygen consumption as compared with the placebo phase (13.5 vs 13.7 mL/kg/min; difference, -0.20 [95% CI, -0.56 to 0.16]; P = .27). There were no significant between-treatment phase differences in daily activity levels (5497 vs 5503 accelerometry units; difference, -15 [95% CI, -264 to 234]; P = .91), Kansas City Cardiomyopathy Questionnaire Clinical Summary Score (62.6 vs 61.9; difference, 1.1 [95% CI, -1.4 to 3.5]; P = .39), functional class (2.5 vs 2.5; difference, 0.1 [95% CI, -0.1 to 0.2]; P = .43), echocardiographic E/e' ratio (16.4 vs 16.6; difference, 0.1 [95% CI, -1.2 to 1.3]; P = .93), or N-terminal fragment of the prohormone brain natriuretic peptide levels (520 vs 533 pg/mL; difference, 11 [95% CI, -53 to 75]; P = .74). Worsening heart failure occurred in 3 participants (2.9%) during the nitrite phase and 8 (7.6%) during the placebo phase., Conclusions and Relevance: Among patients with HFpEF, administration of inhaled inorganic nitrite for 4 weeks, compared with placebo, did not result in significant improvement in exercise capacity., Trial Registration: ClinicalTrials.gov Identifier: NCT02742129.

Context: Depleted nitric oxide levels in the human body play a major role in cardiovascular disease pathogenesis. Inorganic nitrate/nitrite (rich dietary sources include beetroot and spinach) can act as a nitric oxide donor because nitrate/nitrite can be metabolized to produce nitric oxide., Objective: This review and meta-analysis sought to investigate the role of inorganic nitrate/nitrite in preventing or treating cardiovascular disease risk factors in humans., Data Sources: Electronic databases, including Medline, Embase, Cumulative Index to Nursing and Allied Health Literature, Cochrane, and Scopus, were searched., Data Extraction: Experimental trials examining the effect of oral inorganic nitrate/nitrite intake on cardiovascular disease risk factors were included for systematic analysis., Results: Thirty-four studies were included for qualitative synthesis, 23 of which were eligible for meta-analysis. Included studies measured the following outcomes: blood pressure, endothelial function, arterial stiffness, platelet aggregation, and/or blood lipids. Inorganic nitrate intake was found to significantly reduce resting blood pressure (systolic blood pressure: -4.80 mmHg, P < 0.0001; diastolic blood pressure: -1.74 mmHg, P = 0.001), improve endothelial function (flow-mediated dilatation: 0.59%, P < 0.0001), reduce arterial stiffness (pulse wave velocity: -0.23 m/s, P < 0.0001; augmentation index: -2.1%, P = 0.05), and reduce platelet aggregation by 18.9% (P < 0.0001)., Conclusions: Inorganic nitrate consumption represents a simple strategy for targeting cardiovascular disease risk factors. Future studies investigating the long-term effects of inorganic nitrate on cardiovascular disease outcomes are warranted.

In 1867 the organic nitrite, amyl nitrite, was introduced as a therapeutic agent in the treatment of angina pectoris and was later substituted by the organic nitrate nitroglycerin (NTG). Despite having a highly potent vasodilator capacity in veins>coronary arteries>arterioles, the vasodilator effects NTG are rapidly attenuated by the development of nitrate tolerance. We and others established that NTG treatment stimulates the production of reactive oxygen species such as superoxide and peroxynitrite with subsequent marked attenuation of the NTG vasodilator potency. The nitrite anion (NO 2 - ) has more recently been characterized to possess novel pharmacotherapeutic actions such as modulation of vasodilation under hypoxic conditions, thereby providing protection in ischemia-reperfusion injury. Administration of NO 2 - /NO 3 - has also been shown to improve myocardial function in heart failure and to lower blood pressure. Despite these positive aspects there is still a great need to study inorganic nitrate and nitrite therapy in various cardiovascular diseases in prospective outcome directed studies. In case being successful, this kind of therapy would indeed represent a cheap, therefore affordable, effective cardiovascular therapy without major side effects as observed in response to therapy with organic nitrates., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Crush syndrome is characterized by ischemia/reperfusion injury (IRI). The protective effect of nitrite on experimentally induced IRI has been demonstrated in the heart, kidney, liver, and skeletal muscle. IRI in tissues and systemic organs occurs due to the massive generation of reactive oxygen species and subsequent systemic inflammation. Therefore, ischemic pre and postconditioning are performed in clinical practice. Intravenous administration of nitrite inhibits IRI through nitric oxide-mediated mechanisms. In this paper, we discuss the utility of nitrite as a pharmacological postconditioning agent in the treatment of crush syndrome., (© 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Extracellular vesicles (EVs) are implicated in the pathogenesis of cardiovascular disease (CVD). Specifically, platelet-derived EVs are highly pro-coagulant, promoting thrombin generation and fibrin clot formation. Nitrate supplementation exerts beneficial effects in CVD, via an increase in nitric oxide (NO) bioavailability. Clopidogrel is capable of producing NO-donating compounds, such as S-nitrosothiols (RSNO) in the presence of nitrite and low pH. The aim of this study was to assess the effect of nitrate supplementation with versus without clopidogrel therapy on circulating EVs in coronary artery disease (CAD) patients. In this randomized, double-blind, placebo-controlled study, CAD patients with ( n  = 10) or without ( n  = 10) clopidogrel therapy received a dietary nitrate supplement (SiS nitrate gel) or identical placebo. NO metabolites and platelet activation were measured using ozone-based chemiluminescence and multiple electrode aggregometry. EV concentration and origin were determined using nanoparticle tracking analysis and time-resolved fluorescence. Following nitrate supplementation, plasma RSNO was elevated (4.7 ± 0.8 vs 0.2 ± 0.5 nM) and thrombin-receptor mediated platelet aggregation was reduced (-19.9 ± 6.0 vs 4.0 ± 6.4 U) only in the clopidogrel group compared with placebo. Circulating EVs were significantly reduced in this group (-1.183e 11  ± 3.15e 10 vs -9.93e 9  ± 1.84e 10 EVs/mL), specifically the proportion of CD41+ EVs (-2,120 ± 728 vs 235 ± 436 RFU [relative fluorescence unit]) compared with placebo. In vitro experiments demonstrated clopidogrel-SNO can reduce platelet-EV directly (6.209e 10  ± 4.074e 9 vs 3.94e 11  ±  1.91e 10 EVs/mL). In conclusion, nitrate supplementation reduces platelet-derived EVs in CAD patients on clopidogrel therapy, increasing patient responsiveness to clopidogrel. Nitrate supplementation may represent a novel approach to moderating the risk of thrombus formation in CAD patients., Competing Interests: Conflicts of Interests: None declared., (Schattauer GmbH Stuttgart.)

Objective: This research was aimed to investigate the correct dose of nitrite that would act as a protection against the ischemic effects induced by acute myocardial infarction (AMI)., Methods: Mice were randomly divided into a sham-operation group (sham), an AMI operation group (AMI), and a nitrite pretreatment+AMI operation group (N+AMI). Seven days before the AMI operation, mice in the N+AMI group were pretreated with sodium nitrite in drinking water., Results: One week after the AMI operation, serum lactate dehydrogenase (LDH) and creatine kinase (CK) activities in both AMI and N+AMI group were significantly higher than those in the sham group, but there were no significant differences between AMI and N+AMI mice. Contents of inducible nitric oxide synthase (iNOS) in the noninfarct area of the left ventricle in the N+AMI mice were significantly higher than those in the AMI mice, with no difference in the infarct area. Coagulation necrosis in the cardiomyocytes was observed in both AMI and N+AMI mice; however, it was less severe in the N+AMI mice. Western blot analyses showed that nitrite pretreatment resulted in up-regulation of antiapoptotic factors Bcl-2 and p21waf1/cip1 signal proteins, but down-regulation of the proapoptotic factor Bax signal protein. Furthermore, nitrite pretreatment also showed significant alleviation of AMI-induced signal protein expressions of inflammatory factors of NF-K B and oxidative factors of Hsp 70 and HO-1., Conclusion: These results suggest that nitrite show certain protective effects against the ischemic effects induced by AMI in mice, which might be attributed to the synthesis of NO induced by iNOS through up-regulation of antiapoptotic factors and down-regulation of proapoptotic and inflammatory factors.

Loss of nitric oxide (NO) bioavailability underlies the development of hypertensive heart disease. We investigated the effects of dietary nitrite on N G -nitro-l-arginine methyl ester (l-NAME)-induced hypertension. Sprague-Dawley rats were divided into five groups: an untreated control group, an l-NAME-treated group, and three other l-NAME-treated groups supplemented with 10 mg/L or 100 mg/L of nitrite or 100 mg/L of captopril in drinking water. After the 8-week experimental period, mean arterial blood pressure was measured, followed by sampling of blood and heart tissue for assessment of nitrite/nitrate levels in the plasma and heart, the plasma level of angiotensin II (AT II), and the heart transcriptional levels of AT II type 1 receptor (AT 1 R), transforming growth factor-β1 (TGF-β1), and connective tissue proteins such as type 1 collagen and fibronectin. Heart tissue was analyzed by histopathological morphometry, including assessments of ventricular and coronary vascular hypertrophy and fibrosis, as well as immunohistochemistry analyses of myocardial expression of AT 1 R. l-NAME treatment reduced the plasma nitrate level and led to the development of hypertension, with increased plasma levels of AT II and increased heart transcriptional levels of AT 1 R and TGF-β1-mediated connective tissue proteins, showing myocardial and coronary arteriolar hypertrophy and fibrosis. However, dietary nitrite supplementation inhibited TGF-β1-mediated cardiac remodeling by suppressing AT II and AT 1 R. These results suggest that dietary nitrite levels achievable via a daily high-vegetable diet could improve hypertensive heart disease by inhibiting AT II-AT 1 R-mediated cardiac remodeling., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Septic arthritis is a severe and rapidly debilitating disease associated with severe joint pain, inflammation and oxidative stress. Nitroxyl (HNO) has become a nitrogen oxide of significant interest due to its pharmacological endpoints that are potentially favorable for treating varied diseases. However, whether HNO also serves as a treatment to septic arthritis is currently unknown. The aim of this study was to investigate the effect of the HNO donor, Angeli's salt (AS), in the outcome of chronic Staphylococcus aureus (S. aureus)-induced septic arthritis in mice. Daily treatment with AS inhibited mechanical hyperalgesia and inflammation (edema, leukocyte migration, cytokines release and NF-κB activation, and oxidative stress) resulting in reduced disease severity (clinical course, histopathological changes, proteoglycan levels in the joints, and osteoclastogenesis). In addition, AS decreased the number of S. aureus colony forming unities in synovial tissue, enhanced the bactericidal effect of macrophages and inhibited the worsening of systemic inflammatory response (leukocyte counts in the lung and systemic proinflammatory cytokine concentration). Our results suggest for the first time the therapeutic potential of AS in a model of septic arthritis by mechanisms involving microbicidal effects, anti-inflammatory actions and reduction of disease severity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Menopausal women are at greater risk of developing metabolic syndrome with reduced endothelial nitric oxide synthase (eNOS) activity. Hormone replacement therapy increases eNOS activity and normalizes some characteristics of metabolic syndrome. We hypothesized that nitric oxide (NO) supplementation should have a therapeutic effect on this syndrome. We examined the effect of dietary nitrite in a mouse model with postmenopausal metabolic syndrome induced by ovariectomy (OVX) and a high fat diet (HF). C57BL/6 female mice were divided into five groups, sham+normal fat diet (NF), sham+ HF, OVX+HF with or without sodium nitrite (50 mg and 150 mg/l) in the drinking water. Daily food intake and weekly body weight were monitored for 18 wk. OVX and HF significantly reduced plasma levels of nitrate/nitrite (NOx), and mice developed obesity with visceral hypertrophic adipocytes and increased transcriptional levels of monocyte chemoattractant protein-1, TNF-α, and IL-6 in visceral fat tissues. The proinflammatory state in the adipocytes provoked severe hepatosteatosis and insulin resistance in OVX+HF group compared with sham+NF group. However, dietary nitrite significantly suppressed adipocyte hypertrophy and transcriptions of proinflammatory cytokines in visceral fat in a dose-dependent manner. The improvement of visceral inflammatory state consequently reversed the hepatosteatosis and insulin resistance observed in OVX+HF mice. These results suggest that an endogenous NO defect might underlie postmenopausal metabolic syndrome and that dietary nitrite provides an alternative source of NO, subsequently compensating for metabolic impairments of this syndrome., (Copyright © 2017 the American Physiological Society.)

Many conditions culminate in heart failure (HF), a multi-organ systemic syndrome with an intrinsically poor prognosis. Pharmacotherapeutic agents that correct neurohormonal dysregulation and haemodynamic instability have occupied the forefront of developments within the treatment of HF in the past. Indeed, multiple trials aimed to validate these agents in the 1980s and early 1990s, resulting in a large and robust evidence-base supporting their use clinically. An established treatment paradigm now exists for the treatment of HF with reduced ejection fraction (HFrEF), but there have been very few notable developments in recent years. HF remains a significant health concern with an increasing incidence as the population ages. We may indeed be entering the surgical era for HF treatment, but these therapies remain expensive and inaccessible to many. Newer pharmacotherapeutic agents are slowly emerging, many targeting alternative therapeutic pathways, but with mixed results. Metabolic modulation and manipulation of the nitrate/nitrite/nitric oxide pathway have shown promise and could provide the answers to fill the therapeutic gap between medical interventions and surgery, but further definitive trials are warranted. We review the significant evidence base behind the current medical treatments for HFrEF, the physiology of metabolic impairment in HF, and discuss two promising novel agents, perhexiline and nitrite., (© 2016 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Nitric oxide (NO) is generated endogenously by NO synthases to regulate a number of physiological processes including cardiovascular and metabolic functions. A decrease in the production and bioavailability of NO is a hallmark of many major chronic diseases including hypertension, ischaemia-reperfusion injury, atherosclerosis and diabetes. This NO deficiency is mainly caused by dysfunctional NO synthases and increased scavenging of NO by the formation of reactive oxygen species. Inorganic nitrate and nitrite are emerging as substrates for in vivo NO synthase-independent formation of NO bioactivity. These anions are oxidation products of endogenous NO generation and are also present in the diet, with green leafy vegetables having a high nitrate content. The effects of nitrate and nitrite are diverse and include vasodilatation, improved endothelial function, enhanced mitochondrial efficiency and reduced generation of reactive oxygen species. Administration of nitrate or nitrite in animal models of cardiovascular disease shows promising results, and clinical trials are currently ongoing to investigate the therapeutic potential of nitrate and nitrite in hypertension, pulmonary hypertension, peripheral artery disease and myocardial infarction. In addition, the nutritional aspects of the nitrate-nitrite-NO pathway are interesting as diets suggested to protect against cardiovascular disease, such as the Mediterranean diet, are especially high in nitrate. Here, we discuss the potential therapeutic opportunities for nitrate and nitrite in prevention and treatment of cardiovascular and metabolic diseases., (© 2015 The Association for the Publication of the Journal of Internal Medicine.)

Significance: Nitrite is now recognized as an intrinsic signaling molecule that mediates a number of biological processes. One of the most reproducible effects of nitrite is its ability to mediate cytoprotection after ischemia/reperfusion (I/R). This robust phenomenon has been reproduced by a number of investigators in varying animal models focusing on different target organs. Furthermore, nitrite's cytoprotective versatility is highlighted by its ability to mediate delayed preconditioning and remote conditioning in addition to acute protection., Recent Advances: In the last 10 years, significant progress has been made in elucidating the mechanisms underlying nitrite-mediated ischemic tolerance., Critical Issues: The mitochondrion, which is essential to both the progression of I/R injury and the protection afforded by preconditioning, has emerged as a major subcellular target for nitrite. This review will outline the role of the mitochondrion in I/R injury and preconditioning, review the accumulated preclinical studies demonstrating nitrite-mediated cytoprotection, and finally focus on the known interactions of nitrite with mitochondria and their role in the mechanism of nitrite-mediated ischemic tolerance., Future Directions: These studies set the stage for current clinical trials testing the efficacy of nitrite to prevent warm and cold I/R injury.

Nitrite acts as an endocrine source of bioactive nitric oxide, impacting vascular reactivity, angiogenesis and cytoprotection. Nitrite has recently been shown to have a metabolic role although its effects and mechanisms of action in the obese insulin-resistant state are unknown. We examined glucose tolerance and insulin secretion using the frequently sampled intravenous glucose tolerance test and insulin sensitivity using the hyperinsulinaemic euglycaemic clamp in obese male ob(lep) mice administered nitrite (100 mg kg(-1) day(-1) ) or saline (control) for 7 days and compared responses to the known insulin-sensitizing effects of rosiglitazone (6 mg kg(-1) day(-1) ). Under weight-matched conditions, nitrite lowered blood pressure relative to saline and rosiglitazone, whereas only rosiglitazone was effective at reducing hepatic glucose output and basal blood glucose. Both nitrite and rosiglitazone produced improvements, relative to saline, in glucose tolerance (12,524 ± 602, 12,811 ± 692 vs.14,428 ± 335 mg (dl min)(-1) , respectively; P < 0.05) and insulin sensitivity (8.6 ± 0.7, 7.9 ± 0.3 vs. 6.6 ± 0.5 mg kg(-1) min(-1) , respectively; P < 0.001), but there was no effect on insulin secretion. Nitrite exhibited an uncoupling of mitochondrial respiration and a decrease in ATP generation in muscle that was independent of mitochondrial biogenesis or activation of uncoupling proteins. There was no insulin-stimulated phosphorylation of Akt, but nitrite increased the phosphorylation of AMP-activated protein kinase. We conclude that nitrite improves two key components of the metabolic syndrome, blood pressure and insulin sensitivity, independent of weight and with effectiveness comparable to rosiglitazone., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

Nitric oxide (NO) and hydrogen sulfide (H2S) are gasotransmitter molecules important in numerous physiological and pathological processes. Although these molecules were first known as environmental toxicants, it is now evident that that they are intricately involved in diverse cellular functions with impact on numerous physiological and pathogenic processes. NO and H2S share some common characteristics but also have unique chemical properties that suggest potential complementary interactions between the two in affecting cellular biochemistry and metabolism. Central among these is the interactions between NO, H2S, and thiols that constitute new ways to regulate protein function, signaling, and cellular responses. In this review, we discuss fundamental biochemical principals, molecular functions, measurement methods, and the pathophysiological relevance of NO and H2S., (Copyright © 2015 the American Physiological Society.)

Nitrite has emerged as an important bioactive molecule that can be biotransformed to nitric oxide (NO) related metabolites in normoxia and reduced to NO under hypoxic and acidic conditions to exert vasodilatory effects and confer a variety of other benefits to the cardiovascular system. Abundant research is currently underway to understand the mechanisms involved and define the role of nitrite in health and disease. In this review we discuss the impact of nitrite and dietary nitrate on vascular function and the potential therapeutic role of nitrite in acute heart failure., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Aging is the major risk factor for cardiovascular diseases (CVD). This is attributable primarily to adverse changes in arteries, notably, increases in large elastic artery stiffness and endothelial dysfunction mediated by inadequate concentrations of the vascular-protective molecule, nitric oxide (NO), and higher levels of oxidative stress and inflammation. Inorganic nitrite is a promising precursor molecule for augmenting circulating and tissue NO bioavailability because it requires only a one-step reduction to NO. Nitrite also acts as an independent signaling molecule, exerting many of the effects previously attributed to NO. Results of recent studies indicate that nitrite may be effective in the treatment of vascular aging. In old mice, short-term oral sodium nitrite supplementation reduces aortic pulse wave velocity, the gold-standard measure of large elastic artery stiffness, and ameliorates endothelial dysfunction, as indicated by normalization of NO-mediated endothelium-dependent dilation. These improvements in age-related vascular dysfunction with nitrite are mediated by reductions in oxidative stress and inflammation, and may be linked to increases in mitochondrial biogenesis and health. Increasing nitrite levels via dietary intake of nitrate appears to have similarly beneficial effects in many of the same physiological and clinical settings. Several clinical trials are being performed to determine the broad therapeutic potential of increasing nitrite bioavailability on human health and disease, including studies related to vascular aging. In summary, inorganic nitrite, as well as dietary nitrate supplementation, represents a promising therapy for treatment of arterial aging and prevention of age-associated CVD in humans.

Objectives: Sarcoplasmic reticulum (SR) Ca(2+)-handling proteins play an important role in myocardial dysfunction after acute ischemia/reperfusion injury. We hypothesized that nitrite would improve postresuscitation myocardial dysfunction by increasing nitric oxide (NO) generation and that the mechanism of this protection is related to the modulation of SR Ca(2+)-handling proteins., Methods: We conducted a randomized prospective animal study using male Sprague-Dawley rats. Cardiac arrest was induced by intravenous bolus of potassium chloride (40 µg/g). Nitrite (1.2 nmol/g) or placebo was administered when chest compression was started. No cardiac arrest was induced in the sham group. Hemodynamic parameters were monitored invasively for 90 minutes after the return of spontaneous circulation (ROSC). Echocardiogram was performed to evaluate cardiac function. Myocardial samples were harvested 5 minutes and 1 hour after ROSC., Results: Myocardial function was significantly impaired in the nitrite and placebo groups after resuscitation, whereas cardiac function (i.e., ejection fraction and fractional shortening) was significantly greater in the nitrite group than in the placebo group. Nitrite administration increased the level of nitric oxide in the myocardium 5 min after resuscitation compared to the other two groups. The levels of phosphorylated phospholamban (PLB) were decreased after resuscitation, and nitrite increased the phosphorylation of phospholamban compared to the placebo. No significant differences were found in the expression of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) and ryanodine receptors (RyRs)., Conclusions: postresuscitation myocardial dysfunction is associated with the impairment of PLB phosphorylation. Nitrite administered during resuscitation improves postresuscitation myocardial dysfunction by preserving phosphorylated PLB protein during resuscitation.

Background: Inorganic nitrate and nitrite have emerged as alternative substrates for nitric oxide (NO) generation in the gastrointestinal tract, and have shown to be protective against drug-induced gastric injury. The aim of this study was to investigate the preventive and therapeutic effects of nitrate and nitrite in a model of experimental colitis., Methods: Colitis was induced in mice by administrating dextran sulfate sodium (DSS) with concurrent administration of nitrite (1 mM) or nitrate (10 mM) in the drinking water for 7 days. A therapeutic approach was also investigated by initiating nitrite treatment 3 days after DSS-induced colitis. Clinical and inflammatory markers were assessed and the colonic mucus thickness was measured in vivo. The effect of nitrite on wound healing was evaluated using colon epithelial cells., Results: Concurrent administration of DSS and nitrite (1 mM) alleviated inflammation as determined by reduced disease activity index score (DAI) and increased colon length, while nitrate (10 mM) only reduced the DAI-score. Nitrite also displayed therapeutic effects by ameliorating established colonic inflammation with reduced colonic expression of iNOS and improving histopathology. DSS-induced decrease in colonic mucus thickness was completely prevented by nitrite administration. In addition, goblet cell abundance was lower by DSS treatment, but was increased by addition of nitrite. Further studies using colon epithelial cells revealed an NO-dependent improvement in wound healing with nitrite administration., Conclusion: Nitrite exerts both preventive and therapeutic effects in colonic inflammation. The protective effects involve preservation of an intact adherent mucus layer and regulation of epithelial cell restitution.

According to the CDC, the most common type of heart disease is coronary artery disease, which commonly leads to myocardial infarction (MI). Therapeutic approaches to lessen the resulting cardiovascular injury associated with MI are limited. Recently, MicroRNAs (miRNAs) have been shown to act as negative regulators of gene expression by inhibiting mRNA translation and/or stimulating mRNA degradation. A single miRNA can modulate physiological or disease phenotypes by regulating whole functional systems. Importantly, miRNAs can regulate cardiac function, thereby modulating heart muscle contraction, heart growth and morphogenesis. MicroRNA-499 (miRNA-499) is a cardiac-specific miRNA that when elevated causes cardiomyocyte hypertrophy, in turn preventing cardiac dysfunction during MI. Previous studies revealed that combination treatment with conjugated linoleic acid (cLA) and nitrite preserved cardiovascular function in mice. Therefore, it was hypothesized that cLA and nitrite may regulate miRNA-499, thus providing cardiac protection during MI. To test this hypothesis, 12-week old mice were treated with cLA (10 mg/kg/d-via osmotic mini-pump) or cLA and nitrite (50 ppm-drinking water) 3 days prior to MI (ligation of the left anterior descending artery). Echocardiography and pressure-volume (PV)-loop analysis revealed that cLA and nitrite-treated MI mice had improved heart function (10 days following MI) compared to untreated MI mice. Treatment with cLA and nitrite significantly induced levels of miRNA-499 compared to untreated MI mice. In addition, treatment with cLA and nitrite abolished MI-induced protein expression of p53 and dynamin-related protein-1 (DRP-1). Moreover, the antioxidant enzyme expression of heme oxygenase-1 (HO-1) was elevated in MI mice treated with cLA and nitrite compared to untreated MI mice. Confocal imaging on heart tissue confirmed expression the levels of HO-1 and p53. Taken together, these results suggest that therapeutic treatment with cLA and nitrite may provide significant protection during MI through regulation of both cardiac specific miRNA-499 and upregulation of phase 2 antioxidant enzyme expression.

The history began in the 19th century with Ascanio Sobrero (1812-1888), the discoverer of glycerol trinitrate (nitroglycerine, NTG), and with Angelo Angeli (1864-1931), the discoverer of sodium trioxodinitrate (Angeli's salt). It is likely that Angeli and Sobrero never met, but their two histories will join each other more than a century later. In fact, it has been discovered that both NTG and Angeli's salt are able to induce a preconditioning effect. As NTG has a long history as an antianginal drug its newly discovered property as a preconditioning agent has also been tested in humans. Angeli's salt properties as a preconditioning and inotropic agent have only been tested in animals so far.

Organic nitrates are traditionally felt to be a safe adjuvant in the chronic therapy of patients with coronary artery disease. Despite their long use, progress in the understanding of the pharmacology and mechanism of action of these drugs has been achieved only in the last two decades, with the identification of the role of oxidative stress in the pathophysiology of nitrate tolerance, with, the discovery of the ancillary effects of nitrates, and with the demonstration that nitrate therapy has important chronic side effects that might modify patients' prognosis. These advances are however mostly confined to the molecular level or to studies in healthy volunteers, and the true impact of organic nitrates on clinical outcome remains unknown. Complicating this issue, evidence supports the existence of important differences among the different drugs belonging to the group, and there are reasons to believe that the nitrates should not be treated as a homogeneous class. As well, the understanding of the effects of alternative nitric oxide (NO) donors is currently being developed, and future studies will need to test whether the properties of these new medications may compensate and prevent the abnormalities imposed by chronic nitrate therapy. Intermittent therapy with nitroglycerin and isosorbide mononitrate is now established in clinical practice, but they should neither be considered a definitive solution to the problem of nitrate tolerance. Both these strategies are not deprived of complications, and should currently be seen as a compromise rather than a way fully to exploit the benefits of NO donor therapy.

Nitric oxide modulates pain development. However, there is no evidence on the effect of nitroxyl (HNO/NO⁻) in nociception. Therefore, we addressed whether nitroxyl inhibits inflammatory hyperalgesia and its mechanism using the nitroxyl donor Angeli's salt (AS; Na₂N₂O₃). Mechanical hyperalgesia was evaluated using a modified Randall and Selitto method in rats, cytokine production by ELISA and nitroxyl was determined by confocal microscopy in DAF (a cell permeable reagent that is converted into a fluorescent molecule by nitrogen oxides)-treated dorsal root ganglia neurons in culture. Local pre-treatment with AS (17-450 μg/paw, 30 min) inhibited the carrageenin-induced mechanical hyperalgesia in a dose- and time-dependent manner with maximum inhibition of 97%. AS also inhibited carrageenin-induced cytokine production. AS inhibited the hyperalgesia induced by other inflammatory stimuli including lipopolysaccharide, tumor necrosis factor-α, interleukin-1β and prostaglandin E2. Furthermore, the analgesic effect of AS was prevented by treatment with ODQ (a soluble guanylate cyclase inhibitor), KT5823 (a protein kinase G [PKG] inhibitor) or glybenclamide (an ATP-sensitive K⁺ channel blocker), but not with naloxone (an opioid receptor antagonist). AS induced concentration-dependent increase in fluorescence intensity of DAF-treated neurons in a l-cysteine (nitroxyl scavenger) sensitive manner. l-cysteine did not affect the NO⁺ donor S-Nitroso-N-acetyl-DL- penicillamine (SNAP)-induced anti-hyperalgesia or fluorescence of DAF-treated neurons. This is the first study to demonstrate that nitroxyl inhibits inflammatory hyperalgesia by reducing cytokine production and activating the cGMP/PKG/ATP-sensitive K⁺ channel signaling pathway in vivo., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Significance: Pulmonary arterial hypertension (PAH) is a disorder characterized by increased pulmonary vascular resistance and mean pulmonary artery pressure leading to impaired function of the right ventricle, reduced cardiac output, and death. An imbalance between vasoconstrictors and vasodilators plays an important role in the pathobiology of PAH., Recent Advances: Nitric oxide (NO) is a potent vasodilator in the lung, whose bioavailability and signaling pathway are impaired in PAH. It is now appreciated that the oxidative product of NO metabolism, the inorganic anion nitrite (NO(2)(-)), functions as an intravascular endocrine reservoir of NO bioactivity that can be reduced back to NO under physiological and pathological hypoxia., Critical Issues: The conversion of nitrite to NO is controlled by coupled electron and proton transfer reactions between heme- and molybdenum-containing proteins, such as hemoglobin and xanthine oxidase, and by simple protonation and disproportionation, and possibly by catalyzed disproportionation. The two major sources of nitrite (and nitrate) are the endogenous L-arginine-NO pathway, by oxidation of NO, and the diet, with conversion of nitrate from diet into nitrite by oral commensal bacteria. In the current article, we review the enzymatic formation of nitrite and the available data regarding its use as a therapy for PAH and other cardiovascular diseases., Future Directions: The successful efficacy demonstrated in several animal models and safety in early clinical trials suggest that nitrite may represent a promising new therapy for PAH.

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

Nitrite, previously considered physiologically irrelevant and a simple end product of endogenous nitric oxide (NO) metabolism, is now envisaged as a reservoir of NO to be activated in response to oxygen (O(2)) depletion. In the first part of this review, we summarize and compare the mechanisms of nitrite-dependent production of NO in selected bacteria and in eukaryotes. Bacterial nitrite reductases, which are copper or heme-containing enzymes, play an important role in the adaptation of pathogens to O(2) limitation and enable microrganisms to survive in the human body. In mammals, reduction of nitrite to NO under hypoxic conditions is carried out in tissues and blood by an array of metalloproteins, including heme-containing proteins and molybdenum enzymes. In humans, tissues play a more important role in nitrite reduction, not only because most tissues produce more NO than blood, but also because deoxyhemoglobin efficiently scavenges NO in blood. In the second part of the review, we outline the significance of nitrite in human health and disease and describe the recent advances and pitfalls of nitrite-based therapy, with special attention to its application in cardiovascular disorders, inflammation, and anti-bacterial defence. It can be concluded that nitrite (as well as nitrate-rich diet for long-term applications) may hold promise as therapeutic agent in vascular dysfunction and ischemic injury, as well as an effective compound able to promote angiogenesis.

Nitric oxide (NO) generated by vascular NO synthases can exert anti-inflammatory effects, partly through its ability to decrease leukocyte recruitment. Inorganic nitrate and nitrite, from endogenous or dietary sources, have emerged as alternative substrates for NO formation in mammals. Bioactivation of nitrate is believed to require initial reduction to nitrite by oral commensal bacteria. Here we investigated the effects of inorganic nitrate and nitrite on leukocyte recruitment in microvascular inflammation and in NSAID-induced small-intestinal injury. We show that leukocyte emigration in response to the proinflammatory chemokine MIP-2 is reduced by 70% after 7 days of dietary nitrate supplementation as well as by acute intravenous nitrite administration. Nitrite also reduced leukocyte adhesion to a similar extent and this effect was inhibited by the soluble guanylyl cyclase inhibitor ODQ, whereas the effect on emigrated leukocytes was not altered by this treatment. Further studies in TNF-α-stimulated endothelial cells revealed that nitrite dose-dependently reduced the expression of ICAM-1. In rats and mice subjected to a challenge with diclofenac, dietary nitrate prevented the increase in myeloperoxidase and P-selectin levels in small-intestinal tissue. Antiseptic mouthwash, which eliminates oral nitrate reduction, markedly blunted the protective effect of dietary nitrate on P-selectin levels. Despite attenuation of the acute immune response, the overall ability to clear an infection with Staphylococcus aureus was not suppressed by dietary nitrate as revealed by noninvasive IVIS imaging. We conclude that dietary nitrate markedly reduces leukocyte recruitment to inflammation in a process involving attenuation of P-selectin and ICAM-1 upregulation. Bioactivation of dietary nitrate requires intermediate formation of nitrite by oral nitrate-reducing bacteria and then probably further reduction to NO and other bioactive nitrogen oxides in the tissues., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Beyond an inert oxidation product of nitric oxide (NO) metabolism, current thinking posits a key role for nitrite as a mediator of NO signaling, especially during hypoxia. This concept has been discussed in the context of nitrite serving a role as an endogenous modulator of NO homeostasis, but also from a novel clinical perspective whereby nitrite therapy may replenish NO signaling and prevent ischemic tissue injury. Indeed, the relatively rapid translation of studies delineating mechanisms of action to ongoing and planned clinical trials has been critical in fuelling interest in nitrite biology, and several excellent reviews have been written on this topic. In this article we limit our discussions to current concepts and what we feel are questions that remain unanswered within the paradigm of nitrite being a mediator of NO biology., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Nitrite therapy is more effective in cerebral ischemia when administered earlier. It would be beneficial during the hyperacute stages of stroke if the nitrite effect is demonstrated in intracerebral hemorrhage (ICH). When nitrite is injected intravenously 3h after ICH induction in rats, most doses of nitrite provided no beneficial effects on behavioral deficits, brain edema and hematoma volumes. A high dose of nitrite, however, decreased hematoma volume, but not brain edema. Peri-hematomal apoptosis and inflammation were similar between the control and nitrite groups. Nitrite therapy may be considered a therapeutic option in hyperacute stroke because nitrite therapy is tolerated in ICH as well., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The continually increasing rate of myocardial infarction (MI) in the Western world at least partly can be explained by a poor diet lacking in green vegetables, fruits, and fish and enriched in food that contains saturated fat. In contrast, a number of epidemiologic studies provide strong evidence highlighting the cardioprotective benefits of the Mediterranean diet enriched in green vegetables, fruits, fish, and grape wine. Regular consumption of these products leads to an accumulation of nitrate/nitrite/NO, polyunsaturated fatty acids (PUFA), and polyphenolic compounds, such as resveratrol, in the human body. Studies have confirmed that these constituents are bioactive exogenous mediators, which induce strong protection against MI. The aim of this review is to provide a critical, in-depth analysis of the cardioprotective pathways mediated by nitrite/NO, PUFA, and phenolic compounds of grape wines discovered in the recent years, including cross-talk between different mechanisms and compounds. Overall, these findings may facilitate the design and synthesis of novel therapeutic tools for the treatment of MI., (Copyright © 2011 Elsevier Inc. All rights reserved.)