Inorganic Nitrite Supplementation Improves Endothelial Function With Aging: Translational Evidence for Suppression of Mitochondria-Derived Oxidative Stress.

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Inhaled nebulized nitrite and nitrate therapy in a canine model of hypoxia-induced pulmonary hypertension.

Dysfunction in the nitric oxide (NO) signaling pathway can lead to the development of pulmonary hypertension (PH) in mammals. Discovery of an alternative pathway to NO generation involving reduction from nitrate to nitrite and to NO has motivated the evaluation of nitrite as an alternative to inhaled NO for PH. In contrast, inhaled nitrate has not been evaluated to date, and potential benefits include a prolonged half-life and decreased risk of methemoglobinemia. In a canine model of acute hypoxia-induced PH we evaluated the effects of inhaled nitrate to reduce pulmonary arterial pressure (PAP). In a randomized controlled trial, inhaled nitrate was compared to inhaled nitrite and inhaled saline. Exhaled NO, PAP and systemic blood pressures were continuously monitored. Inhaled nitrite significantly decreased PAP and increased exhaled NO. In contrast, inhaled nitrate and inhaled saline did not decrease PAP or increase exhaled NO. Unexpectedly, we found that inhaled nitrite resulted in prolonged (>5 h) exhaled NO release, increase in nitrate venous/arterial levels and a late surge in venous nitrite levels. These findings do not support a therapeutic role for inhaled nitrate in PH but may have therapeutic implications for inhaled nitrite in various disease states.

Inhaled nebulized sodium nitrite decreases pulmonary artery pressure in ß-thalassemia patients with pulmonary hypertension.

Pulmonary hypertension is a life-threatening complication in ß-thalassemia. Inhaled sodium nitrite has vasodilatory effect on pulmonary vasculature. However, its effect on pulmonary artery pressure (PAP) in ß-thalassemia subjects with pulmonary hypertension has never been reported. In this study, we investigated the change in PAP during inhalation of sodium nitrite in 5 ß-thalassemia patients. We demonstrated that sodium nitrite administered by nebulization rapidly decreased PAP as measured by echocardiography and right heart catheterization. The effect of nitrite was short as PAP returned to baseline at end of inhalation. Our findings support acute pulmonary vasodilation effect of nitrite in ß-thalassemia with pulmonary hypertension.

Hemodynamic effects of IV sodium nitrite in hospitalized comatose survivors of out of hospital cardiac arrest.

BACKGROUND: Patients resuscitated from cardiac arrest have brain and cardiac injury. Recent animal studies suggest that the administration of sodium nitrite after resuscitation from 12min of asystole limits acute cardiac dysfunction and improves survival and neurologic outcomes. It has been hypothesized that low doses of IV sodium nitrite given during resuscitation of out of hospital cardiac arrest (OHCA) will improve survival. Low doses of sodium nitrite (e.g., 9.6mg of sodium nitrite) are safe in healthy individuals, however the effect of nitrite on blood pressure in resuscitated cardiac arrest patients is unknown. METHODS: We performed a single-center, pilot trial of low dose sodium nitrite (1 or 9.6mg dose) vs. placebo in hospitalized out-of-hospital cardiac arrest patient to determine whether nitrite administration reduced blood pressure and whether whole blood nitrite levels increased in response to nitrite administration. RESULTS: This is the first reported study of sodium nitrite in cardiac arrest patients. Infusion of low doses of sodium nitrite in comatose survivors of OHCA (n=7) compared to placebo (n=4) had no significant effects on heart rate within 30min after infusion (70±20 vs. 78±3 beats per minute, p=0.18), systolic blood pressure (103±20 vs 108±15mmHg, p=0.3), or methemoglobin levels (0.92±0.33 vs. 0.70±0.26, p=0.45). Serum nitrite levels of 2-4µM were achieved within 15min of a 9.6mg nitrite infusion. CONCLUSIONS: Low dose sodium nitrite does not cause significant hemodynamic effect in patients with OHCA, which suggests that nitrite can be delivered safely in this critically ill patient population. Higher doses of sodium nitrite are necessary in order to achieve target serum level of 10µM.

Carnosine and N-acetyl cysteine protect against sodium nitrite-induced oxidative stress in rat blood.

Sodium nitrite (NaNO2 ) is widely used in the food industry as a preservative and colorant in meat and fish products. Industrialization and improper agricultural practices have greatly increased human exposure to high nitrite levels, mainly through contaminated drinking water, causing various health disorders. We have investigated the protective effect of carnosine (CAR) and N-acetyl cysteine (NAC) on NaNO2 -induced toxicity in rat blood. CAR is a bioactive dipeptide found in mammalian muscle while NAC is a synthetic sulfhydryl amino acid and an important precursor of glutathione. Animals were given a single acute oral dose of NaNO2 at 60 mg/kg body weight with or without prior administration of either CAR or NAC. Rats were sacrificed after 24 h, blood was withdrawn and plasma and erythrocytes were isolated. Administration of NaNO2 alone increased methemoglobin levels and methemoglobin reductase activity, decreased the activities of antioxidant defense and metabolic enzymes and significantly weakened the total antioxidant capacity of rat erythrocytes. Similar effects were seen in plasma of NaNO2 -treated rats. In contrast, administration of CAR or NAC, prior to NaNO2 treatment, markedly attenuated the NaNO2 -elicited deleterious effects. Thus, CAR and NAC can mitigate nitrite-induced metabolic alterations and oxidative damage probably due to their intrinsic biochemical antioxidant properties. This study suggests that CAR and NAC can be potentially used as therapeutic/protective agents against NaNO2 toxicity.

Short-term treatment with nitrate is not sufficient to induce in vivo antithrombotic effects in rats and mice.

In humans, short-term supplementation with nitrate is hypotensive and inhibits platelet aggregation via an nitric oxide (NO)-dependent mechanism. In the present work, we analyzed whether short-term treatment with nitrate induces antithrombotic effects in rats and mice. Arterial thrombosis was evoked electrically in a rat model in which renovascular hypertension was induced by partial ligation of the left renal artery. In mice expressing green fluorescent protein, laser-induced thrombosis was analyzed intravitally by using confocal microscope. Sodium nitrate (NaNO3) or sodium nitrite (NaNO2) was administered orally at a dose of 0.17 mmol/kg, twice per day for 3 days. Short-term nitrate treatment did not modify thrombus formation in either rats or mice, while nitrite administration led to pronounced antithrombotic activity. In hypertensive rats, nitrite treatment resulted in a significant decrease in thrombus weight (0.50 ± 0.08 mg vs. VEH 0.96 ± 0.09 mg; p < 0.01). In addition, nitrite inhibited ex vivo platelet aggregation and thromboxane B2 (TxB2) generation and prolonged prothrombin time. These effects were accompanied by significant increases in blood NOHb concentration and plasma nitrite concentration. In contrast, nitrate did not affect ex vivo platelet aggregation or prothrombin time and led to only slightly elevated nitrite plasma concentration. In mice, nitrate was also ineffective, while nitrite led to decreased platelet accumulation in the area of laser-induced endothelial injury. In conclusion, although nitrite induced profound NO-dependent antithrombotic effects in vivo, conversion of nitrates to nitrite in rats and mice over short-term 3-day treatment was not sufficient to elicit NO-dependent antiplatelet or antithrombotic effects.

Sodium nitrite enhances generation of reactive oxygen species that decrease antioxidant power and inhibit plasma membrane redox system of human erythrocytes.

Nitrite/nitrate salts are used in fertilizers and as food preservatives. Human exposure to high levels of nitrite results in its uptake and subsequent entry into blood where it can interact with erythrocytes. We show that treatment of human erythrocytes with sodium nitrite (NaNO2 ) results in a dose-dependent increase in the production of reactive oxygen species. This was accompanied by a decrease in the antioxidant power which lowered the free radical quenching and metal-reducing ability. NaNO2 treatment also inhibited plasma membrane redox system (PMRS) of erythrocytes. These changes increase the susceptibility of erythrocytes to oxidative damage, decrease the antioxidant power of whole blood, and can be a major cause of nitrite-induced cellular toxicity.

Improved motor and cognitive performance with sodium nitrite supplementation is related to small metabolite signatures: a pilot trial in middle-aged and older adults.

Advancing age is associated with reductions in nitric oxide bioavailability and changes in metabolic activity, which are implicated in declines in motor and cognitive function. In preclinical models, sodium nitrite supplementation (SN) increases plasma nitrite and improves motor function, whereas other nitric oxide-boosting agents improve cognitive function. This pilot study was designed to translate these findings to middle-aged and older (MA/O) humans to provide proof-of-concept support for larger trials. SN (10 weeks, 80 to 160 mg/day capsules, TheraVasc, Inc.) acutely and chronically increased plasma nitrite and improved performance on measures of motor and cognitive outcomes (all p<0.05 or better) in healthy MA/O adults (62 ± 7 years). Untargeted metabolomics analysis revealed that SN significantly altered 33 (160 mg/day) to 45 (80 mg/day) different metabolites, 13 of which were related to changes in functional outcomes; baseline concentrations of 99 different metabolites predicted functional improvements with SN. This pilot study provides the first evidence that SN improves aspects of motor and cognitive function in healthy MA/O adults, and that these improvements are associated with, and predicted by, the plasma metabolome. Our findings provide the necessary support for larger clinical trials on this promising pharmacological strategy for preserving physiological function with aging.

Protective effect of exogenous nitrite in postoperative ileus.

BACKGROUND AND PURPOSE: As the pathogenesis of postoperative ileus (POI) involves inflammation and oxidative stress, comparable to ischaemia/reperfusion injury which can be ameliorated with nitrite, we investigated whether nitrite can protect against POI and explored the mechanisms involved. EXPERIMENTAL APPROACH: We used intestinal manipulation (IM) of the small intestine to induce POI in C57BL/6J mice. Sodium nitrite (48 nmol) was administered intravenously just before IM. Intestinal transit was assessed using fluorescent imaging. Bethanechol-stimulated jejunal circular muscle contractions were measured in organ baths. Inflammatory parameters, neutrophil infiltration, inducible NOS (iNOS) activity, reactive oxygen species (ROS) levels, mitochondrial complex I activity and cGMP were measured in the intestinal muscularis. KEY RESULTS: Pre-treatment with nitrite markedly improved the delay in intestinal transit and restored the reduced intestinal contractility observed 24 h following IM. This was accompanied by reduced protein levels of TNF-α, IL-6 and the chemokine CCL2, along with reduced iNOS activity and ROS levels. The associated neutrophil influx at 24 h was not influenced by nitrite. IM reduced mitochondrial complex I activity and cGMP levels; treatment with nitrite increased cGMP levels. Pre-treatment with the NO scavenger carboxy-PTIO or the soluble guanylyl cyclase inhibitor ODQ abolished nitrite-induced protective effects. CONCLUSIONS AND IMPLICATIONS: Exogenous nitrite deserves further investigation as a possible treatment for POI. Nitrite-induced protection of POI in mice was dependent on NO and this effect was not related to inhibition of mitochondrial complex I, but did involve activation of soluble guanylyl cyclase.

Oral nitrite therapy improves vascular function in diabetic mice.

AIM: We tested the hypothesis that short-term oral sodium nitrite supplementation would improve vascular dysfunction in obese, diabetic mice. METHODS AND RESULTS: Vascular function was determined in control mice and in db/db mice receiving drinking water with or without sodium nitrite (50 mg/L) for 5 weeks. Nitrite supplementation increased plasma nitrite concentrations in db/db mice (0.19±0.02 µM vs 0.80±0.26 µM; p < 0.05). Db/db mice had lower endothelium-dependent dilation (EDD) in response to increasing doses of acetylcholine versus heterozygous control mice (71.2% ± 14.3% vs 93% ± 7.0%; p < 0.05), and sodium nitrite supplementation restored endothelium-dependent dilation to control levels (92.9% ± 2.3% vs 93% ± 7.0%; p < 0.05). The improvement in endothelial function was accompanied by a reduction in intrinsic stiffness, but not by alterations in plasma or vascular markers of inflammation. CONCLUSION: These data suggest that sodium nitrite may be a novel therapy for treating diabetes-related vascular dysfunction; however, the mechanisms of improvement are unknown.

Low-dose sodium nitrite attenuates myocardial ischemia and vascular ischemia-reperfusion injury in human models.

OBJECTIVES: The aim of this study was to assess the potential benefits of inorganic nitrite in 2 clinical models: stress-induced myocardial ischemia and whole-arm ischemia-reperfusion. BACKGROUND: Inorganic nitrite, traditionally considered a relatively inert metabolite of nitric oxide, may exert vasomodulatory and vasoprotective effects. Despite promising results from animal models, few have shown effectiveness in human model systems, and none have fully translated to the clinical setting. METHODS: In 10 patients with inducible myocardial ischemia, saline and low-dose sodium nitrite (NaNO2) (1.5 µmol/min for 20 min) were administered in a double-blind fashion during dobutamine stress echocardiography, at separate visits and in a random order; long-axis myocardial function was quantified by peak systolic velocity (Vs) and strain rate (SR) responses. In 19 healthy subjects, flow-mediated dilation was assessed before and after whole-arm ischemia-reperfusion; nitrite was given before ischemia or during reperfusion. RESULTS: Comparing saline and nitrite infusions, Vs and SR at peak dobutamine increased in regions exhibiting ischemia (Vs from 9.5 ± 0.5 cm/s to 12.4 ± 0.6 cm/s, SR from -2.0 ± 0.2 s(-1) to -2.8 ± 0.3 s(-1)), whereas they did not change in normally functioning regions (Vs from 12.6 ± 0.4 cm/s to 12.6 ± 0.6 cm/s, SR from -2.6 ± 0.3 s(-1) to -2.3 ± 0.1 s(-1)) (p < 0.001, analysis of variance). With NaNO2, the increment of Vs (normalized for increase in heart rate) increased only in poorly functioning myocardial regions (+122%, p < 0.001). Peak flow-mediated dilation decreased by 43% after ischemia-reperfusion when subjects received only saline (6.8 ± 0.7% vs. 3.9 ± 0.7%, p < 0.01); administration of NaNO2 before ischemia prevented this decrease in flow-mediated dilation (5.9 ± 0.7% vs. 5.2 ± 0.5%, p = NS), whereas administration during reperfusion did not. CONCLUSIONS: Low-dose NaNO2 improves functional responses in ischemic myocardium but has no effect on normal regions. Low-dose NaNO2 protects against vascular ischemia-reperfusion injury only when it is given before the onset of ischemia.

Characterization of erythrocytic uptake and release and disposition pathways of nitrite, nitrate, methemoglobin, and iron-nitrosyl hemoglobin in the human circulation.

Nitrite-hemoglobin reactions have been studied extensively in vitro, but there is a lack of information on the kinetics of nitrite and its metabolites in humans. In this study, we developed a nine-compartment physiological pharmacokinetic model to describe the in vivo erythrocytic uptake and release and disposition pathways of nitrite, nitrate, methemoglobin, and iron-nitrosyl hemoglobin in the human circulation. Our model revealed that nitrite entered erythrocytes rapidly with a rate constant of 0.256 min(-1) (i.e., half-life = 2.71 min). The formation of iron-nitrosyl hemoglobin from nitrite, which involves the reduction of nitrite by deoxyhemoglobin to generate nitric oxide (NO) and reaction of NO with deoxyhemoglobin to form iron-nitrosyl hemoglobin, occurred rapidly as well (k = 2.02 min(-1); half-life = 0.343 min = 21 s). The disposition kinetics of methemoglobin was complex. Nitrate formation occurred primarily in erythrocytes through the nitrite-oxyhemoglobin reaction and was higher when nitrite was administered intra-arterially than intravenously. Nitrate reduction was an insignificant metabolic pathway. This study is the first to comprehensively evaluate the kinetics of nitrite and its metabolites in humans and provides unique insights into the rapid equilibrium of nitrite into erythrocytes and conversion to NO in the red cell, which is kinetically associated with vasodilation.

Dietary nitrite ameliorates renal injury in L-NAME-induced hypertensive rats.

Nitric oxide (NO) has numerous important functions in the kidney, and long-term blockage of nitric oxide synthases in rats by L-NAME results in severe hypertension and progressive kidney damage. On the other hand, NO production seems to be low in patients with chronic kidney disease (CKD), and NO deficiency may play a role in CKD progression. In this review, we summarized the mechanisms of amelioration of renal injury induced by L-NAME treated rats by treatment of nitrite. First, we demonstrate whether orally-administrated nitrite-derived NO can shift to the circulation. When 3mg/kg body weight Na(15)NO(2) was orally administered to rats, an apparent EPR signal derived from Hb(15)NO (A(z)=23.4 gauss) appeared in the blood, indicating that orally ingested nitrite can be a source of NO in vivo. Next, in order to clarify the capacity of nitrite to prevent renal disease, we administered low-dose nitrite (LDN: 0.1mg of sodium nitrite in 1L of drinking water), medium-dose nitrite (MDN: 1mg sodium nitrite/L, which corresponds to the amount of nitrite ingested by vegetarians), or high-dose nitrite (HDN: 10mg sodium nitrite/L) to rats simultaneously with L-NAME (1 g l-NAME/L) for 8 weeks, then examined the blood NO level as a hemoglobin-NO adduct (iron-nitrosyl-hemoglobin) using electron paramagnetic resonance spectroscopy, urinary protein excretion, and renal histological changes at the end of the experiment. It was found that oral administration of MDN and HDN but not LDN increased the blood iron-nitrosyl-hemoglobin concentration to the normal level, ameliorated the L-NAME-induced proteinuria, and reduced renal histological damage. The findings demonstrate that chronic administration of a mid-level dietary dose of nitrite restores the circulating iron-nitrosyl-hemoglobin levels reduced by L-NAME and that maintenance of the circulating iron-nitrosyl-hemoglobin level in a controlled range protects against L-NAME-induced renal injury. Taking these findings together, we propose that dietary supplementation of nitrite is a potentially useful nonpharmacological strategy for maintaining circulating NO level in order to prevent or slow the progression of renal disease. It had been believed that nitrite could result in intragastric formation of nitrosamines, which had been linked to esophageal and other gastrointestinal cancers. However, there is no positive association between the intake of nitrate or nitrite and gastric and pancreatic cancer by recent researches. Furthermore, nitrate-derived NO formation pathway is a possible mechanism for the hypotensive effect of vegetable- and fruit-rich diets, which may explain, at least in part, the mechanism of the Dietary Approach to Stop Hypertension (DASH) diet-induced hypotensive and organ-protective effects. Further research is needed to investigate the interaction between nitrite-nitrate intakes and human health.

Bioavailability of sodium nitrite from an aqueous solution in healthy adults.

Nitrate intake in humans is high through intake of vegetables such as beets, lettuce, and spinach. Nitrate itself is a compound of low toxicity but its metabolite, nitrite, formed by bacteria in the oral cavity and gastrointestinal tract, has been suspected of potential carcinogenic effects. Nitrite can induce systemic toxicity only after having been absorbed from the gastrointestinal tract. The aim of this study was to determine the absolute bioavailability of nitrite following oral administration in humans. In an open, three-way cross-over study, nine subjects received two single oral doses of sodium nitrite (0.12 and 0.06 mmol NaNO(2)/mmol Hb) and one intravenous sodium nitrite dose (0.12 mmol NaNO(2)/mmol Hb). Plasma samples were analysed to assess the nitrite levels, and pharmacokinetic parameters were calculated. Nitrate and methaemoglobin levels in plasma were also measured as oxidation of nitrite results in the formation of these two compounds. Absolute bioavailability of nitrite was 98% after oral administration of 0.12 mmol NaNO(2)/mmol Hb, and 95% after oral administration of 0.06 mmol NaNO(2)/mmol Hb. Minor adverse effects were observed after the 0.12 mmol NaNO(2)/mmol Hb oral dose. In conclusion, nitrite in solution is highly absorbed from the gastrointestinal tract and the first pass effect in the liver is low.

Arteriovenous differences in NO2- kinetics in anesthetized rabbits.

Despite of the importance of plasma NO2- as an index of endothelial nitric oxide (NO) formation and a substrate for NO production, only a limited kinetic knowledge is available in vivo. To address this issue, we intravenously injected NaNO2 into anesthetized rabbits and quantified changes in arterial and venous plasma NO2- levels. Plasma NO2- levels in arterial blood (956 +/- 220 nM) were slightly, but significantly, higher than those in venous blood (889 +/- 214 nM) under control conditions. Although similar half-lives of the NO2- were observed in arterial and venous plasma, significant arteriovenous differences were observed in the volume of distribution of the central compartment (0.158 +/- 0.007 l/kg in arterial blood and 0.295 +/- 0.015 l/kg in venous blood) and the peripheral compartment (0.259 +/- 0.035 l/kg in arterial blood and 0.135 +/- 0.034 l/kg in venous blood). When NOR1 (authentic NO) was injected, increases in NO2- levels were greater in arterial plasma, and decreases in blood pressure significantly correlated with arterial NO2- levels only (r = 0.90, p < 0.01). These results indicate that changes in NO2- are larger and more easily detectable as an index of NO formation in arterial plasma.

Sodium nitrite promotes regional blood flow in patients with sickle cell disease: a phase I/II study.

In addition to vaso-occlusion by sickled erythrocytes, the pathophysiology of sickle cell disease (SCD) is compounded by the diminished bioavailability of nitric oxide (NO), associated with vasoconstriction, endothelial activation and cell adhesion. We tested the ability of sodium nitrite, which can be converted to NO by deoxyhaemoglobin at acid pH and low oxygen tension, to improve blood flow in patients with SCD. In a phase I/II clinical trial, sodium nitroprusside, NG-monomethyl-L-arginine, and sodium nitrite were infused sequentially into the brachial artery in 14 patients at steady state. In a dose-dependent manner, sodium nitrite infusion rates of 0.4, 4 and 40 micromol/min into the brachial artery augmented mean venous plasma nitrite concentrations (P < 0.0001) and stimulated forearm blood flow up to 77 +/- 11% above baseline (P < 0.0001), measured by venous occlusion strain gauge plethysmography. This nitrite response was blunted significantly compared to controls without SCD, as previously seen with other NO donors. Sodium nitrite infusions were well tolerated without hypotension, clinically significant methaemoglobinaemia or other untoward events. The unique pharmacological properties of nitrite as a hypoxia-potentiated vasodilator and cytoprotective agent in the setting of ischaemia-reperfusion injury make this anion a plausible NO donor for future clinical trials in SCD.

Effects of the cannabinoid antagonist AM281 on systemic hemodynamics and mortality rate in streptozotocin-induced diabetic rats with endotoxic shock: comparison between non-diabetic and diabetic rats.

PURPOSE: On the basis of previous findings that the anandamide antagonist AM281, an endogenous cannabinoid receptor antagonist, could restore the hemodynamic and cerebral blood flow changes and improve the mortality rate in non-diabetic rats during sepsis, this study was conducted to examine whether AM281 could restore the hemodynamic variables and improve the mortality rate in streptozotocin-induced diabetic rats during sepsis. METHODS: The study was designed to include three sets of experiments, each set of experiment being conducted in both diabetic and non-diabetic animals: (1) measurement of changes in systemic hemodynamics and carotid artery blood flow, (2) measurement of biochemical variables and (3) assessment of mortality rate. Systemic hemodynamics, carotid artery blood flow changes and biochemical variables were assessed at pre-treatment and 1, 2 and 3 h after the treatment was performed. RESULTS: In both non-diabetic and diabetic rats, administration of lipopolysaccharide (LPS) induced a reduction in hemodynamic variables, these reductions being greater in diabetic than in non-diabetic rats. In diabetic rats, administration of AM281 could only partially prevent these hemodynamic changes, these changes being insufficient to elevate these variables to control values. Significant differences were observed in mortality rates at 6 and 12 h between non-diabetic and diabetic groups with the same treatment. At 12 h, only non-diabetic AM281 group rats were still alive (mortality rate 50%). CONCLUSION: Administration of AM281 only partially prevented the hemodynamic, biochemical and carotid artery blood flow changes associated with LPS-induced septicemia in diabetic rats, as compared with non-diabetic rats in whom these changes were prevented to a greater extent.

Nitrite infusion in humans and nonhuman primates: endocrine effects, pharmacokinetics, and tolerance formation.

BACKGROUND: The recent discovery that nitrite is an intrinsic vasodilator and signaling molecule at near-physiological concentrations has raised the possibility that nitrite contributes to hypoxic vasodilation and to the bioactivity of nitroglycerin and mediates the cardiovascular protective effects of nitrate in the Mediterranean diet. However, important questions of potency, kinetics, mechanism of action, and possible induction of tolerance remain unanswered. METHODS AND RESULTS: In the present study, we performed biochemical, physiological, and pharmacological studies using nitrite infusion protocols in 20 normal human volunteers and in nonhuman primates to answer these questions, and we specifically tested 3 proposed mechanisms of bioactivation: reduction to nitric oxide by xanthine oxidoreductase, nonenzymatic disproportionation, and reduction by deoxyhemoglobin. We found that (1) nitrite is a relatively potent and fast vasodilator at near-physiological concentrations; (2) nitrite functions as an endocrine reservoir of nitric oxide, producing remote vasodilation during first-pass perfusion of the opposite limb; (3) nitrite is reduced to nitric oxide by intravascular reactions with hemoglobin and with intravascular reductants (ie, ascorbate); (4) inhibition of xanthine oxidoreductase with oxypurinol does not inhibit nitrite-dependent vasodilation but potentiates it; and (5) nitrite does not induce tolerance as observed with the organic nitrates. CONCLUSIONS: We propose that nitrite functions as a physiological regulator of vascular function and endocrine nitric oxide homeostasis and suggest that it is an active metabolite of the organic nitrates that can be used therapeutically to bypass enzymatic tolerance.

Hematological responses of the Neotropical teleost matrinxã (Brycon cephalus) to environmental nitrite.

Environmental increase in nitrite impairs the function of several aquatic species, including fishes. Nitrite reacts with hemoglobin yielding the non-functional methemoglobin (metHb), and many physiological disturbances can arise. The physiological mechanisms to cope with nitrite are still unclear in fish. Hematological parameters, the role of NADH-methemoglobin reductase system and the electrolytic balance were studied in the freshwater teleost Brycon cephalus (matrinxã) exposed to 0.2, 0.4 and 0.6 mg/L of nitrite N-NO(2) for 24 and 96 h. Hematocrit, total hemoglobin and the red blood cell (RBC) number decreased. Methemoglobin content increased from 1% to 69% for 24 h of exposure and drastically from 5-6% to 90% for 96 h. The activity of NADH-methemoglobin reductase system displayed a tendency of increase in response to nitrite concentration or time of exposure. In the plasma, nitrite was accumulated to values 30-fold higher than the environmental concentration. The plasma K(+) concentration increased only in fish exposed to NO(2) for 24 h. No changes in plasma protein and Na(+) were observed during nitrite exposure but Cl-presented a punctual increase at 0.2 mg/L N-NO(2)-96 h. The hematological data suggest that nitrite caused functional and hemolytic anemia. Furthermore, the electrolytic balance was relatively undisturbed, and the nitrite clearance in matrinxã is likely depending on other factors than NADH-methemoglobin reductase system.

Pharmacokinetics of sodium nitrite-induced methemoglobinemia in the rat.

A biologically based mathematical model was created to characterize time and dose-dependent relationships between exposure to nitrite and induction of methemoglobinemia. The model includes mass action equations for processes known to occur: oral absorption of nitrite, elimination from the plasma, partitioning between plasma and erythrocytes, binding of nitrite to hemoglobin and methemoglobin, and the free radical chain reaction for hemoglobin oxidation. The model also includes Michaelis-Menten kinetics for methemoglobin reductase-catalyzed regeneration of hemoglobin. Body weight-scaled rate constants for absorption (k(a)) and elimination (k(e)), the effective erythrocyte/plasma partition coefficient (P), and the apparent K(m) for methemoglobin reductase were the only parameters estimated by formal optimization to reproduce the observed time course data. Time courses of plasma nitrite concentrations and blood levels of hemoglobin and methemoglobin in male and female rats that had received single intravenous or oral doses of sodium nitrite were measured. Peak plasma levels of nitrite were achieved in both sexes approximately 30 min after oral exposure, and peak methemoglobin levels were achieved after 100 min. The model predicts that 10% of the hemoglobin is oxidized to the ferric form after oral doses of 15.9 mg/kg in male rats and 11.0 mg/kg in female rats and after intravenous doses of 8.9 and 7.1 mg/kg in male and female rats, respectively. The t(1/2) for recovery from methemoglobinemia was 60 to 120 min depending on dose and route of administration. A sensitivity analysis of the model was performed to identify to which parameters the predictions of the model were most sensitive and guide attempts to simplify the model. Replacement of the V(max) of methemoglobin reductase with a value representative of humans predicted a 10% methemoglobinemia following an intravenous dose of 5.8 mg/kg, in close agreement with an observed value of 5.7 mg/kg for humans.