Nitrite Metabolism Research Articles

P36: Nitrite and nitrate concentrations and metabolism in breast milk, artificial milk, and parenteral nutrition of term and preterm infants

Background In the adult, dietary nitrate is converted to nitrite by bacteria in the mouth. In the acidic environment of the stomach, the ingested nitrite is converted to NO where it is has a protective effect by increasing blood flow and mucus production, and by modulating the microbial flora of the GI tract. Ingested nitrite has also been linked to improved cardiovascular function in adults. However, the intake and importance of nitrate and nitrite in newborn infants is largely unknown. The purpose of this work is to determine nitrite and nitrate concentrations in both the diet and total parenteral nutrition (TPN) of term and preterm infants, and to characterize the means by which nitrite is metabolized in fresh and freeze–thawed breast milk. Methods We measured nitrate and nitrite levels in breast milk collected from mothers of preterm and term infants, ten different brands of commonly used infant formula, and TPN. We also measured the effect of freeze–thawing, temperature and oxygen tension variation, and several metal-containing enzyme antagonists on the metabolism of nitrite in samples of breast milk. Results Nitrite concentrations averaged 0.07 ± 0.01 μM in the milk of mothers of preterm infants, significantly less than in the milk of mothers of term infants (0.13 ± 0.02 μM) ( P N -ethylmaleimide to block thiol groups, or the addition of allopurinol to block xanthine oxidase. The metabolism of nitrite was inhibited by the addition of ferricyanide. Nitrite metabolism was also inhibited through the use of three different blockers of lactoperoxidase, a normal constituent of breast milk, and was increased by the addition of purified lactoperoxidase, suggesting that this enzyme is involved in nitrite metabolism. Nitrite concentrations in infant formula varied widely and ranged from 0.01 to 1.3 μM, and nitrate concentrations ranged from 12 to 67 μM. The nitrite (0.08 ± 0.02 μM) and nitrate (9.5 ± 0.8 μM) concentrations in TPN were similar to those measured in fresh breast milk. Conclusions Overall nitrite and nitrate ingestion by term and preterm infants is low compared to adults, and decreases further in infants receiving freeze–thawed breast milk due to metabolism of nitrite by lactoperoxidase. Given recent evidence for the importance of dietary nitrite in adults, we speculate that low levels of ingestion may negatively impact the gastric and cardiovascular health of the newborn. Disclosure Nothing to disclose.

P76: H2S and Nitrite anion: Partners in preserving ischemic tissue function

Hydrogen sulfide (H2S) therapy has been reported to modulate vascular function during ischemia. Although nitric oxide synthase (NOS) dependent mechanisms of H2S mediated protection during tissue ischemia have been reported, little is known regarding the relationship between H2S and nitrite metabolism under similar conditions. We examined the NOS independent but nitrite dependent molecular mechanisms involved in H2S regulation of NO bioavailability and the importance of these effects during chronic tissue ischemia. Permanent unilateral hind limb ischemia was induced in wild type and eNOSKO mice by left femoral artery ligation and excision. Mice were assigned to four different groups (n = 8, each group) and treated with PBS, 0.1, 0.5 and 1 mg/kg sodium sulfide (Na2S, a H2S donor) twice daily by retro-orbital injection. Hind limb perfusion was determined using laser Doppler flowmetry. Angiogenic and cell proliferation index were detected by the ratio of CD31 to DAPI and Ki67/DAPI positive staining respectively. NO generation by Na2S and nitrite reduction in the presence of xanthine oxidase (XO) in endothelial cells was measured using a NO chemiluminescent analyzer. XO activity, cGMP, and VEGF expression were determined by ELISA. Blood perfusion, angiogenic index, cell proliferation index, and VEGF levels were all significantly increased in mice treated with Na2S compared to PBS control. These effects were blunted by cPTIO treatment both in vivo and in vitro suggesting the involvement of NO in H2S mediated ischemic tissue protection. Na2S and nitrite interaction in hypoxic endothelial cells increased NO production in a XO dependent manner. XO activity was increased in Na2S treated mice compared to PBS control. Both in vitro and in vivo data indicate the involvement of XO mediated nitrite reduction to NO that is dependent on H2S. Na2S therapy also increased cGMP levels, which were still observed in eNOS KO mice. Lastly, VEGF164 aptamer inhibited sulfide induced augmentation of blood flow in mice ischemic tissue indicating VEGF164 as a primary downstream target of H2S mediated nitrite reduction to NO under ischemic conditions. Na2S therapy restores ischemic tissue perfusion through a XO mediated nitrite reduction (NO/cGMP/VEGF) pathway. Thus, H2S mediated NO generation from nitrite would be a novel therapeutic option during ischemic vascular remodeling in the state of metabolic syndrome where NOS dependent pathways are defective. Nothing to disclose.

Effects of Erythrocyte Aging on Nitric Oxide and Nitrite Metabolism

Recent studies have suggested that in addition to oxygen transport, red blood cells (RBC) are key regulators of vascular function by both inhibiting and promoting nitric oxide (NO)-mediated vasodilation. Most studies assume that RBC are homogenous, but, in fact, they comprise cells of differing morphology and biochemical composition which are dependent on their age, parameters that control NO reactions. We tested the hypothesis that distinct RBC populations will have differential effects on NO signaling. Young and old RBC were separated by density gradient centrifugation. Consistent with previous reports, old RBC had decreased levels of surface N-acetyl neuraminic acid and increased oxygen binding affinities. Competition kinetic experiments showed that older RBCs scavenged NO∼2-fold faster compared with younger RBC, which translated to a more potent inhibition of both acetylcholine and NO-donor dependent vasodilation of isolated aortic rings. Moreover, nitrite oxidation kinetics was faster with older RBC compared with younger RBC; whereas no differences in nitrite-reduction kinetics were observed. This translated to increased inhibitory effect of older RBC to nitrite-dependent vasodilation under oxygenated and deoxygenated conditions. Finally, leukodepleted RBC storage also resulted in more dense RBC, which may contribute to the greater NO-inhibitory potential of stored RBC. These results suggest that a key element in vascular NO-homeostasis mechanisms is the distribution of RBC ages across the physiological spectrum (0-120 days) and suggest a novel mechanism for inhibited NO bioavailability in diseases which are characterized by a shift to an older RBC phenotype. Older RBC inhibit NO bioavailability by increasing NO- and nitrite scavenging.

Impact of mitochondria on nitrite metabolism in HL-1 cardiomyocytes

Apart from ATP synthesis mitochondria have many other functions, one being nitrite reductase activity. Nitric oxide (NO) released from nitrite has been shown to protect the heart from ischemia/reperfusion (I/R) injury in a cGMP-dependent manner. However, the exact impact of mitochondria on the release of NO from nitrite in cardiomyocytes is not completely understood. Besides mitochondria, a number of non-mitochondrial metalloproteins have been suggested to facilitate this process. The aim of this study was to investigate the impact of mitochondria on the bioactivation of nitrite in HL-1 cardiomyocytes. The levels of nitrosyl complexes of hemoglobin (NO-Hb) and cGMP levels were measured by electron spin resonance spectroscopy and enzyme immunoassay. In addition the formation of free NO was determined by confocal microscopy as well as intracellular nitrite and S-nitrosothiols by chemoluminescence analysis. NO was released from nitrite in cell culture in an oxygen-dependent manner. Application of specific inhibitors of the respiratory chain, p450, NO synthases (NOS) and xanthine oxidoreductase (XOR) showed that all four enzymatic systems are involved in the release of NO, but more than 50% of NO is released via the mitochondrial pathway. Only NO released by mitochondria activated cGMP synthesis. Cardiomyocytes co-cultured with red blood cells (RBC) competed with RBC for nitrite, but free NO was detected only in HL-1 cells suggesting that RBC are not a source of NO in this model. Apart from activation of cGMP synthesis, NO formed in HL-1 cells diffused out of the cells and formed NO-Hb complexes. In addition nitrite was converted by HL-1 cells to S-nitrosyl complexes. In HL-1 cardiomyocytes, several enzymatic systems are involved in nitrite reduction to NO but only the mitochondrial pathway of NO release activates cGMP synthesis. Our data suggest that this pathway may be a key regulator of myocardial contractility especially under hypoxic conditions.

Insertion of an H‐Bonding Residue into the Distal Pocket of the Ferriheme Protein Nitrophorin 4: Effect on NitriteIron Coordination and Nitrite Disproportionation

Heme proteins are important entities for the metabolism of nitrite. Inspection of the structural features of the reported hemoprotein-nitrite crystal structures reveals that, except for nitrophorin 4 (NP4), H-bonding to the nitrite ligand is accomplished via histidine or arginine residues. These H-bonds probably play an important role for the nitrite coordination and/or reactivities. In nitrophorins, which catalyze the nitrite disproportionation reaction, such a residue is missing. Here, we report on the L130R mutant of the NP isoprotein NP4 that provides the Arg130 residue as part of the flexible G-H loop as a potential H-bonding residue in the distal heme pocket. Similar to the wild-type protein, nitrite remains N-bonded in the crystal structure of NP4(L130R). However, spectroscopic investigations show that, in solution, a second ligand-rotational orientation exists, which is in fast-exchange equilibrium with the normal, parallel ligand orientation. Moreover, the nitrite disproportionation is inhibited in NP4(L130R). Comparison with another, also less active mutant NP4(D30N) suggests that the displacement of H(2)O molecules from the heme cavity prevents the proton donation pathway through Asp30.

Oxygen Regulates Tissue Nitrite Metabolism

Once dismissed as an inert byproduct of nitric oxide (NO) auto-oxidation, nitrite (NO(2)(-)) is now accepted as an endocrine reservoir of NO that elicits biological responses in major organs. While it is known that tissue nitrite is derived from NO oxidation and the diet, little is known about how nitrite is metabolized by tissue, particularly at intermediate oxygen tensions. We investigated the rates and mechanisms of tissue nitrite metabolism over a range of oxygen concentrations. We show that the rate of nitrite consumption differs in each organ. Further, oxygen regulates the rate and products of nitrite metabolism. In anoxia, nitrite is reduced to NO, with significant formation of iron-nitrosyl proteins and S-nitrosothiols. This hypoxic nitrite metabolism is mediated by different nitrite reductases in each tissue. In contrast, low concentrations (∼3.5 μM) of oxygen increase the rate of nitrite consumption by shifting nitrite metabolism to oxidative pathways, yielding nitrate. While cytochrome P(450) and myoglobin contribute in the liver and heart, respectively, mitochondrial cytochrome c oxidase plays a significant role in nitrite oxidation, which is inhibited by cyanide. Using cyanide to prevent artifactual nitrite decay, we measure metabolism of oral and intraperitoneally administered nitrite in mice. These data provide insight into the fate of nitrite in tissue, the enzymes involved in nitrite metabolism, and the role of oxygen in regulating these processes. We demonstrate that even at low concentrations, oxygen is a potent regulator of the rate and products of tissue nitrite metabolism.

Nitrite Oxidase Activities of Cytochrome P450 and Mitochondria

Abstract 5310Once dismissed as an inert byproduct of nitric oxide (NO) auto-oxidation, nitrite (NO2−) is now accepted as an endocrine reserve of NO that elicits a number of fundamental biological responses in all major organ systems. While it is known that tissue nitrite is derived from both oxidation of NO and from dietary nitrite and nitrate, much less is known about how nitrite is metabolized by tissue or about the factors that influence this metabolism. Here we investigate the rates and mechanisms by which nitrite is metabolized by tissue over a range of oxygen tensions in rats and mice. We show that the rate of nitrite metabolism differs in heart, liver, lung and brain tissue. Further, oxygen regulates the rate and products of nitrite metabolism in each of these tissues. In hypoxic tissue, nitrite is predominantly reduced to NO, with significant formation of iron-nitrosyl heme proteins and S-nitrosothiols. Interestingly, this hypoxic nitrite metabolism is mediated by different sets of nitrite reductase enzymes in each tissue. In contrast, tissue consumption of nitrite is more rapid in normoxia and the major end product is nitrate. While cytochrome P450s and myoglobin contributed in the liver and heart respectively, mitochondrial cytochrome c oxidase played a significant role in this normoxic nitrite oxidation, which could be completely inhibited by cyanide in all tissues. We used cyanide-based nitrite preservation solution to measure the pharmacokinetics of oral and intraperitoneally administered nitrite in vivo. Using this methodology, we measured basal levels of nitrite in the major tissues and confirm that the heart contains the greatest concentration of nitrite, followed by the liver and finally the lung. We demonstrate that intraperitoneal administration of nitrite to mice increases nitrite levels most significantly in the liver and heart, where nitrite uptake is rapid (5–10 min) and steadily decreases thereafter, such that levels are back to baseline by 30 min. Little to no increase was observed in the lung. In these studies, changes in nitrate were difficult to detect due to the high levels of basal nitrate present in vivo and low concentration of nitrite administered. However, the rapid metabolism of nitrite in the tissue suggests that oxidation is at least partially responsible. In contrast to intraperitoneal nitrite, oral nitrite increased nitrite levels in all organs of mice, an effect which peaked in all organs except liver at 3 days. Collectively, these data provide insight into the fate of nitrite in tissue, the enzymes involved in hypoxic and normoxic nitrite metabolism and the role of oxygen in regulating these processes. Disclosures:No relevant conflicts of interest to declare.

Metabolism of Nitric Oxide and Nitrite, Nitrate

Metabolism of Nitric Oxide and Nitrite, Nitrate

P10. Nitrite and nitrate concentrations and metabolism in breast milk, infant formula, and saliva of term and preterm infants

P10. Nitrite and nitrate concentrations and metabolism in breast milk, infant formula, and saliva of term and preterm infants

Differential uptake and metabolism of nitrite in normoxic and hypoxic goldfish

Nitrite is a physiologically important nitric oxide donor at low concentrations but becomes toxic at high concentrations, as develops in freshwater fish exposed to environmental nitrite. We hypothesized that nitrite uptake across the gills differs between normoxic and hypoxic fish and that nitrite accumulation causes excess nitric oxide formation and nitrosative stress. Nitrite and its metabolites were measured via chemiluminescence in normoxic and hypoxic goldfish in control conditions and after 1 day of nitrite exposure. Exposure to nitrite produced much higher nitrite levels in plasma, red blood cells (RBCs) and muscle tissue of normoxic than hypoxic goldfish, suggesting that nitrite uptake was augmented by normoxia in spite of a predictable lower gill surface area. Elevation of nitrite was associated with increased concentrations of S-nitroso, N-nitroso and Fe-nitrosyl compounds in both extracellular and intracellular compartments, revealing nitrosative stress with extensive nitros(yl)ation of thiols, amines and heme groups. The degree of nitrosative stress correlated with nitrite load. Nitrate levels increased in all compartments, reflecting that a significant fraction of the nitrite taken up was converted to non-toxic nitrate. The generation of methemoglobin and nitrosylhemoglobin (assessed by spectral deconvolution) was more pronounced during normoxic nitrite exposure than during hypoxic nitrite exposure, in agreement with the higher nitrite load in normoxic fish. However, at any given nitrite load inside RBCs, the formation of S-nitroso compounds was augmented by hypoxia. We conclude that ambient oxygen conditions have profound influence on branchial nitrite uptake and that nitrosative stress is an integral part of nitrite toxicity at high nitrite concentrations.

Stable-isotope dilution GC–MS approach for nitrite quantification in human whole blood, erythrocytes, and plasma using pentafluorobenzyl bromide derivatization: Nitrite distribution in human blood

Previously, we reported on the usefulness of pentafluorobenzyl bromide (PFB-Br) for the simultaneous derivatization and quantitative determination of nitrite and nitrate in various biological fluids by GC–MS using their 15N-labelled analogues as internal standards. As nitrite may be distributed unevenly in plasma and blood cells, its quantification in whole blood rather than in plasma or serum may be the most appropriate approach to determine nitrite concentration in the circulation. So far, GC–MS methods based on PFB-Br derivatization failed to measure nitrite in whole blood and erythrocytes because of rapid nitrite loss by oxidation and other unknown reactions during derivatization. The present article reports optimized and validated procedures for sample preparation and nitrite derivatization which allow for reliable quantification of nitrite in human whole blood and erythrocytes. Essential measures for stabilizing nitrite in these samples include sample cooling (0–4 °C), hemoglobin (Hb) removal by precipitation with acetone and short derivatization of the Hb-free supernatant (5 min, 50 °C). Potassium ferricyanide (K 3Fe(CN) 6) is useful in preventing Hb-caused nitrite loss, however, this chemical is not absolutely required in the present method. Our results show that accurate GC–MS quantification of nitrite as PFB derivative is feasible virtually in every biological matrix with similar accuracy and precision. In EDTA-anticoagulated venous blood of 10 healthy young volunteers, endogenous nitrite concentration was measured to be 486 ± 280 nM in whole blood, 672 ± 496 nM in plasma ( C P), and 620 ± 350 nM in erythrocytes ( C E). The C E-to- C P ratio was 0.993 ± 0.188 indicating almost even distribution of endogenous nitrite between plasma and erythrocytes. By contrast, the major fraction of nitrite added to whole blood remained in plasma. The present GC–MS method is useful to investigate distribution and metabolism of endogenous and exogenous nitrite in blood compartments under basal conditions and during hyperemia.

Low-dose sodium nitrite vasodilates hypoxic human pulmonary vasculature by a means that is not dependent on a simultaneous elevation in plasma nitrite

Inorganic nitrite has recently been recognized to possess vascular activity that is enhanced in hypoxia. This has been demonstrated in humans in the forearm vascular bed. In animal models nitrite reduces pulmonary vascular resistance, but its effects upon the pulmonary circulation of humans have not yet been demonstrated. This paradigm is of particular interest mechanistically since the pulmonary vasculature is known to behave differently to the systemic. To investigate, 18 healthy volunteers were studied in a hypoxic chamber (inspired oxygen, 12%) or while breathing room air. Each received an infusion of sodium nitrite (1 micromol/min) or 0.9% saline. Three protocols were performed: nitrite/hypoxia (n = 12), saline/hypoxia (n = 6), and nitrite/normoxia (n = 6). Venous blood was sampled for plasma nitrite, forearm blood flow was measured by strain-gauge plethysmography, and pulmonary arterial pressure was measured by transthoracic echocardiography. Plasma nitrite doubled and clearance kinetics were similar whether nitrite was infused in hypoxia or normoxia. During hypoxia, nitrite increased forearm blood flow (+36%, P < 0.001) and reduced three separate indirect indexes of pulmonary arterial pressure by 16%, 12%, and 17% (P < 0.01). Pulmonary, but not systemic, arterial effects persisted 1 h after stopping the infusion, at a time when plasma nitrite had returned to baseline. No effects were observed during normoxia. Therefore, in hypoxic but not normoxic subjects, sodium nitrite causes arterial and pulmonary vasodilatation. In addition, hypoxia-induced pulmonary vasoconstriction was attenuated for a prolonged period and not dependent on a simultaneous elevation of plasma nitrite. This finding is consistent with the direct extravascular metabolism of nitrite to nitric oxide to effect hypoxia-associated bioactivity.

Dietary sources of nitrite as a modulator of ischemia/reperfusion injury

The nitrite anion is an endogenous product of nitric oxide (NO) metabolism, a key intermediate in the nitrogen cycle in plants and bacteria, and a constituent of many foods. Research over the past 6 years has revealed a surprising biological and cytoprotective activity of this anion. Its ability to restore NO homeostasis throughout the physiological oxygen gradient in vivo has transformed this once-thought to be inert anion into a critical molecule in health and disease. Ischemia-reperfusion (I/R) injury is a major clinical problem worldwide. NO has been shown to be one of the most important molecules for the prevention of injury from I/R. Paradoxically, however, enzymatic NO formation from NO synthase (NOS) is inactive during conditions of inadequate oxygen and substrate delivery, such as in ischemia. Nitrite has emerged as a viable alternative source of NO under ischemic conditions. As nitrite is known to be derived not only from the oxidation of NO but also through diet, understanding nitrite metabolism and mechanisms of cytoprotection may offer novel and natural means to prevent disease or at least limit injury from an I/R event. Here, we review the current body of knowledge regarding dietary sources of nitrite and its modulation of cytoprotection in an I/R injury.

Nitrite as undesirable substances in animal feed ‐ Scientific Opinion of the Panel on Contaminants in the Food Chain

Nitrite as undesirable substances in animal feed ‐ Scientific Opinion of the Panel on Contaminants in the Food Chain

Transpulmonary Plasma ET-1 and Nitrite Differences in High Altitude Pulmonary Hypertension

Berger, Marc M., Christoph Dehnert, Damian M. Bailey, Andrew M. Luks, Elmar Menold, Christian Castell, Guido Schendler, Vitalie Faoro, Heimo Mairbäurl, Peter Bärtsch, and Eric R. Swenson. Transpulmonary plasma ET-1 and nitrite differences in high altitude pulmonary hypertension. High Alt. Med. Biol. 10:17-24, 2009.- Thirty-four mountaineers were studied at low (110 m) and high altitude (4559 m) to evaluate if increased pulmonary artery systolic pressure (PASP) at high altitude is associated with increased pulmonary endothelin-1 (ET-1) availability and alterations in nitrite metabolism across the lung. Blood samples were obtained using central venous and radial artery catheters for plasma ET-1 and nitrite. Pulmonary blood flow was measured by inert gas rebreathing to calculate transpulmonary exchange of plasma ET-1 and nitrite, and PASP was assessed by transthoracic Doppler echocardiography. After ascent to high altitude, PASP increased from 23 +/- 4 to 39 +/- 10 mmHg. Arterial and central venous plasma ET-1 increased, while plasma nitrite did not change significantly. At low altitude there was a transpulmonary loss of plasma ET-1, but a transpulmonary gain at high altitude. In contrast was a transpulmonary gain of plasma nitrite at low altitude and a transpulmonary loss at high altitude. PASP positively correlated with a transpulmonary gain of plasma ET-1 and negatively correlated with a transpulmonary loss of plasma nitrite. These results suggest that a transpulmonary gain of plasma ET- 1 is associated with higher PASP at high altitude. Transpulmonary loss of plasma nitrite indicates either less pulmonary nitric oxide (NO) production, which contributes to higher PASP, or increased NO bioavailability arising from nitrite reduction, which may oppose ET-1-mediated vasoconstriction.

Cardioprotective actions of nitrite therapy and dietary considerations

Nitrite is an intrinsic signaling molecule. Its ability to form nitric oxide (NO) under hypoxic conditions as well as its ability to form S-nitrosothiols under normoxic conditions has transformed this once inert anion into a critical molecule in maintaining NO and nitroso homeostasis throughout the entire physiological oxygen gradient in vivo in addition to serving as the endocrine mediator of NO. Steady state plasma nitrite has historically been used as a biomarker of NO availability. However, tissue specific metabolism of nitrite along with differences in nitrite and nitrate intake from diet warrant a careful assessment of plasma nitrite as an index of NO biochemistry. Metabolism and regulation of NO and nitrite are at the local cellular and tissue level dependent upon cellular redox status, redox active metal and thiol availability. Understanding nitrite metabolism and mechanisms of cytoprotection may offer novel means to prevent cardiovascular disease or limit injury from a cardiovascular event. Here the state of the art of nitrite in cytoprotection as well as maintenance of steady state blood and tissue nitrite is reviewed.

Nitrite reductase in Streptoccocus mutans plays a critical role in the survival of this pathogen in oral cavity

The mechanisms of nitric oxide (NO) production by bacteria in the oral cavity are still not clearly defined but salivary streptococci have been reported to generate NO. The aim of this study was to clarify the mechanism of nitrite metabolism and generation of NO by Streptococcus mutans, a major pathogen of dental caries. We searched the genomic database of oral pathogens for nitrite reductase and used a polymerase chain reaction (PCR) to clone the nirJ gene from S. mutans GS5. His-tagged recombinant NirJ protein was expressed in Escherichia coli BL21 and characterized. We constructed a nirJ gene-disrupted mutant strain of S. mutans (DeltanirJ) to analyze the physiological significance of nirJ. S. mutans generates NO from nitrite, probably as a result of the possession of nitrite reductase. We cloned the nirJ gene from S. mutans GS5 by PCR. The recombinant NirJ protein catalyzed the reduction of nitrite with a K(m) value of 3.37 microM and a specific activity of 2.5 micromol/min/mg of protein at 37 degrees C. Biochemical analysis revealed that the nitrite-reducing activity of the mutant (DeltanirJ) strain was significantly lower than that of the wild-type strain. The growth of the mutant strain, but not of the wild-type strain, was strongly suppressed by the presence of physiological levels of nitrite ( approximately 0.2 mM) in saliva. These observations suggest that the elimination of nitrite and/or the generation of NO are important for the survival of S. mutans in the oral cavity.

In vitro and in vivo kinetic handling of nitrite in blood: effects of varying hemoglobin oxygen saturation

Growing evidence suggests that nitrite, acting via reduction to nitric oxide by deoxyhemoglobin, may play an important role in local control of blood flow during hypoxia. To investigate the effect of hypoxia (65 Torr arterial Po(2)) on the kinetic properties of nitrite, a bolus injection of sodium nitrite (10 mg/kg iv) was given to normoxic or hypoxic newborn lambs, and the time course of plasma nitrite and methemoglobin (MetHb) concentrations was measured. The in vivo kinetics of nitrite disappearance from plasma were biphasic and were not affected by hypoxia. Changes in MetHb, a product of the nitrite-hemoglobin reaction, also did not differ with the level of oxygenation. Hypoxia potentiated the hypotensive effects of nitrite on pulmonary and systemic arterial pressures. The disappearance of nitrite from plasma was equivalent to the increase in MetHb on a molar basis. In contrast, nitrite metabolism in sheep blood in vitro resulted in more than one MetHb per nitrite equivalent under mid- and high-oxygenation conditions: oxyhemoglobin (HbO(2)) saturation = 50.3 +/- 1.7% and 97.0 +/- 1.3%, respectively. Under the low-oxygenation condition (HbO(2) saturation = 5.2 +/- 0.9%), significantly less than 1 mol of MetHb was produced per nitrite equivalent, indicating that a significant portion of nitrite is metabolized through pathways that do not produce MetHb. These data support the idea that the vasodilating effects of nitrite are potentiated under hypoxic conditions due to the reduction of nitrite to nitric oxide by deoxyhemoglobin.

Nitrite metabolism in Debaryomyces hansenii TOB-Y7, a yeast strain involved in tobacco fermentation

The Italian cigar manufacturing process includes a fermentation step that leads to accumulation of nitrite and tobacco-specific nitrosamines (TSNA), undesirable by-products due to their negative impact on health. In this study, growth and biochemical properties of Debaryomyces hansenii TOB-Y7, a yeast strain that predominates during the early phase of fermentation, have been investigated. With respect to other D. hansenii collection strains (Y7426, J26, and CBS 1796), TOB-Y7 was characterized by the ability to tolerate very high nitrite levels and to utilize nitrite, but not nitrate, as a sole nitrogen source in a chemically defined medium, a property that was enhanced in microaerophilic environment. The ability to assimilate nitrite was associated to the presence of YNI1, the gene encoding the assimilatory NAD(P)H:nitrite reductase (NiR), absent in Y7426, J26, and CBS 1796 by Southern blot data. YNI1 from TOB-Y7 was entirely sequenced, and its expression was analyzed in different media by Northern blot and reverse transcriptase polymerase chain reaction. The evidence that, in D. hansenii TOB-Y7, YNI1 was transcriptional active also in the presence of high ammonia concentration typical of tobacco fermentation, stimulated the development of an improved process that, on a laboratory scale, was proved to be effective in minimizing nitrite and TSNA accumulation.

Evidence for Mutagenesis by Nitric Oxide during Nitrate Metabolism in Escherichia coli

In Escherichia coli, nitrosative mutagenesis may occur during nitrate or nitrite respiration. The endogenous nitrosating agent N2O3 (dinitrogen trioxide, nitrous anhydride) may be formed either by the condensation of nitrous acid or by the autooxidation of nitric oxide, both of which are metabolic by-products. The purpose of this study was to determine which of these two agents is more responsible for endogenous nitrosative mutagenesis. An nfi (endonuclease V) mutant was grown anaerobically with nitrate or nitrite, conditions under which it has a high frequency of A:T-to-G:C transition mutations because of a defect in the repair of hypoxanthine (nitrosatively deaminated adenine) in DNA. These mutations could be greatly reduced by two means: (i) introduction of an nirB mutation, which affects the inducible cytoplasmic nitrite reductase, the major source of nitric oxide during nitrate or nitrite metabolism, or (ii) flushing the anaerobic culture with argon (which should purge it of nitric oxide) before it was exposed to air. The results suggest that nitrosative mutagenesis occurs during a shift from nitrate/nitrite-dependent respiration under hypoxic conditions to aerobic respiration, when accumulated nitric oxide reacts with oxygen to form endogenous nitrosating agents such as N2O3. In contrast, mutagenesis of nongrowing cells by nitrous acid was unaffected by an nirB mutation, suggesting that this mutagenesis is mediated by N2O3 that is formed directly by the condensation of nitrous acid.