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PubMed2004

The role of Ynt1 in nitrate and nitrite transport in the yeast Hansenula polymorpha.

Ynt1 is the only high-affinity nitrate uptake system in Hansenula polymorpha. Nitrate uptake was directly correlated with the Ynt1 levels and shown to be independent of nitrate reductase (NR) activity levels. Ynt1 failed to transport chlorate and, as a result, strains lacking YNT1 were sensitive to chlorate, as is the wild-type. Nitrite uptake in a wild-type strain was partially inhibited by nitrate to levels shown by a YNT1-disrupted strain in which, in turn, nitrite transport was not inhibited by nitrate. It is concluded that nitrite uptake takes place by two different transport systems: Ynt1 and a nitrite-specific transporter(s). The nitrite-specific transport system was induced by nitrate; consistently, no induction was observed in strains lacking the transcription factor YNA1, which is involved in nitrate and nitrite induction of the nitrate assimilatory structural genes. Ynt1 presents its optimal rate for nitrite uptake at pH 6, while pH 4 was optimal for the specific nitrite uptake system(s). At pH 5.5, the contribution of Ynt1 to high-affinity nitrate and nitrite uptake was around 95% and 60%, respectively. The apparent Km of Ynt1 for nitrate and nitrite is in the microM range, as is the specific nitrite uptake system for nitrite. The analysis of the effect of the reduced nitrogen sources on nitrate assimilation revealed that glutamine inactivates nitrate and nitrite transport, dependent on Ynt1, but not the nitrite-specific system.

Anion Transport Proteins↗

PubMed1996

Metabolism and detoxification of nitrite by trout hepatocytes.

Nitrite (NO2-) is one of the most important toxicants to fish. Freshwater fish are especially sensitive, particularly salmonids. Nitrite uptake is thought to occur via the HCO3-, Cl- -exchanger at the gill epithelia with nitrite substituting for chloride. In this way freshwater fish accumulate nitrite in the blood up to 100-fold from the surrounding water. Another source, endogenous nitrite as a degradation product of nitric oxide, rarely leads to pharmacologically relevant concentrations. We developed a new method for the determination of nitrate (NO3-) in biological samples and used it to measure nitrite oxidation in isolated rainbow trout (Oncorhynchus mykiss) hepatocytes which were found to detoxify nitrite to the almost non-toxic nitrate. Detoxification is inhibited by 0.05 mM bumetanide and 0.1 mM furosemide but not by SITS and DITS, suggesting the involvement of the Na+, K+, 2Cl- -cotransporter with nitrite or nitrate substituting for chloride. Oxidation of nitrite is strongly accelerated by 0.05 mM uric acid. The efficacy of this antioxidant suggests that similar reactions are involved as known for haemoglobin [33]. However, in the case of trout liver also membrane bound detoxificating activity can be observed which is also enhanced by uric acid. ATP concentrations remained constant in the hepatocytes during all experiments demonstrating that hepatocyte energy status was not influenced by nitrite oxidation. Thus nitrite resistance in fish is governed by at least two mechanisms, nitrite uptake and the rate of detoxification. It is unknown whether fish actually differ in their ability to distinguish between chloride and nitrite during branchial uptake, but evidence presented in this paper suggests a significant contribution of detoxification pathways to a possible nitrite tolerance of fish.

Animals↗

PubMed2006

Low-dose intravenous nitrite improves hemodynamics in a canine model of acute pulmonary thromboembolism.

Acute pulmonary thomboembolism (APT)-induced pulmonary hypertension can be counteracted by activating the nitric oxide (NO)-cGMP pathway. Recent studies have demonstrated that the naturally occurring anion nitrite (NO(2)(-)) is a bioactive storage reservoir for NO, and is reduced to NO under conditions of hypoxia and acidosis. We hypothesized that nitrite infused intravenously could attenuate the hemodynamic changes associated with APT. APT was induced with autologous blood clots injected into the right atrium in mongrel dogs. After APT (or saline), the dogs received an intravenous nitrite (or saline) infusion (6.75 micromol/kg over 15 min and then 0.28 micromol/kg/min) and hemodynamic evaluations were carried out for 2 h. Plasma nitrite concentrations were measured using ozone-based reductive chemiluminescence methodologies. APT decreased cardiac index (CI) and increased pulmonary vascular resistance index (PVRI); these effects were improved during infusions of sodium nitrite. Accordingly, nitrite infusion increased cardiac index by 28%, reduced the PVRI by 48%, and the systemic vascular resistance index (SVRI) by 21% in embolized dogs, suggesting a greater effect on the ischemic embolized vascular system than the systemic circulation following embolization. Interestingly, in nonembolized control dogs the same nitrite infusion decreased MAP and CI (all P<0.05). The nitrite infusion increased plasma nitrite concentrations by approximately 2 microM, and produced dose-dependent effects on PVRI, MAP, and SVRI. Remarkably, blood levels of nitrite as low as 500 nM decreased PVRI and SVRI in this model, suggesting a potential role of nitrite in physiological blood flow regulation. These results suggest that a low-dose nitrite infusion produces beneficial hemodynamic effects in a dog model of APT. These findings suggest a new therapeutic application for nitrite and support emerging evidence for a surprisingly potent and potentially physiological vasoactivity of nitrite.

Acute Disease↗

PubMed2003

Nitrite disrupts multiple physiological functions in aquatic animals.

Nitrite is a potential problem in aquatic environments. Freshwater fish actively take up nitrite across the gills, leading to high internal concentrations. Seawater fish are less susceptible but do take up nitrite across intestine and gills. Nitrite has multiple physiological effects. Its uptake is at the expense of chloride, leading to chloride depletion. Nitrite also activates efflux of potassium from skeletal muscle and erythrocytes, disturbing intracellular and extracellular K(+) levels. Nitrite transfer across the erythrocytic membrane leads to oxidation of haemoglobin to methaemoglobin (metHb), compromising blood O(2) transport. Other haem proteins are also oxidised. Hyperventilation is observed, and eventually tissue O(2) shortage becomes reflected in elevated lactate concentrations. Heart rate increases rapidly, before any significant elevations in metHb or extracellular potassium occur. This suggests nitrite-induced vasodilation (possibly via nitric oxide generated from nitrite) that is countered by increased cardiac pumping to re-establish blood pressure. Nitrite can form and/or mimic nitric oxide and thereby interfere with processes regulated by this local hormone. Steroid hormone synthesis may be inhibited, while changes in ammonia and urea levels and excretion rates reflect an influence of nitrite on nitrogen metabolism. Detoxification of nitrite occurs via endogenous oxidation to nitrate, and elimination of nitrite takes place both via gills and urine. The susceptibility to nitrite varies between species and in some cases also within species. Rainbow trout fall into two groups with regard to susceptibility and physiological response. These two groups are not related to sex but show significant different nitrite uptake rates.

Animals↗

PubMed1998

Determination of five abused drugs in nitrite-adulterated urine by immunoassays and gas chromatography-mass spectrometry.

The adulteration of urine specimens with nitrite ion hasseen shown to mask the gas chromatography-mass spectrometry (GC-MS) confirmation testing of marijuana use. This study was designed to further investigate the effect of nitrite adulteration on the detection of five commonly abused drugs by immunoassay screening and GC-MS analysis. The drugs tested are cocaine metabolite (benzoylecgonine), morphine, 11-nor-delta-tetrahydrocannabinol-9-carboxylic acid (THCCOOH), amphetamine, and phencyclidine. The immunoassays evaluated included the instrument-based Abuscreen ONLINE assays, the on-site Abuscreen ONTRAK assays, and the one-step ONTRAK TESTCUP-5 assay. Multianalyte standards containing various levels of drugs were used to test the influence of both potassium and sodium nitrite. In the ONLINE immunoassays, the presence of up to 1.0M nitrite in the multianalyte standards had no significant effect for benzoylecgonine, morphine, and phencyclidine assays. With a high concentration of nitrite, ONLINE became more sensitive for amphetamine (detected more drug than what was expected) and less sensitive for THCCOOH (detected less drug than what was expected). No effects of nitrite were observed on the results of the Abuscreen ONTRAK assays. Similarly, no effects were observed on the absolute qualitative results of the TESTCUP-5 when testing the nitrite-adulterated standards. However, the produced intensities of the signals that indicate the negative test results were slightly lowered in the THC and phencyclidine assays. The presence of 1.0M of nitrite did not show dramatic interference with the GC-MS analysis of benzoylecgonine, morphine, amphetamine, and phencyclidine. In contrast, nitrite ion significantly interfered with the detection of THCCOOH by GC-MS. The presence of 0.03M of nitrite ion resulted in significant loss in the recovery of THCCOOH and its internal standard by GC-MS. The problem of nitrite adulteration could be alleviated by sodium bisulfite treatment even when the specimens were spiked with 1.0M of nitrite ion. Although bisulfite treatment decomposed all nitrite ions in the sample to recover the remaining THCCOOH by GC-MS, the net recovery of THCCOOH depended on urinary pH and time and conditions of sample storage. The presence of nitrite concentrations that might arise from all possible natural sources, including microorganisms, pathological conditions, and medications, did not interfere with the GC-MS analysis of THCCOOH.

Amphetamine↗

PubMed2002

Nitrous oxide formation in the Colne estuary, England: the central role of nitrite.

Nitrate and nitrite concentrations in the water and nitrous oxide and nitrite fluxes across the sediment-water interface were measured monthly in the River Colne estuary, England, from December 1996 to March 1998. Water column concentrations of N(2)O in the Colne were supersaturated with respect to air, indicating that the estuary was a source of N(2)O for the atmosphere. At the freshwater end of the estuary, nitrous oxide effluxes from the sediment were closely correlated with the nitrite concentrations in the overlying water and with the nitrite influx into the sediment. Increases in N(2)O production from sediments were about 10 times greater with the addition of nitrite than with the addition of nitrate. Rates of denitrification were stimulated to a larger extent by enhanced nitrite than by nitrate concentrations. At 550 microM nitrite or nitrate (the highest concentration used), the rates of denitrification were 600 micromol N.m(-2).h(-1) with nitrite but only 180 micromol N.m(-2).h(-1) with nitrate. The ratios of rates of nitrous oxide production and denitrification (N(2)O/N(2) x 100) were significantly higher with the addition of nitrite (7 to 13% of denitrification) than with nitrate (2 to 4% of denitrification). The results suggested that in addition to anaerobic bacteria, which possess the complete denitrification pathway for N(2) formation in the estuarine sediments, there may be two other groups of bacteria: nitrite denitrifiers, which reduce nitrite to N(2) via N(2)O, and obligate nitrite-denitrifying bacteria, which reduce nitrite to N(2)O as the end product. Consideration of free-energy changes during N(2)O formation led to the conclusion that N(2)O formation using nitrite as the electron acceptor is favored in the Colne estuary and may be a critical factor regulating the formation of N(2)O in high-nutrient-load estuaries.

Bacteria, Anaerobic↗

PubMedDate not supplied

Sodium nitrite, a potent relaxant of rat stomach fundus: in vitro evidence.

The effects of sodium nitrite (0.1, 1, 10 mM) on mechanical activity of isolated rat stomach fundus muscle and the influence of guanylate cyclase activity inhibitor (methylene blue) and channel inhibitors (tetrodotoxin, charybdotoxin, apamin) were studied. Nitrite evoked dose-dependent relaxation in the longitudinal and circular muscle layers. The lowest effective concentration of sodium nitrite was 0.1 mM, which is comparable with the NOAEL (no observed adverse effect level). Tetrodotoxin (1 microM) markedly inhibited electrically induced contraction and rebound relaxation, but did not influence the nitrite-induced relaxation. Charybdotoxin (100 nM) decreased the relaxation evoked by 10 mM nitrite to 52.3 and 65.7% of control reaction in the circular and longitudinal muscle layer, respectively. Apamin (100 nM) did not influence the nitrite-induced relaxation. Methylene blue (10 microM) decreased relaxation induced by nitrite in the longitudinal and circular muscle layer, respectively, to 66.7 and 54.3% of the response to 1 mM nitrite alone. Relaxation induced by nitrite was decreased in the presence of L-cysteine (5 mM), and in the circular and longitudinal muscle layer reached 29.6 and 23.1%, respectively, of the response to 1 mM nitrite alone. We conclude that the relaxing effect of nitrite on gastric fundus results from its direct action on smooth muscle cells and probably the enteric nervous system is not involved in this action. The nitrite-elicited relaxation depends on activation of guanylate cyclase and high conductance Ca2+-activated potassium channels; however, activation of potassium channels might be a part of or might act in parallel with the mechanism involving the cyclic GMP system. Effects of nitrite observed in the presence of L-cysteine suggest that nitrosothiols are not responsible for nitrite-evoked activation of guanylate cyclase.

Acetylcholine↗

PubMed1981

The isolation of a hexaheme cytochrome from Desulfovibrio desulfuricans and its identification as a new type of nitrite reductase.

Desulfovibrio desulfuricans (ATCC 27774), a strictly anaerobic sulfate-reducing bacteria, is able to perform anaerobic nitrate respiration in which nitrate is first reduced to nitrite by the action of nitrate reductase, and nitrite reductase then catalyzes the six-electron reduction of nitrite to ammonia. The nitrite reductase was found to be a membrane-bound enzyme and has been purified to electrophoretic homogeneity. The purified enzyme has a minimal Mr = 66,000 as judged by sodium dodecyl sulfate gel electrophoresis and contains 6 c-type heme groups/molecule. Pure nitrite reductase exhibits a typical c-type cytochrome absorption spectrum with reduced alpha-band at 552.5 nm. NADH and NADPH do not function as direct electron donors for the nitrite reductase. Desulfovibrio vulgaris hydrogenase, however, is able to transfer electrons from H2 to the nitrite reductase using FAD as the electron transfer mediator. The dithionite-reduced nitrite reductase was demonstrated to be auto-oxidizable even in the presence of potassium cyanide. On addition of nitrite, the dithionite-reduced enzyme is re-oxidized immediately. Hydroxylamine, however, can only partially re-oxidize the reduced enzyme. Ascorbate reduces the enzyme to a limited extent and the partially reduced enzyme is neither auto-oxidizable nor re-oxidizable by nitrite or hydroxylamine. Purified nitrite reductase has a pH optimum in the range of 8.0-9.5 and optimal activity at 57 degrees C. Purified nitrite reductase also has hydroxylamine reductase activity, and the Km for nitrite was determined to be 1.14 mM and that for hydroxylamine is 113.5 mM. The difference in Km values seems to exclude the possibility of hydroxylamine being a free intermediate in the reduction of nitrite.

Amino Acids↗

PubMed1965

THE REDUCTION OF NITRATE, NITRITE AND HYDROXYLAMINE TO AMMONIA BY ENZYMES FROM CUCURBITA PEPO L. IN THE PRESENCE OF REDUCED BENZYL VIOLOGEN AS ELECTRON DONOR.

1. Enzyme systems from Cucurbita pepo have been shown to catalyse the reduction of nitrite and hydroxylamine to ammonia in yields about 90-100%. 2. Reduced benzyl viologen serves as an efficient electron donor for both systems. Activity of the nitrite-reductase system is directly related to degree of dye reduction when expressed in terms of the function for oxidation-reduction potentials, but appears to decrease to negligible activity below about 9% dye reduction. 3. NADH and NADPH alone produce negligible nitrite loss, but NADPH can be linked to an endogenous diaphorase system to reduce nitrite to ammonia in the presence of catalytic amounts of benzyl viologen. 4. The NADH- or NADPH-nitrate-reductase system that is also present can accept electrons from reduced benzyl viologen, but shows relationships opposite to that for the nitrite-reductase system with regard to effect of degree of dye reduction on activity. The product of nitrate reduction may be nitrite alone, or nitrite and ammonia, or ammonia alone, according only to the degree of dye reduction. 5. The relative activities of nitrite-reductase and hydroxylamine-reductase systems show different relationships with degree of dye reduction and may become reversed in magnitude when effects of degree of dye reduction are tested over a suitable range. 6. Nitrite severely inhibits the rate of reduction of hydroxylamine without affecting the yield of ammonia as a percentage of total substrate loss, but hydroxylamine has a negligible effect on the activity of the nitrite-reductase system. 7. The apparent K(m) for nitrite (1 mum) is substantially less than that for hydroxylamine, for which variable values between 0.05 and 0.9mm (mean 0.51 mm) have been observed. 8. The apparent K(m) values for reduced benzyl viologen differ for the nitrite-reductase and hydroxylamine-reductase systems: 60 and 7.5 mum respectively. 9. It is concluded that free hydroxylamine may not be an intermediate in the reduction of nitrite to ammonia by plants, and a possible mechanism for reduction of both compounds by the same enzyme system is discussed in the light of current ideas relating to other organisms.

Ammonia↗

PubMed2000

Recovery of Penaeus monodon from functional anaemia after exposure to sublethal concentration of nitrite at different pH levels.

Tiger shrimp Penaeus monodon (14.79+/-0.19 g) which had been exposed individually to 0.72 mM nitrite at pH 6.8, 8.2 and 9.8 after 3, 6, 12, 24 and 48 h were examined for the water nitrite concentration, hemolymph nitrite, oxyhemocyanin, protein and osmolality levels. Same parameters were examined for shrimp depurated in nitrite-free water after 3, 6, 12 and 24 h following 48 h exposure to nitrite. In the nitrite-exposed test, nitrite influx, hemolymph nitrite and osmotic differential (medium osmolality - hemolymph osmolality) increased with exposure time, and were higher at pH 6.8, whereas water nitrite concentration, oxyhemocyanin, protein, ratio of oxyhemocyanin to protein and hemolymph osmolality decreased with exposure time and were lower at pH 6.8. In the depuration test, water nitrite concentration increased with depuration time, and was higher at pH 6.8, whereas, hemolymph oxyhemocyanin, ratio of oxyhemocyanin to protein and hemolymph osmolality increased with depuration time and were higher at pH 9.8. Hemolymph nitrite decreased with depuration time, and was 2.52, 2.19 and 0.24 µmol ml(-1) after 6 h at pH 6.8, 8.2 and 9.8, respectively. Nitrite entry is considered to occur mainly via the form of nitrous acid (HNO(2)) in water uptake. The fact that the hemolymph nitrite decreased to non-detectable level with 72-88% recovery of oxyhemocyanin after 24 h depuration suggests an occurrence of reductase for nitrite-exposed shrimps.

Journal Article↗

PubMed1988

Role of nitrite and nitrate as a redox couple in the rat colon. Implications for diarrheal conditions.

Colonic levels of nitrite and circulating levels of nitrate are elevated in subjects with chronic diarrhea. The role of colonic epithelial cells in oxidation-reduction of nitrite and consequent control of nitrite and nitrate levels is unknown. Isolated rat colonocytes and isolated loops of colon were used to study oxidation and reduction of nitrate and nitrite. Colonocytes oxidized nitrite to nitrate at a rate of 162 +/- 33 nmol/min.g (dry wt) (n = 6) over 0-20 min, a value increased by addition of 5 mM glucose and 1 mM nicotinamide adenine dinucleotide (p = less than 0.01), but not altered by antibiotics. Nitrite oxidation correlated linearly with the dry weight of isolated colonocytes (r = 0.92), indicating a cellular reaction. Nitrite was absorbed at a rate of 0.28 nmol/min.cm2 (n = 9) from the colonic lumen, which reflected 74% absorption of the available nitrite. Of the absorbed nitrite, 14.3% reappeared in the colonic lumen as nitrate. Colonocytes may be important in oxidizing nitrite to nitrate, thus preventing excess nitrite from entering into the circulation and returning some nitrate to the colonic lumen for respiration by anaerobic bacteria. The interaction of nitrites and colonocytes deserves further study with regard to the oxidation-reduction of nitrite and to the development of colonic neoplasia in chronic diarrheal conditions.

Animals↗

PubMed1994

Quantification of nitrite and nitrate in human urine and plasma as pentafluorobenzyl derivatives by gas chromatography-mass spectrometry using their 15N-labelled analogs.

For the quantification of nitrite and nitrate, the stable metabolites of L-arginine-derived nitric oxide (NO) in human urine and plasma, we developed a gas chromatographic-mass spectrometric (GC-MS) method in which [15N]nitrite and [15N]nitrate were used as internal standards. Endogenous nitrite and [15N]nitrite added to acetone-treated plasma and urine samples were converted into their pentafluorobenzyl (PFB) derivatives using PFB bromide as the alkylating agent. For the analysis of endogenous nitrate and [15N]nitrate they were reduced to nitrite and [15N]nitrite, respectively, by cadmium in acidified plasma and urine samples prior to PFB alkylation. Reaction products were extracted with toluene and 1-microliter aliquots were analyzed by selected-ion monitoring at m/z 46 for endogenous nitrite (nitrate) and m/z 47 for [15N]nitrite ([15N]nitrate). The intra- and inter-assay relative standard deviations for the determination of nitrite and nitrate in urine and plasma were below 3.8%. The detection limit of the method was 22 fmol of nitrite. Healthy subjects (n = 12) excreted into urine 0.49 +/- 0.25 of nitrite and 109.5 +/- 61.7 of nitrate (mean +/- S.D., mumol/mmol creatinine) with a mean 24-h output of 5.7 mumol for nitrite and 1226 mumol for nitrate. The concentrations of nitrite and nitrate in the plasma of these volunteers were determined to be (mean +/- S.D., mumol/l) 3.6 +/- 0.8 and 68 +/- 17, respectively.

Female↗

PubMed2006

Nitrite in nitric oxide biology: cause or consequence? A systems-based review.

All life requires nitrogen compounds. Nitrite is such a compound that is naturally occurring in nature and biology. Over the years, the pharmacological stance on nitrite has undergone a surprising metamorphosis, from a vilified substance that generates carcinogenic nitrosamines in the stomach to a life-saving drug that liberates a protective agent (nitric oxide or NO) during hypoxic events. Nitrite has been investigated as a vasodilator in mammals for over 125 years and is a known by-product of organic nitrate metabolism. There has been a recent rediscovery of some of the vasodilator actions of nitrite in physiology along with novel discoveries which render nitrite a fundamental molecule in biology. Until recently nitrite was thought to be an inert oxidative breakdown product of endogenous NO synthesis but the past few years have focused on the reduction of nitrite back to NO in the circulation as a possible mechanism for hypoxic vasodilatation. Nitrite has evolved into an endogenous signaling molecule and regulator of gene expression that may not only serve as a diagnostic marker but also find its role as a potential therapeutic agent of cardiovascular disease. These data therefore warrant a reevaluation on the fate and metabolism of nitrite in biological systems. This review serves to encompass the history and recent evolution of nitrite, the compartment-specific metabolism of nitrite and its role in plasma as a biomarker for disease, the role of nitrite as a potential regulator of NO homeostasis, and the future of nitrite-based research.

Animals↗

PubMed1986

Mechanisms for nitrite loss from the stomach.

Nitrite loss from the stomach was studied using dogs equipped with Thomas cannulas for direct access to the stomach lumen. Solutions containing sodium nitrite and non-absorbable volume marker (polyethylene glycol, PEG) were infused into the stomach, and samples were taken over 60 min to determine the concentration of 'total nitrite' (including NO2-, HNO2 and other species in equilibrium with NO2-) and rate of dilution of the stomach contents as a function of time. Changes in stomach volume were also measured. Nitrite loss was found to be very rapid, with total nitrite concentrations declining to less than half the initial levels in 10 min. The decay in total nitrite concentrations was due predominantly to gastric absorption, with small additional contributions from dilution of the stomach contents (inferred from PEG concentrations) and chemical reactions (from in vitro kinetic data). Results for initial nitrite concentrations varying over a range of 0.15-4.5 mM showed absorption to be first order in total nitrite. The permeability-area product for nitrite absorption (PA) was about 0.6 l/h, and was unaffected by the addition of 1 mM SCN- or Cl-. All of these results are consistent with nitrite absorption in the form of NO2- or HNO2. Buffering the infusate with HCO3- to increase luminal pH from approximately 2 to 7 caused a three-fold reduction in the apparent value of PA. When pentagastrin was used to stimulate acid secretion, nitrite absorption was only half as fast as when acid secretion was inhibited with cimetidine, or when no drug was given. This effect could not be explained by variations in luminal pH, and suggests that acid secretion either decreases PA or is accompanied by active secretion of nitrite. Based on these data, a mathematical model was developed to stimulate the physical and chemical factors governing nitrite concentrations in the stomach.

Animals↗

PubMedDate not supplied

Investigation of nitrite adulteration on the immunoassay and GC-MS analysis of cannabinoids in urine specimens.

Nitrite ion has been identified as the active ingredient of two commercial adulterants that could cause discrepant results between the immunoassay screening and gas chromatographic-mass spectrometric (GC-MS) confirmation of 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in urine. Procedures to chemically convert the nitrite ion at the beginning of sample preparation for GC-MS analysis may not overcome all nitrite adulteration cases because portions of the THCCOOH might have been lost between the time of sample collection and the time of analysis. This study was conducted to further investigate the influence of both urine sample matrix and the duration of nitrite exposure on nitrite interference of THCCOOH detection. Forty clinical "THC-positive samples" that had been screened and confirmed positive for the presence of THCCOOH were spiked with 0.15M or 0.3M of nitrite. The levels of THCCOOH at various time intervals after nitrite spiking were monitored by instrument-based cannabinoids immunoassays (Syva EMIT d.a.u. and/or Roche Abuscreen ONLINE assays) and by an onsite THC immunoassay (Roche ONTRAK TESTSTIK). Results from this report demonstrate that the two outstanding "urine specimen factors" that dictated the effectiveness of the nitrite adulteration were urinary pH and the original drug concentration before nitrite spiking. Significant decreases in the immunoassay results could be observed within 4 h of nitrite treatment in the majority of samples with acidic urinary pH values. Regardless of their original concentration of THCCOOH (GC-MS ranging from 33 to 488 ng/mL), all of the 20 samples that had acidic pH values gave negative immunoassay results 1 day after nitrite adulteration. In contrast, the immunoassay results of samples with neutral or basic pH values were less affected by nitrite exposure in the same studies. Approximately two-thirds of the samples with pH values greater than 7.0 remained immunoassay-positive 3 days after nitrite spiking. Nevertheless, some of the adulterated urine that showed no change in immunoassay results might exhibit significant decrease in GC-MS recoveries even with bisulfite treatment, collaborating with the observations that a portion of samples screened positive with THC immunoassay in the laboratory could fail to confirm with GC-MS analysis. The decrease or loss of immunoassay detectable cannabinoid cross-reactives in acidic "THC-positive samples" can be attenuated by chemically increasing the pH value of the samples to the basic pH range.

Dronabinol↗

PubMed1988

Evidence that inhibitory factor extracted from bovine retractor penis is nitrite, whose acid-activated derivative is stabilized nitric oxide.

1. Unactivated extracts of bovine retractor penis (BRP) contains 3-7 microM nitrite. Acid-activation of these extracts at pH 2 for 10 min followed by neutralization generates the active form of inhibitory factor (IF; assayed by its vasodilator action on rabbit aorta), and is associated with partial loss of nitrite. 2. Increasing the time of acid-activation at pH 2 from 10 to 60 min with intermittent vortex mixing generates greater vasodilator activity and increases nitrite loss. 3. When acid-activated and neutralized extracts are incubated at 37 degrees C or 30 min or boiled for 5 min, vasodilator activity is lost and nitrite content increased. Reactivation of these samples at pH 2 for 10 min followed by neutralization leads to partial recoveries of vasodilator activity with loss in nitrite content. 4. Addition of sodium nitrite to BRP extracts increases acid-activatable vasodilator activity pro rata. 5. Acid-activation of aqueous sodium nitrite solutions results in less loss of nitrite and generation of less vasodilator activity than BRP extracts. Vasodilatation is only transient and is rapidly abolished on neutralization, whereas responses to acid-activated BRP extracts are more prolonged and activity is stable on ice. 6. Bovine aortic endothelial cells yield vasodilator activity that is indistinguishable from that isolated from BRP. It is activated by acid, stable on ice, abolished by boiling or by haemoglobin, and appears to be due to the generation of nitric oxide (NO) from nitrite. 7. The data provide confirmatory evidence that nitrite in BRP extracts is IF, that acid-activation of BRP extracts yields NO which is responsible for its vasodilator action, and that inactivation occurs by decay of NO to nitrite and nitrate. They further suggest that BRP extracts contain a NO-stabilizing agent which favours conversion of nitrite to NO. 8. The finding that bovine aortic endothelial cells yield an agent indistinguishable from IF suggests that nitrite in endothelial cells may likewise be the precursor of endothelium-derived relaxing factor (EDRF), itself identified as NO.

Animals↗

PubMed1991

NarK enhances nitrate uptake and nitrite excretion in Escherichia coli.

narK mutants of Escherichia coli produce wild-type levels of nitrate reductase but, unlike the wild-type strain, do not accumulate nitrite when grown anaerobically on a glucose-nitrate medium. Comparison of the rates of nitrate and nitrite metabolism in cultures growing anaerobically on glucose-nitrate medium revealed that a narK mutant reduced nitrate at a rate only slightly slower than that in the NarK+ parental strain. Although the specific activities of nitrate reductase and nitrite reductase were similar in the two strains, the parental strain accumulated nitrite in the medium in almost stoichiometric amounts before it was further reduced, while the narK mutant did not accumulate nitrite in the medium but apparently reduced it as rapidly as it was formed. Under conditions in which nitrite reductase was not produced, the narK mutant excreted the nitrite formed from nitrate into the medium; however, the rate of reduction of nitrate to nitrite was significantly slower than that of the parental strain or that which occurred when nitrite reductase was present. These results demonstrate that E. coli is capable of taking up nitrate and excreting nitrite in the absence of a functional NarK protein; however, in growing cells, a functional NarK promotes a more rapid rate of anaerobic nitrate reduction and the continuous excretion of the nitrite formed. Based on the kinetics of nitrate reduction and of nitrite reduction and excretion in growing cultures and in washed cell suspensions, it is proposed that the narK gene encodes a nitrate/nitrite antiporter which facilitates anaerobic nitrate respiration by coupling the excretion of nitrite to nitrate uptake. The failure of nitrate to suppress the reduction of trimethylamine N-oxide in narK mutants was not due to a change in the level of trimethylamine N-oxide reductase but apparently resulted from a relative decrease in the rate of anaerobic nitrate reduction caused by the loss of the antiporter system.

Dithionite↗

PubMed1996

Physiology and interaction of nitrate and nitrite reduction in Staphylococcus carnosus.

Staphylococcus carnosus reduces nitrate to ammonia in two steps. (i) Nitrate was taken up and reduced to nitrite, and nitrite was subsequently excreted. (ii) After depletion of nitrate, the accumulated nitrite was imported and reduced to ammonia, which again accumulated in the medium. The localization, energy gain, and induction of the nitrate and nitrite reductases in S. carnosus were characterized. Nitrate reductase seems to be a membrane-bound enzyme involved in respiratory energy conservation, whereas nitrite reductase seems to be a cytosolic enzyme involved in NADH reoxidation. Syntheses of both enzymes are inhibited by oxygen and induced to greater or lesser degrees by nitrate or nitrite, respectively. In whole cells, nitrite reduction is inhibited by nitrate and also by high concentrations of nitrite (> or = 10 mM). Nitrite did not influence nitrate reduction. Two possible mechanisms for the inhibition of nitrite reduction by nitrate that are not mutually exclusive are discussed. (i) Competition for NADH nitrate reductase is expected to oxidize the bulk of the NADH because of its higher specific activity. (ii) The high rate of nitrate reduction could lead to an internal accumulation of nitrite, possibly the result of a less efficient nitrite reduction or export. So far, we have no evidence for the presence of other dissimilatory or assimilatory nitrate or nitrite reductases in S. carnosus.

Cytosol↗