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PubMed1984

Source of the oxygen atoms of nitrate in the oxidation of nitrite by Nitrobacter agilis and evidence against a P-O-N anhydride mechanism in oxidative phosphorylation.

15N, 18O Tracer studies were applied to the aerobic oxidation of nitrite to nitrate by the chemolithotrophic bacterium, Nitrobacter agilis. It was established that, in conversion of nitrite to nitrate, one oxygen atom of nitrate arose from water and none from O2 or inorganic phosphate. This result confirms that of Kumar et al. [(1983) FEBS Lett. 152, 71-74]. Oxygen exchange between water and inorganic phosphate was small and that between water and nitrite or nitrate or any reaction intermediates between these two was not detected. Oxidation of nitrite was, therefore, effectively irreversible under the conditions employed. The uptake of extracellular phosphate was sufficient to allow significant transfer of 18O from phosphate to nitrate if oxidative phosphorylation had occurred by way of a P-O-N anhydride between phosphate (or ADP) and nitrate. The results are, therefore, inconsistent with the occurrence of a reaction of this type during nitrite oxidation.

Anhydrides↗

PubMed1991

A reexamination of the properties of spinach nitrite reductase: protein and siroheme content heterogeneity in purified preparations.

Recent preparations of nitrite reductase do not display the heterodimeric quaternary structure obtained previously (total molecular weight 85,000; subunit molecular weights 24,000 and 61,000), but rather yield only the 61,000 molecular weight subunit, even when buffers containing the protease inhibitor phenylmethylsulfonyl fluoride are used. Nevertheless, such preparations retain the high ratio of ferredoxin-linked to methyl viologen-linked enzyme activity which has been previously taken as a characteristic of only the heterodimeric form. These preparations display a siroheme prosthetic group to protein ratio of 1.1. When nitrite reductase samples are frozen during the purification scheme, even though the ferredoxin-linked specific activity does not significantly decrease, enzyme activity-stained native gel electrophoresis of the subsequently purified protein reveals that gels with several bands of activity can be obtained. Further evidence of protein heterogeneity in these preparations comes from N-terminal amino acid analysis which reveals that even nonfrozen preparations contain two major peptides with valine and cysteine as the N-termini. Formation of complexes of purified nitrite reductase with ferredoxin resulted in siroheme difference electronic spectra which resembled those observed previously for monomeric preparations. However, the siroheme midpoint potential of recent preparations of nitrite reductase (-287 mV) is close to that of the heterodimeric preparations. Ultrafiltration studies of crude extracts of the enzyme indicate that, at least at certain stages of the preparation, higher molecular weight forms of the enzyme may exist. We conclude that the 24,000 molecular weight polypeptide is a contaminant and that the heterodimeric quaternary structure model for spinach nitrite reductase is incorrect. Furthermore, the monomeric preparations we do obtain display both significant protein heterogeneity and facile loss of siroheme upon gel filtration.

Chromatography, Gel↗

PubMed1978

The mechanism of the control of carbon fixation by the pH in the chloroplast stroma. Studies with nitrite-mediated proton transfer across the envelope.

1. CO2 fixation of intact spinach chloroplasts is inhibited by nitrite in a pH-dependent mode. At pH 7.3 in the medium 1 mM NaNO2 and at pH 7.9 5 mM NaNO2 were required for 50% inhibition. 2. The addition of nitrite leads to an acidificiation in the stroma. It appears that nitrite renders the envelope permeable for protons resulting in a breakdown of the pH gradient between the external space and the stroma. 3. In view of earlier results on the pH sensitivity of C02 fixation it is concluded that this pH shift in the stroma is responsible for the observed inhibition of CO2 fixation by nitrite. 4. Octanoate and to some extent also high concentrations of bicarbonate and acetate have a similar effect as nitrite in inhibiting CO2 fixation through an acidification in the stroma. 5. The levels of the intermediates of the CO2 fixation cycle were measured. A strong rise of the levels of fructose- and sedoheptulose biphosphates and a concomitant decrease of the corresponding monophosphates was observed during inhibition of CO2 fixation. It appears that the enzymatic steps of the CO2 fixation cycle responsible for the overall inhibition of CO2 fixation caused by lowering of the H+ concentration in the stroma are fructose- and sedopheptulose bisphosphatase. These two enzymes have an important function in the light regulation of CO2 fixation.

Ammonium Chloride↗

PubMed1986

Inactivation of B12 and folate coenzymes by butyl nitrite as observed by NMR: implications on one-carbon transfer mechanism.

The effects of butyl nitrite, a frequently used recreational drug, on methyl cobalamin and 5-methyl tetrahydrofolate were investigated by using 1H and 31P NMR spectroscopies. While no effect could be observed in organic solvents, strong interactions of butyl nitrite with the methyl cobalamin and 5-methyl tetrahydrofolate were found to occur in water. Butyl nitrite decomposes in water generating H+ and NO-2. The former protonates to give the "base-off" configuration of methyl cobalamin while the Co-CH3 bond is cleaved. Similarly, the methyl group at the 5N position and the pyrazine ring of 5-methyl tetrahydrofolate were found to be affected by butyl nitrite. The overall interaction of butyl nitrite with both coenzymes shows displacement of the methyl group and derivatization or destruction of the coenzymes that may lead to deficiencies of both B-12 and/or folates.

Magnetic Resonance Spectroscopy↗

PubMed1984

Biochemical and ultrastructural effects of nitrite in rainbow trout: liver hypoxia as the root of the acute toxicity mechanism.

In Salmo gairdneri specimens exposed for 12, 24, 48, and 72 hr to nitrite (450 micrograms NO2-N) the main biochemical parameters of cerebral and hepatic hypoxia were studied. Observations on liver ultrastructure were also made to follow nitrite hepatotoxicity. Furthermore, some physiological alterations in liver mitochondria incubated in a medium containing a nitrite concentration similar to that found in the liver of in vivo exposed trout were studied. Results suggest that tissue hypoxia, due to the nitrite-induced high methemoglobinemia, is too low to be directly responsible for animal death. Nevertheless liver hypoxia is thought to be at the root of nitrite acute toxicity mechanism by producing suitable conditions for toxic potentialities. Thus irreversible and deadly damages arise in liver biochemistry and ultrastructure, particularly at the mitochondrial level.

Adenosine Triphosphate↗

PubMed1991

Comparison of isobutyl nitrate and isobutyl nitrite: tolerance and cross-tolerance to glyceryl trinitrate.

This study includes the first systematic comparison of an organic nitrate with the corresponding organic nitrite-isobutyl nitrate and isobutyl nitrite. The spasmolytic activity of the nitrite on isolated rabbit aortic strips was stronger, more rapid in onset, but less stable than the activity of the nitrate. In vitro tolerance to glyceryl trinitrate and isobutyl nitrite greatly weakened the activity of isobutyl nitrate, respectively, but had much less effect on isobutyl nitrite. Possible reasons for these differences are discussed.

Animals↗

PubMed1988

Nitrite-induced iron deficiency in the neonatal rat.

Neonatal rats from dams receiving 2 or 3 g NaNO2/liter in the drinking water through -gestation and lactation suffered severe microcytic anemia as well as growth retardation and high mortality. Lipemia, fatty liver damage, decreased erythropoiesis of spleen and bone marrow, and reduced plasma and tissue iron levels were noted in affected pups. These effects were all consistent with and characteristic of iron deficiency. Experiments presented here were designed to show that the maternally mediated toxicity of nitrite is actually an iron deficiency syndrome in the pups caused by inadequate iron transfer from dam to pup. It was found that administration of exogenous iron supplement to pups of treated mothers reversed the anemia and other effects of nitrite toxicity noted both in previous studies and in unsupplemented littermates. Mothers of affected pups were themselves anemic. Finally, we fully documented severe iron deficiency in pups of nitrite-treated mothers and showed that these mothers produced milk of reduced iron content. It appears then that nitrite-consuming dams have a reduced capacity to transfer iron to their pups. The nitrite-associated toxicities in the pups are actually a result of an iron deficiency.

Anemia↗

PubMed1990

Pretreatment effects on nitrite-induced methemoglobinemia: saline and calcium channel antagonists.

This study was undertaken to evaluate the effects of pretreatment with calcium channel antagonists (nimodipine or verapamil) on the formation of methemoglobin produced by sodium nitrite. Unexpectedly, the pretreatment of animals with control injections of physiological saline 2 hours before the nitrite administration reduced the amount of methemoglobin found in the blood 25 minutes later. When either of the calcium channel antagonists was given 2 hours before the administration of sodium nitrite, the saline effect was eliminated. When the injections of physiological saline or either of the calcium channel blockers were divided 24 hours before the nitrite administration, all reduced the amount of methemoglobin formed relative to rats that received no pretreatment. A tentative hypothesis is that the reduction of the nitrite-induced methemoglobin can be induced by the stress of handling and intraperitoneal injection and that this stress effect can last at least 24 hours. It is likely that whatever stress-related mechanism is involved in reducing methemoglobin levels, this effect can be reduced by the presence of "L channel" voltage-sensitive calcium antagonists or their active metabolites.

Animals↗

PubMed1985

An example of interaction between environmental pollutants: modification of thiram toxicity to freshwater organisms by nitrites or nitrates in relation to nitrosamine synthesis.

Thiram, a dithiocarbamate fungicide, is known to evolve to dimethylnitrosamine (DMNA) when associated with nitrites. Conditions of appearance of that carcinogenic compound have been studied in short-term experiments by association of the fungicide, nitrates or nitrites, and species representative of freshwater biota. DMNA has been estimated by GLC equipped with a specific detector. Chlorella vulgaris can rapidly produce nitrites from nitrates and DMNA is obtained in presence of thiram. Daphnia magna can also synthesize DMNA but nitrites have to be added to the medium. Increased toxicity of thiram is observed. The same results are obtained on Cyprinus carpio and for a part on Brachydanio rerio. When the species are associated in a 15-day experimental food chain, and intoxicated algae feed the two other levels, no significant transfer is observed. Nevertheless, some DMNA hazard may exist for particular species exposed to thiram associated with nitrites or even nitrates if algae are present.

Animals↗

PubMed1986

Mutagenicities of indole and 30 derivatives after nitrite treatment.

Indole and 7-derivatives, L- and D-tryptophan and 9 derivatives, and beta-carboline (norharman) and 11 derivatives were tested for mutagenicity to Salmonella typhimurium TA100 and TA98 after nitrite treatment. 1-Methylindole, which is present in cigarette smoke condensate (Grob and Voellmin, 1970; Hoffmann and Rathkamp, 1970), was the most mutagenic to TA100 without S9 mix after nitrite treatment, inducing 615,000 revertants/mg. 2-Methylindole, 1-methyl-DL-tryptophan, harmaline and (-)-(1S,3S)-1,2-dimethyl-1,2,3,4-tetrahydro-beta-carboline-3- carboxylic acid also showed strong mutagenicity after nitrite treatment, inducing 129,000, 184,000, 103,000 and 197,000 revertants/mg, respectively. These mutagenic potencies were comparable with those of benzo[alpha]pyrene, 3-methylcholanthrene and 2-amino-9H-pyrido[2,3-b]indole (A alpha C) (Sugimura, 1982). Of 31 compounds tested, 22 were mutagenic after nitrite treatment. Since various indole compounds are ubiquitous in our environment, especially in plants, the presence of their mutagenicities after nitrite treatment warrants further studies, including those on their in vivo carcinogenicities.

Alkaloids↗

PubMed1994

Influence of pH, salt and nitrite on the heme-dependent catalase activity of lactic acid bacteria.

A screening of commercial starter cultures used for the production of dry sausage showed a maximum heme-dependent catalase activity in the range of 60 mumol/l hematin for Lactobacillus sake, Lactobacillus plantarum, Lactobacillus pentosus and Pediococcus acidilactici. Pseudocatalase activity was not detected. In standard dry sausage production, 2-3% (w/w) nitrite salt (0.6% sodium nitrite per 100 g NaCl) is normally added, which corresponds to 4-6% salt in the water phase. In vitro experiments with L. sake and L. plantarum have shown that such a high concentration of salt caused a significant reduction of catalase activity and bacterial growth. In the case of P. acidilactici, the catalase activity remained constant at a salt concentration up to 6% (w/w); at 7% (w/w) the activity decreased sharply. The pH also affected the catalase activity, which remained constant up to pH 5.1 and decreased dramatically at lower values. The effect of nitrite has also been investigated. L. pentosus and P. acidilactici were not affected by the addition of 160 ppm nitrite (NO2-); L. plantarum, on the other hand, showed a significantly reduced catalase activity. In practice, optimum fermentation characteristics combined with an optimum catalase activity which are not inhibited by salt concentrations higher than 6% (w/v) and a residual nitrite content of about 160 ppm (w/v), are of the utmost importance in screening and selection of lactic acid bacteria for starter cultures.

Catalase↗

PubMed1983

The formation of nitrite and N-nitroso compounds in salivas in vitro and in vivo.

The considerable differences in salivary nitrite levels observed previously in subjects who had abstained overnight from dietary nitrate as completely as possible were again found in tests on ten separate days. A 25-fold variation was observed in the rate of production of nitrite in vitro during the incubation of salivas from different subjects with 16 mM-nitrate under standardized conditions. N-Nitroso compounds were detected as a group on 11/100 and 14/100 occasions after incubations in vitro without and with added nitrate, respectively. No significant changes in salivary nitrite level were found 1 hr after the volunteers had ingested water-borne nitrate at approximately the World Health Organization recommended limit for continuous use (50 mg NO-3/litre), but increases in nitrite concentration were consistently found after volunteers ingested nitrate in water at four times this concentration. Although the nitrite concentrations were markedly increased following the intake of nitrate at the higher level, the occurrence of N-nitroso compounds in the salivas of the volunteers was greater immediately prior to the ingestion of water-borne nitrate than 1 hr afterwards.

Female↗

PubMed1985

Excretion of nitrates and nitrites in saliva and bile in the dog.

The kinetics of salivary and biliary excretion of inorganic nitrates and nitrites was studied in mongrel dogs after iv injection of these compounds labelled with 15N. There was strong salivary excretion of 15NO-3 after injection of Na15NO3 but little or no nitrites were excreted in the saliva, indicating that the nitrites present in the mouth are mainly the result of in situ transformation of nitrates into nitrites. In the bile, large quantities of nitrates were excreted when the dogs received 15NO-2, indicating endogenous oxidation of the nitrite.

Animals↗

PubMed1988

Interaction of nitrite with catalase in the perfused rat liver.

The interaction of nitrite with catalase was investigated spectrophotometrically in the perfused rat liver. Real-time spectral changes were obtained using a reflectance scanning spectrophotometer in the liver perfused with a haemoglobin-free medium in a non-recirculating system. Administration of sodium nitrite caused a specific pattern of spectral change indicating the decomposition of catalase compound I to free catalase. The spectral change due to the interaction with nitrite did not occur during potassium cyanide or ethanol infusion, nor in the aminotriazole-pretreated rat liver. The spectral change was observed at concentrations of nitrite in the perfusate over 0.01 mM, and the K0.5 value (the concentration producing half the maximum spectral change) was 0.06 mM. It was concluded that relatively low concentrations of nitrite caused decomposition of catalase compound I in the physiologically functioning liver cell.

Animals↗

PubMed1977

Effect of nitrite upon leghemoglobin and interaction with nitrogen fixation.

Nitrite (0.4 mM) added to soybean bacteroid preparations strongly inhibited C2H2 reduction. In the presence of leghemoglobin (0.1mM), a 3-fold enhancement of nitrogen fixation occurred but the inhibitory effect of nitrite was delayed. Spectra of leghemoglobin showed a rapid disappearance of the 574 nm and 541 nm peaks of oxyleghemoglobin the presence of nitrite. Concomitant oxidation of this hemoprotein gave ferric leghemoglobin as the single final product. High nitrite levels could depress nitrogen fixation both by inactivation of nitrogenase and by conversion of leghemoglobin into an inactive form. Nitrite present at low concentrations reacts with this hemoprotein and is then no longer able to penetrate into bacteroids.

Hemeproteins↗

PubMed1983

Effect of intra-pregastric administration of nitrite on erythrocyte GSH and GSSG contents of male golden hamster (Mesocricetus auratus).

Effect of single dose of nitrite on the formation of methaemoglobin, the erythrocyte content and redox state of glutathione of male golden hamster were investigated up to 20 min. The marked depletion of GSH following the oxidation of haemoglobin was observed in nitrite treated animals. Conversely, the GSSG contents of animals given 20 and 25 mg of nitrite were slightly higher than that of normal but not difference in total glutathione. The respective difference of the redox electrode potential between saline and nitrite treated animals was -84 mV at 20 mg and -125 mV at 25 mg of nitrite 20 min after administration.

Animals↗

PubMed1993

Rapid and simple method for the determination of nitrite in synaptosomal superfusates by liquid chromatography with electrochemical detection.

A rapid and simple technique using reversed-phase high-performance liquid chromatography with electrochemical detection (HPLC-ED) was developed for the determination of nitrite. Standard solutions of sodium nitrite produced a linear current response over the concentration range 1.5-30 microM. The chromatographic peaks of sodium nitrite were superimposable with peaks obtained after injection into the chromatograph of supernatants from rat cerebellar synaptosomes. The concentrations of nitrite in synaptosomes measured with HPLC-ED were similar to those found with a widely used colorimetric procedure. Therefore, this new method may represent a useful means of measuring nitrite in biological samples.

Animals↗

PubMed1993

Application of a leukocyte and nitrite urine test strip to the management of children with neurogenic bladder.

A urine leukocyte count of > or = 50/mm3 together with a bacterial count of > or = 10(5) colony-forming units (CFUs) per milliliter was used to define significant infection in 160 children with neurogenic bladder and evaluate the leukocyte and nitrite components of the Chemstrip 9 test. A Chemstrip 9 leukocyte reading of < or = 25 together with a negative nitrite reaction occurred in 99 children and had a sensitivity of 83.5% and a negative predictive value for infection of 97.0%. A Chemstrip 9 reading of > or = 500 leukocytes together with a positive nitrite reaction occurred in 18 children and had a sensitivity of 40% with a 100% positive predictive value for infection. Other combinations of Chemstrip 9 leukocyte and nitrite reactions were unhelpful or of uncertain value. Selection of up to three specimens from each patient increased the number of samples to 360 and provided general confirmation of the above conclusions. Nitrofurantoin may reduce the sensitivity of the nitrite strip reaction.

Child↗