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PubMed2001

Lack of association between CSF nitrate and sera nitrate in falciparum malaria infection.

Nitrate levels in CSF and sera from 16 coma and 19 noncoma falciparum malaria patients were determined using nitric oxide colorometric assay. The medians (range lower, upper limits) of nitrate in sera of comatose and noncomatose patients were 0.28 (0.11, 1.24) and 0.23 (0.05, 0.87) microM, respectively. The medians of nitrate level in CSF of coma and noncoma cases were 0.09 (0.01, 0.28) and 0.15 (0, 1.18) microM, respectively. There was no difference of nitrate level in sera and CSF from comatose or noncomatose patients compared to that in normal sera and CSF. The amount of nitrate in sera and CSF of both groups was not significantly correlated with coma depth, parasitemia, parasite clearance time and time to recovery. Contrast to our in vitro study using immunoperoxidase staining, we found inducible nitric oside synthase production by brain endothelial cells during 4-24 hours of coculturing with late stage of P. falciparum infected red blood cells. These results suggests that malaria severity can not be differentiated by nitrate level in body fluid.

Adolescent↗
PubMed1998

Engineering of pyridine nucleotide specificity of nitrate reductase: mutagenesis of recombinant cytochrome b reductase fragment of Neurospora crassa NADPH:Nitrate reductase.

The cytochrome b reductase fragment of Neurospora crassa NADPH:nitrate reductase (EC 1.6.6.3) was overexpressed in Escherichia coli with a His-tag for purification after mutation of the NADPH binding site. The recombinant enzyme fragment was altered by site-directed mutagenesis guided by the three-dimensional structure of cytochrome b reductase fragment of corn NADH:nitrate reductase (EC 1.6.6.1). Substitution of Asp for Ser920 (using residue numbering for holo-NADPH:nitrate reductase of N. crassa) greatly increased preference for NADH. This mutant had nearly the same NADH:ferricyanide reductase kcat as wild-type with NADPH. Substitutions for Arg921 had little influence on coenzyme specificity, while substitution of Ser or Gln for Arg932 did. The cytochrome b reductase mutant with greatest preference for NADH over NADPH was the doubly substituted form, Asp for Ser920/Ser for Arg932, but it had low activity and low affinity for coenzymes, which indicated a general loss of specificity in the binding site. Steady-state kinetic constants were determined for wild type and mutants with NADPH and NADH. Wild type had a specificity ratio of 1100, which was defined as the catalytic efficiency (kcat/Km) for NADPH divided by catalytic efficiency for NADH, while Asp for Ser920 mutant had a ratio of 0.17. Thus, the specificity ratio was reversed by over 6000-fold by a single mutation. Preference for NADPH versus NADH is strongly influenced by presence/absence of a negatively charged amino acid side chain in the binding site for the 2' phosphate of NADPH in nitrate reductase, which may partially account for existence of bispecific NAD(P)H:nitrate reductases (EC 1.6.6.2).

Amino Acid Sequence↗
PubMed2003

Characterisation and expression analysis of a nitrate transporter and nitrite reductase genes, two members of a gene cluster for nitrate assimilation from the symbiotic basidiomycete Hebeloma cylindrosporum.

Symbiotic ectomycorrhizal fungi contribute to the nitrogen nutrition of their host-plants but little information is available on the molecular control of their nitrogen metabolism. We cloned and characterised genes encoding a nitrite reductase and a nitrate transporter in the ectomycorrhizal basidiomycete Hebeloma cylindrosporum. These two genes are divergently transcribed and linked to a previously cloned nitrate reductase gene, thus demonstrating that nitrate assimilation gene clusters occur in homobasidiomycetes. The nitrate transporter polypeptide (NRT2) is characterised by 12 transmembrane domains and presents both a long putative intracellular loop and a short C-terminal tail, two structural features which distinguish fungal high-affinity transporters from their plant homologues. In different wild-type genetic backgrounds, transcription of the two genes was repressed by ammonium and was strongly stimulated not only in the presence of nitrate but also in the presence of organic nitrogen sources or under nitrogen deficiency.

Anion Transport Proteins↗
PubMed1984

A rapid method for the assay of nitrate in urine using the nitrate reductase enzyme of Escherichia coli.

A method was developed for urinary nitrate analysis utilizing an enzyme of Escherichia coli for the reduction of nitrate to nitrite. The resulting nitrite was assayed by a standard diazotization procedure. Under the experimental conditions stoichiometric conversion of nitrate to nitrite was achieved. Crude enzyme present in bacterial suspensions was used without any initial purification and no prior treatment of the urine samples was necessary. The bacteria were cultured under conditions producing high nitrate reductase activity and used formate as an exogenous electron donor without demonstrating any nitrite reductase activity. The procedure was subsequently automated to produce rapid, simultaneous determination of urinary nitrate and nitrite at the rate of 45 analyses/hr.

Autoanalysis↗
PubMed2005

Electrochemical determination of nitrate with nitrate reductase-immobilized electrodes under ambient air.

Nitrate monitoring biosensors were prepared by immobilizing nitrate reductase derived from yeast on a glassy carbon electrode (GCE, d = 3 mm) or screen-printed carbon paste electrode (SPCE, d = 3 mm) using a polymer (poly(vinyl alcohol)) entrapment method. The sensor could directly determine the nitrate in an unpurged aqueous solution with the aid of an appropriate oxygen scavenger: the nitrate reduction reaction driven by the enzyme and an electron-transfer mediator, methyl viologen, at -0.85 V (GCE vs Ag/AgCl) or at -0.90 V (SPCE vs Ag/AgCl) exhibited no oxygen interference in a sulfite-added solution. The electroanalytical properties of optimized biosensors were measured: the sensitivity, linear response range, and detection limit of the sensors based on GCE were 7.3 nA/microM, 15-300 microM (r2 = 0.995), and 4.1 microM (S/N = 3), respectively, and those of SPCE were 5.5 nA/microM, 15-250 microM (r2 = 0.996), and 5.5 microM (S/N = 3), respectively. The disposable SPCE-based biosensor with a built-in well- or capillary-type sample cell provided high sensor-to-sensor reproducibility (RSD < 3.4% below 250 microM) and could be used more than one month in normal room-temperature storage condition. The utility of the proposed sensor system was demonstrated by determining nitrate in real samples.

Air↗
PubMed1981

[The problem of nitrate and nitrite in human food. II. The occurrence of nitrate, nitrite, and thiocyanate in human saliva (author's transl)].

When the alimentary nitrate intake is in the range of 50 mg NO3- the nitrate values in saliva of adults and children follow a Gaussian normal distribution. With high and very high nitrate intakes the individual fluctuations of nitrate in saliva are distributed randomly in a wide range with a factor of 1:4. The nitrite values in saliva fluctuate even more, so that a proper judgment should be made only in individual cases. The average content of thiocyanate in saliva of adults was found to be 134 ppm SCN-, the saliva of children contained 50 ppm SCN-. The estimated molecular ratio of thiocyanate to nitrite in saliva of adults and children may fluctuate between 8:1 and 1:1 depending on the nitrate content of food.

Adult↗
PubMed1988

Protein-related differences in the excretion of nitrosoproline and nitrate by the rat--possible modification of de novo nitrate synthesis.

Male Ola:SD rats were fed purified diets containing 5 or 20% lactalbumin as the source of protein, and the daily urinary excretion of nitrate and nitrosoproline was measured. Animals fed the high-protein diet consistently excreted more nitrate and nitrosoproline than littermates fed the low-protein ration, despite a similar, negligible amount of nitrate in both diets. Furthermore, whereas nitrite administration enhanced nitrosoproline excretion in both diet groups, nitrate administration increased nitrosamine output in the low-protein animals but did not affect nitrosation by rats given the 20% lactalbumin ration. Animals fed the 5% lactalbumin diet produced a smaller volume of urine than did the 20% diet group but other measurements of renal function were comparable for both treatments. The results suggest differences in endogenous nitrosation between rats fed diets high or marginal in protein, possibly reflecting decreased nitrate synthesis in the low-protein group.

Animals↗
PubMed2003

Enzymatic nitration of phytophenolics: evidence for peroxynitrite-independent nitration of plant secondary metabolites.

Peroxynitrite (ONOO(-)), a reactive nitrogen species, is capable of nitrating tyrosine residue of proteins. Here we show in vitro evidence that plant phenolic compounds can also be nitrated by an ONOO(-)-independent mechanism. In the presence of NaNO(2), H(2)O(2), and horseradish peroxidase (HRP), monophenolic p-coumaric acid (p-CA, 4-hydroxycinnamic acid) was nitrated to form 4-hydroxy-3-nitrocinnamic acid. The reaction was completely inhibited by KCN, an inhibitor for HRP. The antioxidant ascorbate suppressed p-CA nitration and its suppression time depended strongly on ascorbate concentration. We conclude that nitrogen dioxide radical (NO(2)(radical)), but not ONOO(-), produced by a guaiacol peroxidase is the intermediate for phytophenolic nitration.

Antioxidants↗
PubMed1995

Plasma nitrate concentration and urinary nitrate excretion in patients with gastroenteritis.

The concentration of nitrate (the stable oxidation product of nitric oxide) in plasma and its excretion in urine was measured in 20 patients with a diagnosis of gastroenteritis. On day 1 of the illness plasma nitrate concentration was significantly elevated compared with a healthy control population (92.7 +/- 17.0 mumol/l vs. 33.1 +/- 1.6 mumol/l; P < 0.001) and continued to be elevated on days 2 and 3. Urinary nitrate excretion was also elevated. The plasma nitrate concentration correlated with disease severity as assessed by stool frequency and plasma urea concentration. Plasma nitrate concentration may be a sensitive and clinically useful indicator of severity of gastroenteritis.

Adult↗
PubMed1999

Interference by S-nitroso compounds with the measurement of nitrate in methods requiring reduction of nitrate to nitrite by cadmium.

Various methods suited for the measurement of nitrate require its reduction to nitrite by cadmium under acidic or alkaline conditions. N(G)-Nitroarginine analogs have been shown to interfere with the measurement of nitrate by such assays. In the present work we show by gas chromatography-mass spectrometry that under alkaline reduction conditions the S-nitroso compounds S-nitrosoglutathione and S-nitrosohomocysteine but not S-nitroso-N-acetylcysteine and S-nitroso-N-acetylpenicillamine can considerably contribute to nitrate and thus interfere with its measurement. Our results suggest that S-nitroso compounds may interfere with the measurement of nitrate in methods requiring cadmium-catalyzed reduction of nitrate to nitrite.

Cadmium↗
PubMed2002

An accurate and stable nitrate-selective electrode for the in situ determination of nitrate in agricultural drainage waters.

A field evaluation of a novel nitrate-ion selective electrode (ISE) was undertaken by continuous immersion over a period of 5 months in agricultural drainage weirs. The nitrate sensor N,N,N-triallyl leucine betaine was covalently attached to polystyrene-block-polybutadiene-block-polystyrene (SBS) using a free radical initiated co-polymerisation, to produce a rubbery membrane which was incorporated into a commercially available electrode body. A measurement unit was constructed comprising the nitrate-ISEs, a reference electrode and a temperature probe connected through a pre-amplifier to a data-logger and battery supply. A temperature correction algorithm was developed to accomodate the temperature changes encountered in the drainage weirs. The nitrate results obtained with the ISEs at hourly intervals compared very favourably (R2 = 0.99) with those obtained with laboratory automated chemical determinations made on contemporaneous samples of drainage in a concentration range 0.47-16 ppm nitrate-N. The ISEs did not require re-calibration and no deterioration in performance or fouling of the membrane surface was observed over four months of deployment.

Electrodes↗
PubMed2004

Effect of low molecular size humic substances on nitrate uptake and expression of genes involved in nitrate transport in maize (Zea mays L.).

In this study, a detailed characterization of earthworm low molecular size humic substances (LMS) was performed and these substances were used to study their effect on the nitrate influx in roots, tissue nitrate content, and expression of maize genes putatively involved in nitrate uptake in maize (Zea mays L.). The results show that the humic fraction with low molecular size used in this study is endowed with the characteristic structural network described for most humic substances so far isolated and confirm the presence of IAA in this fraction. The results also show that the LMS fraction of humic substances stimulates the uptake of nitrate by roots and the accumulation of the anion at the leaf level. Moreover, the analysis of the expression of genes encoding two putative maize nitrate transporters (ZmNrt2.1 and ZmNrt1.1) and of two maize H(+)-ATPase isoforms (Mha1 and Mha2) show that these substances may exert direct effects on gene transcription in roots, as shown for the Mha2 gene, and long-distance effects in shoots, as observed for the ZmNrt2.1 gene.

Actins↗
PubMed2001

Numerical analysis of the transport and fate of nitrate in the soil and nitrate leaching to drains.

In this study, the transport and fate of nitrate within the soil profile and nitrate leaching to drains were analyzed by comparing historic field data with the simulation results of the DRAINMOD model. The nitrogen version of DRAINMOD was used to simulate the performance of the nitrogen transport and transformation of the Hooibeekhoeve experiment, situated in the sandy region of the Kempen (Belgium) and conducted for a 30-year (1969-1998) period. In the analysis, a continuous cropping with maize was assumed. Comparisons between experimentally measured and simulated state variables indicate that the nitrate concentrations in the soil and nitrate leaching to drains are controlled by the fertilizer practice, the initial conditions, and the rainfall depth and distribution. Furthermore, the study reveals that the model used gives a fair description of the nitrogen dynamics in the soil and subsurface drainage at field scale. From the comparative analysis between experimental data and simulation results it can also be concluded that the model after calibration is a useful tool to optimize as a function of the combination "climate-crop-soil-bottom boundary condition" the nitrogen application strategy resulting in an acceptable level of nitrate leaching for the environment.

Agriculture↗
PubMed2002

Scandium(III) trifluoromethanesulfonate catalyzed aromatic nitration with inorganic nitrates and acetic anhydride.

The rare earth metal(III) trifluoromethanesulfonate (rare earth metal(III) triflate, RE(OTf)3) was found to be an efficient catalyst for aromatic nitration with carboxylic anhydride-inorganic nitrate as the nitrating agent. In the presence of a catalytic amount of RE(OTf)3, the nitration of substituted benzenes proceeded to afford the corresponding nitrobenzenes. Especially, scandium(III) trifluoromethanesulfonate (scandium(III) triflate, Sc(OTf)3) is the most active catalyst among our tested Lewis acids. It was also found that acetic anhydride-Al(NO3).9H2O is the most active nitrating agent in this system.

Acetic Anhydrides↗
PubMedDate not supplied

[Dynamics of methemoglobin levels in the blood of calves in relation to the ingested quantity of nitrates and the transrenal transport of nitrates].

Changes of methaemoglobin levels were investigated in the blood of suckling calves. Transrenal passage of nitrates was determined in dependence on the ingested amount of nitrates. The experiments were conducted under defined husbandry conditions; no excessive nitrate and nitrite supplementation of the calves by feeds and water could be stated. Imitation of possible field conditions when mainly water, but feeds too, may contain higher nitrate and nitrite levels, was carried out by peroral administration of an aquaeous solution of KNO3 to calves. The administered dose was increased from one to 2, 5 and 10 g per animal and day, respectively, in weekly intervals. MtHb determination in the blood of experimental calves on day 1 and 5 of the administration of 1 g KNO3 revealed no significant values. On day 1 and 5 of the administration of 2, 5 and 10 g KNO3 per animal and day, respectively, a significant increase of MtHb levels in the blood of calves was observed 2 and 3 hours after administration, followed by a decrease 4 hours after administration. The maximum values of MtHb in the blood of experimental calves, observed 3 hours following application of the respective KNO3 dose, were within the tolerance limits of the reference values. In urine, 3 hours after the administration of 10 g of KNO3 a mean nitrate value of 941.40.

Age Factors↗
PubMedDate not supplied

A combination of ion exchange and electrochemical reduction for nitrate removal from drinking water. Part I: nitrate removal using a selective anion exchanger in the bicarbonate form with reuse of the regenerant solution.

The process of selective nitrate removal from drinking water by means of ion exchange was studied. A commercial strong base anion exchanger with triethylammonium (-N+Et3) functional groups was used in the bicarbonate (HCO3-) and carbonate (CO3(2-)) form. The aim of this study was to optimize ion-exchanger regeneration in view of the subsequent electrochemical reduction of nitrates in the spent regenerant solution. The effects of ion-exchanger form, concentration of regenerant solution, and presence of nitrates, chlorides, and sulphates in the regenerant solution were studied. The strong base anion exchanger in HCO3- form that was investigated was able to treat 270 bed volumes of model water solution containing 124 mg dm(-3) nitrates. To achieve adequate regeneration of the saturated anion exchanger, it is necessary to use approximately 30 bed volumes of fresh 1-M sodium bicarbonate (NaHCO3) regenerant solution. The presence of residual 50-mg dm(-3) nitrates in the regenerant solution, treated by electrolysis, resulted in an increase in the dose of regenerant solution to 35 bed volumes and a decrease in the subsequent sorption run of approximately 13%. The volume of applied regenerant solution was high, but the consumption of NaHCO3 for regeneration was low.

Anions↗
PubMed1990

[Nitrates and nitrites and the utilization of beta-carotene. II. Determining the threshold of the tolerated dose of nitrates and nitrites in the diet of rats receiving beta-carotene].

The limit levels of potassium nitrate and sodium nitrite were determined in the diet of rats receiving orally beta-carotene. Two 30-day biological experiments were done on male Wistar rats standardized for vitamin A level in the liver. The animals were given semisynthetic diet, without vitamin A, containing definite amounts of potassium nitrate or sodium nitrite: 4.0; 4.5; 5.0; 5.5; 6.0% of the nitrate, and 0.5; 0.6; 0.7; 0.8; 0.9; 1.0% of the nitrite. The rats were given also orally two drops of an oil solution of beta-carotene in doses of 30 micrograms per rat daily. The limit of tolerance was established measuring beta-carotene utilization according to the method of balance studies and liver function test. Diet consumption and body weight of the animals were considered as well. It was found that in this experiment the limit dose, that is the dose of nitrates and nitrites over which the rats died, was 5.0% for nitrate and 0.7% for nitrite.

Animal Feed↗
PubMed1988

Oxygen inhibition of nitrate uptake is a general regulatory mechanism in nitrate respiration.

It has recently been demonstrated that oxygen inhibits nitrate uptake by denitrifying Pseudomonas aeruginosa. The purpose of the present investigation was to determine if this novel mechanism of regulation is universal for the regulation of nitrate respiration in other widely divergent species of bacteria. Nitrate transport by whole cell suspensions was completely and reversibly inhibited in 11 out of 12 species tested, whereas nitrate reduction by cell-free extracts was not affected by oxygen or was only partially inhibited in some cases. These results indicate that oxygen inhibition of nitrate uptake is a general regulatory phenomenon.

Enterobacter↗