Gas chromatographic–tandem mass spectrometric quantification of human plasma and urinary nitrate after its reduction to nitrite and derivatization to the pentafluorobenzyl derivative

Gas chromatography–mass spectrometry (GC–MS) of nitrite as its pentafluorobenzyl derivative in the negative-ion chemical ionization mode is a useful analytical tool to quantify accurately and sensitively nitrite and nitrate after its reduction to nitrite in various biological fluids. In the present study we demonstrate the utility of GC–tandem MS to quantify nitrate in human plasma and urine. Our present results verify human plasma and urine levels of nitrite and nitrate measured previously by GC–MS.     

The effect of ammonium on assimilatory nitrate reduction in the haloarchaeon Haloferax mediterranei

Physiology, regulation and biochemical aspects of the nitrogen assimilation are well known in Prokarya or Eukarya but they are poorly described in Archaea domain. The haloarchaeon Haloferax mediterranei can use different nitrogen inorganic sources (NO₃⁻, NO₂⁻ or NH₄⁺) for growth. Different approaches were considered to study the effect of NH₄⁺ on nitrogen assimilation in Hfx. mediterranei cells grown in KNO₃ medium. The NH₄⁺ addition to KNO₃ medium caused a decrease of assimilatory nitrate (Nas) and nitrite reductases (NiR) activities. Similar effects were observed when nitrate-growing cells were transferred to NH₄⁺ media. Both activities increased when NH₄⁺ was removed from culture, showing that the negative effect of NH₄⁺ on this pathway is reversible. These results suggest that ammonium causes the inhibition of the assimilatory nitrate pathway, while nitrate exerts a positive effect. This pattern has been confirmed by RT-PCR. In the presence of both NO₃⁻ and NH₄⁺, NH₄⁺ was preferentially consumed, but NO₃⁻ uptake was not completely inhibited by NH₄⁺ at prolonged time scale. The addition of MSX to NH₄⁺ or NO₃⁻ cultures results in an increase of Nas and NiR activities, suggesting that NH₄⁺ assimilation, rather than NH₄⁺ per se, has a negative effect on assimilatory nitrate reduction in Hfx. mediterranei. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

施氮水平对高产麦田土壤硝态氮时空变化及氨挥发的影响

研究了不同施氮水平对高产麦田土壤硝态氮时空变化和氨挥发的影响.结果表明,高产麦田土壤硝态氮在播种至冬前阶段不断向深层移动,并在140cm以下土层积累.施纯氮96～168 kg·hm^-2处理,增加了60 cm以上土层土壤硝态氮含量,降低了土壤氮素表观损失量占施氮量的比例,提高了小麦籽粒蛋白质含量和籽粒产量,且土壤氨挥发损失较低,基施氮氨挥发损失占基施氮量的4.23%～5.51%;施氮量超过240 kg N·hm^-2,促进了土壤硝态氮向深层的移动和积累,基施氮氨挥发损失、土壤氮素表观损失量及其占施氮量的比例均显著升高,对小麦籽粒蛋白质含量无显著影响,但籽粒产量降低.高产麦田适宜的氮素用量为132～204 kg N·hm^-2.     

基质氮素对大球盖菇生长及亚硝酸盐含量的影响

目的探索氮源对大球盖菇生长及亚硝酸盐、硝酸盐含量的影响。方法在培养基中添加不同含氮化合物,培养菌丝,定时测定生长量,并采用重氮偶合分光光度法测定其亚硝酸盐、硝酸盐含量。结果添加亚硝酸盐的基质,菌丝生长速度比对照慢。尿素、硫酸铵、硝酸铵和硝酸钾四处理之间差异无显著性,其生长速度最快。各处理间亚硝酸盐、硝酸盐含量差异均存在非常显著性。亚硝酸钾处理,亚硝酸盐含量高达402.03 mg/kg,不宜食用。硝酸钾处理,亚硝酸盐含量为30.87 mg/kg,食用100 g,就会超出亚硝酸盐日限量;硝酸铵处理,硝酸盐含量远远超过日常蔬菜,也不宜食用。结论在栽培大球盖菇时氮源不宜使用亚硝酸盐、硝酸盐,应使用尿素或硫酸铵,有机氮源也可以。     

Simple and rapid method for the measurement of nitrite and nitrate in human plasma and cerebrospinal fluid by capillary electrophoresis

Nitrite and nitrate levels in physiological fluids are commonly used as an index of nitric oxide production. We developed simple and rapid method for the determination of these anions by capillary zone electrophoresis employing borate buffer (pH 10, 100 mmol/l) as running electrolyte. The anions were analyzed in plasma and cerebrospinal fluid (CSF) without deproteinization of the samples. Electrophoresis was carried out in a capillary (36.5 cm×75 μm) at a potential of 15 kV, with on-column UV detection at 214 nm. Mean retention times for nitrite and nitrates were 4.631 and 5.152 min, respectively. The method was linear (r=0.999) within a 1–500 μmol/l concentration range. Physiological levels of nitrate in plasma (40.2 μmol/l) and CSF (15.3 μmol/l) could be determined with good precision (coefficients of variation <6%) and accuracy (recoveries of added nitrate to plasma and CSF were 97.4 and 104.5%, respectively). Measurements of the physiological levels of nitrite in plasma (6.1 μmol/l) and CSF (0.9 μmol/l) were less precise and accurate.     

The influence of oxygen concentrations on denitrification in soil

Summary The influence of different O₂ concentrations on denitrification was studied in an agricultural soil. In both nitrate and nitrite amended soil, denitrification was not observed until the O₂ concentration decreased to 0.20 and 0.21 mol/ml, respectively. Denitrification was not observed in soil samples with O₂ concentrations above 0.28 mol/ml in the gas phase. These findings suggest that a completely anoxic environment is not required for denitrification to occur in soil.     

几种硝化抑制剂对土壤和小白菜硝酸盐含量及产量的影响

通过田间试验,对湖南长沙地区的红菜园土和冲积菜园土两种土壤条件下氢醌 (HQ)、双氰胺 (DCD)和硫脲 (TU) 3种硝化抑制剂对土壤和小白菜硝酸盐含量及小白菜产量的影响进行研究.结果表明,3种硝化抑制剂在试验的各个时期均不同程度地降低了土壤和小白菜的硝酸盐含量,其中以双氰胺的效果最好.但不同供试土壤条件下的试验结果不完全一致.3种硝化抑制剂均可不同程度提高小白菜产量,其中双氰胺效果最好,且在红菜园土的增产效果比冲积菜园土显著.     

Accumulation and reduction of nitrate in cereal plants dependent on N supply

Summary In pot experiments the NO ₃ ^– accumulation and the occurrence of nitrate reductase (NR) capacity of wheat plants were investigated depending on late N applications at tillering, shooting and heading. NO ₃ ^– is preferentially accumulated in the stems, while NR dominates in the leaves. NO ₃ ^– accumulation is enhanced by late N treatments especially if N supply at seeding is sufficient. NR capacity of the plants is stimulated by late nitrogen supply, but its increment rates decrease with increasing NO ₃ ^– accumulation.     

Inhibition by phenylglyoxal of nitrate transport in Paracoccus denitrificans: a comparison with the effect of a protonophorous uncoupler

The amino acid modifier phenylglyoxal (PG) gradually inactivated the methyl viologen-coupled nitrate reductase activity of the anoxically grown whole cells of Paracoccus denitrificans. A double log plot of the pseudo-first-order inactivation rate constant versus PG concentration was linear with a mean slope of 1.4 (0.1M sodium phosphate) or 0.87 (0.1M sodium borate). Phenylglyoxalation of cells lowered the limiting velocity (V), while hardly affecting the apparent half-saturation concentration (K(m)) of nitrate. Nitrate afforded no protection against inactivation. The inhibition by PG could be removed by the detergent Triton X-100 or by the lipid-soluble tetraphenylphosphonium countercation, suggesting that PG exerts its effect at the level of nitrate transport. Based on studies with membrane potential- and pH-sensitive fluorescent probes, the inhibition was shown not to be due to changes in the electrochemical gradient of hydrogen ions. Both K(m) and V values for nitrate uptake increased in a hyperbolic fashion in response to exogenously added nitrite. Nitrite promoted a bypass of the inhibition caused by low concentrations of the proton-conducting agent carbonyl cyanide m-chlorophenylhydrazone (CCCP), but was almost ineffective in the case of the PG block. These results are rationalized in terms of two nitrate import pathways that are comparably inhibited by PG and differ in their sensitivities to CCCP. A simplified kinetic model for phenylglyoxalation is proposed to account for the observed nonintegral reaction orders.     

秸秆在土壤内分解初期氮素矿化与固持的模拟测定

利用模拟软件Modelmaker对3种作物秸秆在土壤内分解初期氮素循环转化过程进行了模拟,取得了土壤铵态氮、硝态氮、微生物氮及其¹⁵N丰度等个变量模拟值和测定值的良好一致性.模型模拟对氮转化速率测定的结果表明,土壤微生物主要固持铵态氮,对硝态氮固持非常微弱.氮矿化主要发生于作物秸秆,腐殖质氮的矿化极其微弱.一级动力学方程对秸秆氮素矿化过程的描述优于零级动力学方程.微生物固持氮的再矿化过程落后于氮固持过程,假定再矿化不发生或认为再矿化与固持化同时进行可导致氮矿化与固持速率测定的严重误差.忽略氮硝化过程和挥发损失将导致氮矿化和固持速率的测定值偏低.净固持或净矿化的产生不仅与秸秆碳氮比有关,而且与秸秆在土壤内分解时间有关.     

The effect of an intermittent water-table gradient on soil and xylem nitrate in cotton

Summary Cotton (Gossypium hirsutum cv Deltapine 61) was grown in a sloping plot of soil in the field to examine the effect of a gradient of water-table depth on soil nitrate availability and plant uptake during two periods of the growing season. Before the water-table was imposed NO₃ was less concentrated at the lower end of the sloping plot. This was attributed to slow denitrification at microsites within the soil at the lower end which was wetter than further up the plot. At flooding NO₃ disappeared only slowly due to a carbon substrate limitation to denitrification in the soil. This loss occurred primarily in areas where the water-table was high and oxygen concentration in the soil solution was low. Plant NO₃ uptake, assessed by measuring the concentration in the xylem, parallelled the distribution of NO₃ in the soil solution. Under high water-tables xylem NO₃ levels fell but it was not possible to say whether this was due to impaired root function or to the reduced concentration of NO₃ observed in the soil solution. At intermediate water-table depths where soil NO₃ availability remained high xylem NO₃ concentration fell relative to the well drained control plants, suggesting that flooding had damaged the root system.Manufactured by Merck. Mention of commerical names does not imply endorsement by either CSIRO or USDA.     

Abiotic immobilization of nitrate in two soils of relic <Emphasis Type="Italic">Abies pinsapo</Emphasis>-fir forests under Mediterranean climate

Evidence for abiotic immobilization of nitrogen (N) in soil is accumulating, but remains controversial. Identifying the fate of N from atmospheric deposition is important for understanding the N cycle of forest ecosystems. We studied soils of two Abies pinsapo fir forests under Mediterranean climate seasonality in southern Spain—one with low N availability and the other with symptoms of N saturation. We hypothesized that biotic and abiotic immobilization of nitrate (NO₃ ⁻) would be lower in soils under these forests compared to more mesic temperate forests, and that the N saturated stand would have the lowest rates of NO₃ ⁻ immobilization. Live and autoclaved soils were incubated with added ¹⁵NO₃ ⁻ (10 μg N g⁻¹ dry soil; 99% enriched) for 24 h, and the label was recovered as total dissolved-N, NO₃ ⁻, ammonium (NH₄ ⁺), or dissolved organic-N (DON). To evaluate concerns about possible iron interference in analysis of NO₃ ⁻ concentrations, both flow injection analysis (FIA) and ion chromatography (IC) were applied to water extracts, soluble iron was measured in both water and salt extracts, and standard additions of NO₃ ⁻ to salt extracts were analyzed. Good agreement between FIA and IC analysis, low concentrations of soluble Fe, and 100% (±3%) recovery of NO₃ ⁻ standard additions all pointed to absence of an interference problem for NO₃ ⁻ quantification. On average, 85% of the added ¹⁵NO₃ ⁻ label was recovered as ¹⁵NO₃ ⁻, which supports our hypothesis that rates of immobilization were generally low in these soils. A small amount (mean = 0.06 μg N g⁻¹ dry soil) was recovered as ¹⁵NH₄ ⁺ in live soils and none in sterilized soils. Mean recovery as DO¹⁵N ranged from 0.6 to 1.5 μg N g⁻¹ dry soil, with no statistically significant effect of sterilization or soil type, indicating that this was an abiotic process that occurred at similar rates in both soils. These results demonstrate a detectable, but modest rate of abiotic immobilization of NO₃ ⁻ to DON, supporting our first hypothesis. These mineral soils may not have adequate carbon availability to support the regeneration of reducing microsites needed for high rates of NO₃ ⁻ reduction. Our second hypothesis regarding lower expected abiotic immobilization in soils from the N-saturated site was not supported. The rates of N deposition in this region may not be high enough to have swamped the capacity for soil NO₃ ⁻ immobilization, even in the stand showing some symptoms of N saturation. A growing body of evidence suggests that soil abiotic NO₃ ⁻ immobilization is common, but that rates are influenced by a combination of factors, including the presence of plentiful available carbon, reduced minerals in anaerobic microsites and adequate NO₃ ⁻ supply.     

Effect of changing the nanoscale environment on activity and stability of nitrate reductase

Nitrate reductase (NR) is employed for fabrication of nitrate sensing devices in which the enzyme in immobilized form is used to catalyze the conversion of nitrate to nitrite in the presence of a suitable cofactor. So far, instability of immobilized NR due to the use of inappropriate immobilization matrices has limited the practical applications of these devices. Present study is an attempt to improve the kinetic properties and stability of NR using nanoscale iron oxide (nFe₃O₄) and zinc oxide (nZnO) particles. The desired nanoparticles were synthesized, surface functionalized, characterized and affixed onto the epoxy resin to yield two nanocomposite supports (epoxy/nFe₃O₄ and epoxy/nZnO) for immobilizing NR. Epoxy/nFe₃O₄ and epoxy/nZnO support could load as much as 35.8 ± 0.01 and 33.20 ± 0.01 μg/cm² of NR with retention of about 93.72 ± 0.50 and 84.81 ± 0.80% of its initial activity respectively. Changes in surface morphology and chemical bonding structure of both the nanocomposite supports after addition of NR were confirmed by scanning electron microscopy (SEM) and fourier transform infrared spectroscopy (FTIR). Optimum working conditions of pH, temperature and substrate concentration were ascertained for free as well as immobilized NR preparations. Further, storage stability at 4 °C and thermal stability between 25–50 °C were determined for all the NR preparations. Analytical applications of immobilized NR for determination of soil and water nitrates along with reusability data has been included to make sure the usefulness of the procedure.     

Interspecies differences in the preference of ammonium and nitrate in vascular plants

Three solution experiments were performed to test the importance of NH ₄ ⁺ versus NO ₃ ^- +NH ₄ ⁺ to growth of 23 wild-forest and open-land species, using field-relevant soil solution concentrations at pH 4.5. At N concentrations of 1–200 M growth increased with increasing N supply in Carex pilulifera, Deschampsia flexuosa, Elymus caninus and Bromus benekenii. Geum urbanum was the most N demanding species and had little growth below 200 M. The preference for NH ₄ ⁺ or NO ₃ ^- +NH ₄ ⁺ was tested also at pH 4.0; no antagonism was found between NH ₄ ⁺ and H⁺, as indicated by similar relative growth in both of the N treatments at both pH levels. Growth in solution with NH ₄ ⁺ relative to NO ₃ ^- +NH ₄ ⁺ , 200 M, was negatively related to the mean pH of the field occurrence of the species tested; acid-tolerant species grew equally well with only NH ₄ ⁺ as with NO ₃ ^- +NH ₄ ⁺ (Oxalis acetosella, Carex pilulifera, Festuca gigantea, Poa nemoralis, Deschampsia flexuosa, Stellaria holostea, Rumex acetosella), while species of less acid soils were favoured by NO ₃ ^- +NH ₄ ⁺ (Urtica dioica, Ficaria verna, Melandrium rubrum, Aegopodium podagraria, Geum urbanum, Bromus benekenii, Sanguisorba minor, Melica ciliata, Silene rupestris, Viscaria vulgaris, Plantago lanceolata). Intermediate species were Convallaria majalis, Elymus caninus, Hordelymus europaeus and Milium effusum. No antagonism between NH ₄ ⁺ and Ca²⁺, Mg²⁺ and K⁺ was indicated by the total uptake of the elements during the experiment.     

Diverse effects of formate on the dissimilatory metabolism of nitrate in Pseudomonas denitrificans ATCC 13867: Growth,nitrite accumulation in culture,cellular activities of nitrate and nitrite reductases

The growth of Pseudomonas denitrificans ATCC 13867 under denitrifying conditions was significantly stimulated by adding an appropriate amount of formate (2.5 mM or above) to the growth medium. The accumulation of nitrite in the culture was markedly depressed so long as formate remained in the culture above a certain level. Cellular activities of enzymes participating in denitrification also changed. The cells grown in the presence of formate exhibited a lower nitrate reductase activity and, in contrast, a higher nitrite reductase activity than the cells grown without added formate.     

Influence of paddy and corn in different rotations on wheat yield,nutrient removal and soil properties

Summary In a 3 year field study, the effect of two rotations, paddy (Oryza sativa L.), wheat (Triticum aestivum L.), mung (Phaseolus aureus L.) and corn (Zea mays L.), wheat (Triticum aestivum L.), mung (Phaseolus aureus L.) on wheat yield and chemical, physical and biological properties of soil was evaluated. Grain yield of the wheat sown after paddy during 1972–73, 1973–74 and 1974–75 was lesser by 0,300 and 390 kg/ha respectively than that of the wheat following corn. The reduction in the yield of wheat was attributed to relatively higher NPK removal by paddy, greater immobilization of N applied to wheat and deterioration of soil physical conditions in the paddy field. Total NPK removal by paddy average 131 kg/ha more than that by corn. Sharp increase in bacterial population of soil during early growth of wheat that followed paddy and the concurrent less available N in soil and low N content in wheat plants suggested that the N applied to wheat was immobilised to organic form. The increase in soil bulk density and particle dispersion ratio and decrease in water storage in deeper soil layers in the paddy field probably restricted the root proliferation and growth of wheat.     

Influence of soil applications of nitrogen on nitrate reductase activity,leaf and grain protein content in sorghum

Summary Three sorghum genotypes, L.187, SK5912 and RCFA×L.187 (the latter being a hybrid) were field grown under four nitrogen application rates and replicated four times. The experiments were of complete randomized plot design and conducted in 1976, 1977 and 1978.Nitrate Reductase Activity (NRA) was measured at 5, 7, 9, 11, 13, and 15 weeks and at 18, 19, 20 and 21 weeks after planting in 4th vegetative leaf and flag leaf respectively. Flag leaf, 4th leaf and grain protein contents were also measured.Nitrogen application generally increased NRA in both 4th leaf and flag leaf in the 3 genotypes at all the sampling dates.NRA of the hybrid, RCFA×L.187 was consistently higher than those of SK5912 and L.187 (both straight varieties).NRA of 4th leaf was negatively correlated with leaf protein but flag leaf protein tended to increase with applied nitrogen.Flag leaf NRA was positively and significantly correlated with grain protein indicating an important part played by the flag leaf relative to protein accumulation of the developing grain.     

The effect of ammonium and nitrate nitrogen sources on copper uptake and amino acid status of cereals

Summary The effects of different nitrogen sources (NH₄, NO₃, and NH₄ NO₃) on the uptake of copper by wheat and barley growing in solution culture were compared in three experiments. Both the copper concentration and weight gain of shoots and roots were found to decrease in the order NO₃>NH₄ NO₃>NH₄ irrespective of the solution copper concentration. Ammonium nitrogen was also found to decrease the copper concentration of wheat grown on a copper deficient soil compared with a nitrate source of nitrogen. Increasing concentrations of ammonium ions in solution culture caused ammonium toxicity and reduced both plant copper concentrations and vegetative yield. Biochemical investigations using paper chromatography revealed that the amino acid asparagine was the major detoxification product of ammonia in wheat. Copper deficient plants were found to have elevated levels of amino acids compared with controls, irrespective of the nitrogen source.     

Effects of light quantity and quality and soil nitrogen status on nitrate reductase activity in rainforest species of the genus Piper

Summary We studied nitrate reductase (NR) activity in six species of the genus Piper (Piperaceae) growing under a broad range of light availabilities. Field measurements were made on plants growing naturally in rainforest at the Los Tuxtlas Tropical Biological Preserve, Veracruz, Mexico at high- and lowlight extremes for each species. Foliar nitrogen on an area basis was positively related to the average daily photosynthetically active photon flux density (PFD) received by the leaf (r=0.76, p<0.01). In vivo NR activity was highly correlated with PFD (r=0.95, p<0.001) and less so with total leaf nitrogen (r=0.68, p<0.05). In vivo NR activity was always higher in high-light plants than in low-light plants within a species. Similarly, gap species such as P. auritum had much higher in vivo NR activities than shade species such as P. aequale. Soil NO ₃ ^– and NH ₄ ⁺ pools and nitrogen-mineralization rates at Los Tuxtlas were similar between high- and low-light sites, indicating that the elevated NR activities in high-light plants were not the result of higher NO ₃ ^– availabilities in high-light microsites. We performed additional experiments at Stanford, California, USA on Piper plants grown at high- and low-light. Foliar NR was highly inducible by nitrate in the gap species (auritum) but not in the generalist (hispidum) or shade (aequale) species. Root NR activities were, in general, an order of magnitude lower than foliar activities. In total, these studies suggest that Piper gap species are inherently more competent to assimilate NO ₃ ^– and are better able to respond to sudden increases in NO ₃ ^– availability than are shade species.CJWDPB publication # 1097