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

For the quantification of nitrite and nitrate, the stable metabolites of -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-μl 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., μmol/mmol creatinine) with a mean 24-h output of 5.7 μmol for nitrite and 1226 μmol for nitrate. The concentrations of nitrite and nitrate in the plasma of these volunteers were determined to be (mean ± S.D., μmol/l) 3.6 ± 0.8 and 68 ± 17, respectively.     

Production of succinate by a <Emphasis Type="Italic">pfl</Emphasis>B <Emphasis Type="Italic">ldh</Emphasis>A double mutant of <Emphasis Type="Italic">Escherichia coli</Emphasis> overexpressing malate dehydrogenase

The gene encoding malate dehydrogenase (MDH) was overexpressed in a pflB ldhA double mutant of Escherichia coli, NZN111, for succinic acid production. With MDH overexpression, NZN111/pTrc99A-mdh restored the ability to metabolize glucose anaerobically and 0.55 g/L of succinic acid was produced from 3 g/L of glucose in shake flask culture. When supplied with 10 g/L of sodium bicarbonate (NaHCO₃), the succinic acid yield of NZN111/pTrc99A-mdh reached 1.14 mol/mol glucose. Supply of NaHCO₃ also improved succinic acid production by the control strain, NZN111/pTrc99A. Measurement of key enzymes activities revealed that phosphoenolpyruvate (PEP) carboxykinase and PEP carboxylase in addition to MDH played important roles. Two-stage culture of NZN111/pTrc99A-mdh was carried out in a 5-L bioreactor and 12.2 g/L of succinic acid were produced from 15.6 g/L of glucose. Fed-batch culture was also performed, and the succinic acid concentration reached 31.9 g/L with a yield of 1.19 mol/mol glucose.     

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 (NO₂^-) 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 μmol/kg over 15 min and then 0.28 μmol/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 μM, 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.     

Increased NADPH availability in <Emphasis Type="Italic">Escherichia coli</Emphasis>: improvement of the product per glucose ratio in reductive whole-cell biotransformation

A basic requirement for the efficiency of reductive whole-cell biotransformations is the reducing capacity of the host. Here, the pentose phosphate pathway (PPP) was applied for NADPH regeneration with glucose as the electron-donating co-substrate using Escherichia coli as host. Reduction of the prochiral β-keto ester methyl acetoacetate to the chiral hydroxy ester (R)-methyl 3-hydroxybutyrate (MHB) served as a model reaction, catalyzed by an R-specific alcohol dehydrogenase. The main focus was maximization of the reduced product per glucose yield of this pathway-coupled cofactor regeneration with resting cells. With a strain lacking the phosphoglucose isomerase, the yield of the reference strain was increased from 2.44 to 3.78 mol MHB/mol glucose. Even higher yields were obtained with strains lacking either phosphofructokinase I (4.79 mol MHB/mol glucose) or phosphofructokinase I and II (5.46 mol MHB/mol glucose). These results persuasively demonstrate the potential of NADPH generation by the PPP in whole-cell biotransformations.     

Anaerobic energy metabolism of the sulfur-reducing bacterium “Spirillum” 5175 during dissimilatory nitrate reduction to ammonia

In a batch culture experiment the microaerophilic Campylobacter-like bacterium “Spirillum” 5175 derived its energy for growth from the reduction of nitrate to nitrite and nitrite to ammonia. Hereby, formate served as electron donor, acetate as carbon source, and l-cysteine as sulfur source. Nitrite was quantitatively accumulated in the medium during the reduction of nitrate; reduction of nitrite began only after nitrate was exhausted from the medium. The molar growth yield per mol formate consumed, Y_m, was 2.4g/mol for the reduction of nitrate to nitrite and 2.0 g/mol for the conversion of nitrite to ammonia. The gain of ATP per mol of oxidized formate was 20% higher for the reduction of nitrate to nitrite, compared to the reduction of nitrite to ammonia. With succinate as carbon source and nitrite as electron acceptor, Y_m was 3.2g/mol formate, i.e. 60% higher than with acetate as carbon source. No significant amount of nitrous oxide or dinitrogen was produced during growth with nitrate or nitrite both in the presence or absence of acetylene. No growth on nitrous oxide was found. The hexaheme c nitrite reductase of “Spirillum” 5175 was an inducible enzyme. It was present in cells cultivated with nitrate or nitrite as electron acceptor. It was absent in cells grown with fumarate, but appeared in high concentration in “Spirillum” 5175 grown on elemental sulfur. Furthermore, the dissimilatory enzymes nitrate reductase and hexaheme c nitrite reductase were localized in the periplasmic part of the cytoplasmic membrane.     

Biodegradation of 2,4,6-trinitrophenol (picric acid) in a biological aerated filter (BAF)

This study demonstrated the microbial purification of a model wastewater containing 2,4,6-trinitrophenol (TNP), which was carried out in a continuously working biological aerated filter (BAF). The main emphasis was on the operating performance of the reactor as a function of the pollution load. TNP was degraded at a maximum volumetric removal rate of 2.53 g TNP/L d, with low residual COD and TNP concentration. Overloading of TNP inhibited the nitrite-oxidizing activity, resulting in poor TNP degradation performance in the BAF system. The inhibition depended on some factors, such as influent concentrations and flow rates of the influent. It is assumed that nitrite-oxidizing occurred spontaneously during TNP degradation in the BAF system, could have significant influence on TNP wastewater treatment. One year after the reactor start-up, the dominance of Rhodococcus, which was initially inoculated in the reactor, was confirmed by analysis of 16S rDNA sequence of the PCR products separated by DGGE.     

The <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> species is abundant among hydrogen-producing facultative anaerobic bacteria in Lake Averno sediment

Lake Averno sediment was used to isolate the facultative anaerobic bacteria having the potential for H₂ production. Twenty-five out of 35 isolates recovered from the sediment sample produced hydrogen under anaerobic conditions from glucose with yields ranging from 0.1 to 0.49 mol H₂/mol glucose. Identification based on 16S rRNA gene sequence analysis revealed that most of them belong to the Firmicutes group, with a prevalence of the Paenibacillus polymyxa species. Seven distinct genomic fingerprints among the 11 P. polymyxa isolates were obtained using the random amplified polymorphic DNA (RAPD) technique. Glucose fermentation by P. polymyxa isolates was investigated. Glucose was totally consumed after 3 days of fermentation. The fermentation products were hydrogen (0.18–0.47 mol H₂/mol glucose), ethanol (0.1–0.5 mol ethanol/mol glucose), and 2,3-butanediol (0.1 mol 2,3-butanediol/mol glucose). Lower amounts of acetic, butyric, formic, lactic, and propionic acids were detected. All metabolic data concerning P. polymyxa isolates were analyzed by cluster analysis to reveal similarities and/or differences with clustering based on RAPD profiles. Despite the high metabolic similarity among almost all P. paenibacillus isolates, results of cluster analyses of metabolic and genetic data do not match completely.     

An efficient succinic acid production process in a metabolically engineered <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> strain

A Corynebacterium glutamicum strain (ΔldhA-pCRA717) that overexpresses the pyc gene encoding pyruvate carboxylase while simultaneously exhibiting a disrupted ldhA gene encoding l-lactate dehydrogenase was investigated in detail for succinic acid production. Succinic acid was shown to be efficiently produced at high-cell density under oxygen deprivation with intermittent addition of sodium bicarbonate and glucose. Succinic acid concentration reached 1.24 M (146 g l⁻¹) within 46 h. The yields of succinic acid and acetic acid from glucose were 1.40 mol mol⁻¹ (0.92 g g⁻¹) and 0.29 mol mol⁻¹ (0.10 g g⁻¹), respectively. The succinic acid production rate and yield depended on medium bicarbonate concentration rather than glucose concentration. Consumption of bicarbonate accompanied with succinic acid production implied that added bicarbonate was used for succinic acid synthesis.     

Flow‐injection determination of cysteine in pharmaceuticals based on luminol–persulphate chemiluminescence detection

A flow injection (FI) method is reported for the determination of l‐ cysteine, based on its enhancement on chemiluminescence (CL) emission of luminol oxidized by sodium persulphate in alkaline solution. The calibration graph was linear over the range 1.0 × 10^–9–5.0 × 10^–7 mol/L (r² = 0.9992), with relative standard deviations (RSDs) in the range 1.1–2.3% (n = 4). The limit of detection (3σ blank) was 5.0 × 10^–10 mol/L with a sample throughput of 120/h. The method was applied to pharmaceuticals and the results obtained were in reasonable agreement with the amount labelled. The proposed method was also applied to cysteine in synthetic amino acid mixtures. Calibration graphs of N‐acetylcysteine and glutathione over the range 1.0–50 × 10^–8 and 0.5–7.5 × 10^–7 mol/L were also established (r² = 0.998 and 0.9986) with RSDs in the range 1.0–2.0% (n = 4), and the limits of detection (3σ blank) were 5.0 × 10^–9 and 1.0 × 10^–8 mol/L, respectively. Copyright © 2008 John Wiley & Sons, Ltd.     

Free nitrous acid selectively inhibits and eliminates nitrite oxidizers from nitrifying sequencing batch reactor

In a complete nitrification sequencing batch reactor (CNSBR), where ammonium containing wastewater (200–1,000 mg N/L) is completely oxidized to nitrate up to 2.4 kg NH₄ ⁺–N/m³ d, both ammonia oxidizers and nitrite oxidizers were enriched in the sludge granules. Quantitative fluorescence in situ hybridization analyses of the sludge granules of the CNSBR showed that ammonia oxidizers and nitrite oxidizers occupied 31 and 4.2% of total bacteria, respectively. Most of the nitrite oxidizers were Nitrobacter species (95% of the nitrite oxidizers) and the remainder was Nitrospira species. The population of nitrite oxidizers was significantly higher than that of partial nitrification SBR (PNSBR) where most of the ammonium was oxidized to nitrite. The PNSBR had 37% (ammonia oxidizers) and 0.4% (nitrite oxidizers) of total bacteria. Comparative study with CNSBR and PNSBR revealed that free nitrous acid, rather than free ammonia, played a critical inhibition role to wash out nitrite oxidizers from the reactor. The concentrations of free ammonia and nitrite as well as free nitrous acid in the CNSBR selected Nitrobacter as the dominant nitrite oxidizers rather than Nitrospira.     

Asymmetric dimethylarginine in children with homocystinuria or phenylketonuria

Plasma concentration of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis from l-arginine and a cardiovascular risk factor, was found to be elevated in plasma of homocysteinemic adults. Enhanced cardiovascular risk due to homocystinuria and impaired renal function has been found in patients with phenylketonuria (PKU) on protein-restricted diet. However, it is still unknown whether ADMA synthesis is also elevated in children with homocystinuria due to cystathionine beta-synthase deficiency (classical homocystinuria), and whether ADMA may play a role in phenylketonuria in childhood. In the present study, we investigated the status of the l-arginine/NO pathway in six young patients with homocystinuria, in 52 young phenylketonuria patients on natural protein-restricted diet, and in age- and gender-matched healthy children serving as controls. ADMA in plasma and urine was determined by GC–MS/MS. The NO metabolites nitrate and nitrite in plasma and urine, and urinary dimethylamine (DMA), the dimethylarginine dimethylaminohydrolase (DDAH) metabolite of ADMA, were measured by GC–MS. Unlike urine ADMA excretion, plasma ADMA concentration in patients with homocystinuria was significantly higher than in controls (660 ± 158 vs. 475 ± 77 nM, P = 0.035). DMA excretion rate was considerably higher in children with homocystinuria as compared to controls (62.2 ± 24.5 vs. 6.5 ± 2.9 μmol/mmol creatinine, P = 0.068), indicating enhanced DDAH activity in this disease. In contrast and unexpectedly, phenylketonuria patients had significantly lower ADMA plasma concentrations compared to controls (512 ± 136 vs. 585 ± 125 nM, P = 0.009). Phenylketonuria patients and controls had similar l-arginine/ADMA molar ratios in plasma. Urinary nitrite excretion was significantly higher in phenylketonuria as compared to healthy controls (1.7 ± 1.7 vs. 0.7 ± 1.2 μmol/mmol creatinine, P = 0.003). Our study shows that the l-arginine/NO pathway is differently altered in children with phenylketonuria and homocystinuria. Analogous to hyperhomocysteinemic adults, elevated ADMA plasma concentrations could be a cardiovascular risk factor in children with homocystinuria. In phenylketonuria, the l-arginine/NO pathway seems not be altered. Delineation of the role of ADMA in childhood phenylketonuria and homocystinuria demands further investigation.     

Effect of nitrite on growth and microcystins production of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> PCC7806

As a common pollutant, nitrite concentrations can approach 15 mg NO₂⁻-N L⁻¹ in some aquatic systems. Microcystis aeruginosa blooms are common and widespread in eutrophic freshwater bodies. In this study, M. aeruginosa was exposed to nitrite concentrations ranging from 0 to 15 mg NO₂⁻-N L⁻¹, and the responses of M. aeruginosa were investigated. The specific growth rates, maximum cell densities, light-saturated photosynthetic rates (P_m ^chla ), dark respiration rates (R_d ^chla ), and apparent photosynthetic efficiencies (α^chla ) showed a significant decline with nitrite concentrations increasing. Electrical conductivity and malondialdehyde contents investigation revealed cell membrane damage and apparent leakage of intracellular contents under high nitrite level conditions due to oxidative stress enhancement. Intracellular microcystin (MC)-LR content reached the highest value at 10 mg NO₂⁻-N L⁻¹; however, extracellular MC-LR contents showed a continuous increase until 15 mg NO₂⁻-N L⁻¹ owing to the increasing leakage of intracellular contents. These results elucidated that the high-level nitrite inhibited M. aeruginosa growth by rising oxidative stress, damaging cell membrane, and reducing photosynthesis. However, the moderate increase in nitrite concentrations promoted toxin production and release of toxin.     

Substrate binding and activation in the active site of cytochrome <Emphasis Type="Italic">c</Emphasis> nitrite reductase: a density functional study

Cytochrome c nitrite reductase is a homodimeric enzyme, containing five covalently attached c-type hemes per subunit. Four of the heme irons are bishistidine-ligated, whereas the fifth, the active site of the protein, has an unusual lysine coordination and calcium site nearby. A fascinating feature of this enzyme is that the full six-electron reduction of the nitrite is achieved without release of any detectable reaction intermediate. Moreover, the enzyme is known to work over a wide pH range. Both findings suggest a unique flexibility of the active site in the complicated six-electron, seven-proton reduction process. In the present work, we employed density functional theory to study the energetics and kinetics of the initial stages of nitrite reduction. The possible role of second-sphere active-site amino acids as proton donors was investigated by taking different possible protonation states and geometrical conformations into account. It was found that the most probable proton donor is His₂₇₇, whose spatial orientation and fine-tuned acidity lead to energetically feasible, low-barrier protonation reactions. However, substrate protonation may also be accomplished by Arg₁₁₄. The calculated barriers for this pathway are only slightly higher than the experimentally determined value of 15.2 kcal/mol for the rate-limiting step. Hence, having proton-donating side chains of different acidity within the active site may increase the operational pH range of the enzyme. Interestingly, Tyr₂₁₈, which was proposed to play an important role in the overall mechanism, appears not to take part in the reaction during the initial stage.     

Changes in growth and photosynthetic patterns of oil palm (<Emphasis Type="Italic">Elaeis guineensis</Emphasis> Jacq.) seedlings exposed to short-term CO<Subscript>2</Subscript> enrichment in a closed top chamber

Three varieties of oil palm seedlings (Deli Yangambi, Deli Urt, Deli AVROS) were exposed to three levels of CO₂ (400, 800, 1,200 μmol/mol) in split plot design to determine growth (net assimilation rate, NAR; relative growth rate, RGR) and photosynthetic patterns of the seedlings under short-term CO₂ exposure of 15 weeks. Increasing CO₂ from 400 to 800 and 1,200 μmol/mol significantly enhanced total biomass and leaf area, net photosynthesis (A) and water use efficiency (WUE) especially from weeks 9 to 15. By the end of week 15, total biomass increased by 113%, and A and WUE by one- and fivefold, respectively, while specific leaf area decreased by 37%. Both enhanced biomass and A under elevated CO₂ were effective in modifying NAR and RGR as shown by high correlation coefficient values (r ² = 0.68 and 0.72; r ² = 0.63 and 0.67, respectively), although WUE seemed to have more influence over the NAR (r ² = 0.97) and RGR (r ² = 0.93). Neither interspecific preference nor its interaction with CO₂ imposed any significant effect on parameters observed. Growth improvement with CO₂ seemed able to produce healthy, bigger and vigorous oil palm seedlings, and the technique may have potential to be developed for use to reduce nursery period.     

Purification and Characterization of Albumin from Frog Skin of <Emphasis Type="Italic">Duttaphrynus melanostictus</Emphasis>

Following determination of trypsin inhibitory activity, a serine protease inhibitor was purified and characterized from frog Duttaphrynus melanostictus serum. It was identified as serum albumin, with molecular weight of 67 kDa (DmA-serum). Different from bovine serum albumin, DmA-serum potently inhibited trypsin with similar K _i values around 1.6 × 10⁻⁷ M. No inhibitory effect on thrombin, chymotrypsin, elastase and subtilisin was observed under the assay conditions. The N-terminal amino acid is EAEPHSRI. Subsequently, a protein with same N-terminal amino acid was purified from skin, termed as DmA-skin. However, DmA-skin is distinct from DmA-serum by binding of a haem b (0.5 mol/mol protein), and with low trypsin inhibitory activity. Frog albumin is distributed in frog skin and exhibited trypsin inhibitory activity, suggesting that it plays important roles in skin physiological functions, like water economy, metabolite exchange and osmoregulation, etc.     

Chemiluminescence method for the determination of piroxicam by the enhancement of the tris‐(4,7‐diphenyl‐1,10‐phenanthrolinedisulphonic acid) ruthenium(II) (RuBPS)–cerium(IV) system and its application

A simple, rapid chemiluminescence (CL) method was described for the determination of piroxicam, a commonly used analgesic agent drug. A strong CL signal was detected when cerium(IV) sulphate was injected into tris‐(4,7‐diphenyl‐1,10‐phenanthrolinedisulphonic acid) ruthenium(II) (RuBPS)–piroxicam solution. The CL signal was proportional to the concentration of piroxicam in the range 2.8 × 10^–8–1.2 × 10^–5 mol/L. The detection limit was 2 × 10^–8 mol/L and the relative standard deviation (RSD) was 3.7% (c = 7.0 × 10^–7 mol/L piroxicam; n = 11). The proposed method was applied to the determination of piroxicam in pharmaceutical preparations in capsules, spiked serum and urine samples with satisfactory results. Copyright © 2008 John Wiley & Sons, Ltd.     

Highly sensitive spectrofluorimetric determination of trace amounts of prulifloxacin using the aluminium(III)–prulifloxacin system

The fluorescence of the prulifloxacin (PUFX)–Al(III) system was investigated . Experiments indicated that the fluorescence intensity of prulifloxacin could be greatly enhanced by Al(III) and sensitized by sodium dodecylbenzene sulphonate (SDBS). Accordingly, a sensitive spectrofluorimetric method for the determination of prulifloxacin was established. While excited at 275 nm, the enhanced fluorescence intensity at 412 nm of the system (ΔF) showed a good linear relationship with the concentration of prulifloxacin within the range 4.0 × 10^–8–3.0 × 10^–6 mol/L. The regression equation was ΔF = 9.83 + 10.8 × 10⁷c (mol/L); the correlation coefficient and detection limit (3σ/k) were 0.99901 and 2.0 × 10^–8 mol/L, respectively. The proposed method has been successfully applied to determine prulifloxacin in real pharmaceutical samples. The luminescence mechanism of the system is also discussed in detail. Copyright © 2008 John Wiley & Sons, Ltd.     

A new method to measure nitrate/nitrite with a NO-sensitive electrode

There are different methods to measure the unstable moleculenitric oxide (NO). We will describe a new sensitive method to measure NO by reconversion of nitrate/nitrite to NO, which will bedetermined with an amperometric Clark-type electrode. Nitrate andnitrite are the degradation products of NO. First, nitrate isenzymatically converted to nitrite with the use of the nitrate reductase. Second, nitrite is reduced to equimolar NO concentrations byan acidic iodide solution. The detection limit of the electrode in anaqueous solution was 2 nmol/l NO (meaning the threshold was dependingon the volume added: 500 µl of a 0.2 µmol/l nitrite solution addedto a 10-ml bath). This method provides the ability to assess basal andagonist-stimulated NO releases of different biological models. Wemeasured basal and carbachol-stimulated NO release of nativeendothelial cells from porcine coronary arteries and porcine aorticendothelial cell cultures. Moreover, it was possible to measure thenitrate/nitrite concentration in the coronary effluent of a guinea pigheart. In conclusion, we present a valid, highly sensitive new methodof measuring nitrite/NO in biological systems with a commerciallyavailable electrode.