Evaluation of methods for the extraction of nitrite and nitrate in biological fluids employing high-performance anion-exchange liquid chromatography for their determination

Measurements of nitrite (NO(2)(-)) and nitrate (NO(3)(-)) in biological fluids are proposed as indices of cellular nitric oxide (NO) production. Determination of NO(2)(-) and NO(3)(-) in standard solutions is not difficult, however, determinations which reflect accurately cellular NO synthesis represent a considerable analytical challenge. Problems are often encountered arising from background NO(2)(-)/NO(3)(-) contamination in experimental solutions and laboratory hardware, and with methods for sample extraction. We investigated potential procedures for the extraction and determination of NO(2)(-) and NO(3)(-) in biological samples. Consequently, a protocol was devised which yielded acceptable results regarding extraction efficiency, assay reproducibility, sample throughput and contaminant minimisation. It entailed rigorous washing of all equipment with water of low NO(2)(-) and NO(3)(-) content, sample deproteinisation by centrifugal ultrafiltration through a 3K filter and analysis by high-performance anion-exchange liquid chromatography with UV detection. Retention times for NO(2)(-) and NO(3)(-) in standards and plasma were 4.4 and 5.6 min, respectively. Assay linearity for standards ranged between 31 nM and 1 mM. The limit of detection for NO(2)(-) and NO(3)(-) in standards was 3 pmol. Recoveries of NO(2)(-) and NO(3)(-) from spiked plasma (1-100 microM KNO(2)/KNO(3)) and from extracted standards (1-250 microM) were approximately 100%. Intra-assay and inter-assay RSDs for NO(2)(-) and NO(3)(-) in spiked and unspiked plasma were 10.6% or less. Assays on washed platelet supernatants demonstrated collagen-induced platelet generation of NO products and analysis of murine and rat cardiac perfusates was achieved. Our procedure may be suitable for routine determination of NO(2)(-) and NO(3)(-) in various biological fluids, e.g., plasma.     

Nitrate in exhaled breath condensate of patients with different airway diseases.

There is an increasing interest in the measurement of nitric oxide (NO.) in the airways. NO. is a free radical that reacts rapidly with reactive oxygen species in aqueous solution to form peroxynitrite which can then break down to nitrite (NO(2)(-)) and nitrate (NO(3)(-)). NO(3)(-) is considered a stable oxidative end product of NO. metabolism. The aim of this study was to assay NO(3)(-) in exhaled breath condensate (EBC) of normal nonsmoking and smoking subjects, asthmatics, patients with obstructive pulmonary disease (COPD), and patients with community-acquired pneumonia (CAP). EBC was collected using a glass condenser and samples were assayed for NO(3)(-) by ion chromatography followed by conductivity measurement. NO(3)(-) was detectable in EBC of all subjects. NO(3)(-) was elevated in smokers [median (range)] [62.5 (9.6-158.0) microM] and in asthmatics [68.0 (25.8-194.6) microM] compared to controls [9.6 (2.6-119.4) microM; p=0.003 and p=0.006, respectively], whereas NO(3)(-) was not elevated in COPD patients [24.1 (1.9-337.0 microM]. The concentration of NO(3)(-) in patients with CAP [243.4 (26.1-584.5) microM] was higher than that in controls (p=0.002) and NO(3)(-) values decreased after treatment and recovery from illness [40.0 (4.1-167.0) microM, p=0.009]. This study shows that NO(3)(-) is detectable in EBC of healthy subjects and it varies in patients with inflammatory airway diseases.     

Direct electrochemistry and electrocatalysis of myoglobin immobilized on a hexagonal mesoporous silica matrix

The direct electrochemistry of myoglobin (Mb) immobilized on a hexagonal mesoporous silica (HMS)-modified glassy carbon electrode was described. The interaction between Mb and HMS was investigated by using Fourier transfer infrared spectroscopy, nitrogen adsorption isotherm, and cyclic voltammetry. Two couples of redox peaks corresponding to Fe(III) to Fe(II) conversion of the Mb intercalated in the mesopores and adsorbed on the surface of the HMS were observed with the formal potentials of -0.167 and -0.029V in 0.1M, pH 7.0, phosphate buffer solution, respectively. The electrode reaction showed a surface-controlled process with one proton transfer. The immobilized Mb displayed good electrocatalytic responses to the reduction of both hydrogen peroxide (H(2)O(2)) and nitrite (NO(2)(-)), which were used to develop novel sensors for H(2)O(2) and NO(2)(-). The apparent Michaelis-Menten constants of the immobilized Mb for H(2)O(2) and NO(2)(-) were 0.065 and 0.72mM, respectively, showing good affinity. Under optimal conditions, the sensors could be used for the determinations of H(2)O(2) ranging from 4.0 to 124microM and NO(2)(-) ranging from 8.0 to 216microM. The detection limits were 6.2x10(-8) and 8.0x10(-7)M at 3 sigma, respectively. The HMS provided a novel matrix for protein immobilization and the construction of biosensors via the direct electron transfer of immobilized protein.     

Anticoagulants and other preanalytical factors interfere in plasma nitrate/nitrite quantification by the Griess method.

Nitric oxide (NO) is a signal molecule with functions such as neurotransmission, local vascular relaxation, and anti-inflammation in many physiological and pathological processes. Various factors regulate its intracellular lifetime. Due to its high reactivity in biological systems, it is transformed in the bloodstream into nitrates (NO(-)(3)) by oxyhemoglobin. The Griess reaction is a technically simple method (spectrophotometric, 540 nm) for the analysis of nitrites (NO(-)(2)) in aqueous solutions. We studied the interference of common anticoagulants in the quantification of nitrate and nitrite in plasma samples by the Griess method. We obtained rat plasma using heparin or sodium EDTA as anticoagulants, then added, or otherwise, known NO(-)(3) amounts in order to calculate their recovery. We also studied the effect of ultra-filtration performed before Griess reaction on plasma and aqueous solutions of various anticoagulants (heparin, EDTA, and also sodium citrate) to compare the recoveries of added NO(-)(3) or NO(-)(2). We used standards of NO(-)(3) or NO(-)(2) for quantification. We conclude that: (i) The bacterial nitrate reductase used to reduce NO(-)(3) to NO(-)(2) is unstable in certain storage conditions and interferes with different volumes of plasma used. (ii) The ultrafiltration (which is sometimes performed before the Griess reaction) of plasma obtained with EDTA or citrate is not recommended because it leads to overestimation of NO(minus sign)(3). In contrast, ultrafiltration is necessary when heparin is used. (iii) The absorbance at 540 nm attributed to plasma itself (basal value or background) interferes in final quantification, especially when ultrafiltration is not performed. For the quantification of plasma NO(-)(3) we recommend: sodium EDTA as anticoagulant, no ultrafiltration of plasma, and measurement of the absorbance background of each sample.     

HIV gp120 enhances NO production by cardiac myocytes through p38 MAP kinase-mediated NF-kappaB activation

Human immunodeficiency virus (HIV) infection is associated with a surprisingly high frequency of myocardial dysfunction. Potential mechanisms include direct effects of HIV, indirect effects mediated by cytokines, or a combination. We have previously reported that interleukin-1beta (IL-1beta) (500 U/ml) alone induced nitric oxide (NO) production by neonatal rat cardiac myocytes (CM). Effects of the HIV-1 envelope, glycoprotein120 (gp120), on inducible NO synthase (iNOS) in CM have not been previously reported. Unlike IL-1beta, recombinant HIV-gp120 (1 microgram/ml) alone failed to enhance NO production in CM (0.5 +/- 0.4 vs. 0.4 +/- 0.5 micromol/1.25 x 10(5) cells/48 h, gp120 vs. control, respectively; n = 12, P = not significant). However, the addition of gp120 to IL-1beta significantly enhanced iNOS mRNA expression (70 +/- 1.5 vs. 26 +/- 2.4 optical units, IL-1beta + gp120 vs. IL-1beta, respectively; n = 3), iNOS protein synthesis (42 +/- 1.4 vs. 18 +/- 0.8 optical units, IL-1beta + gp120 vs. IL-1beta, respectively; n = 3), and NO production (NO(2)(-)) (6.6 +/- 0.6 vs. 4.1 +/- 0.8 micromol/1.25 x 10(5) cells/48 h, IL-1beta + gp120 vs. IL-1beta, respectively; n = 12, P 相似文献   

硝酸盐供应对玉米侧根生长的影响

以两个玉米(Zea mays L.)自交系478和Wu312为研究材料,采用琼脂培养方法,研究不同浓度NO-3对侧根生长的影响.结果表明,在外部浓度0.01～1.0mmol/L范围内,NO-3供应能显著增加侧根的长度及根生物量.但当NO-3供应超过1.0 mmo1/L后,侧根长度开始下降.当NO-3供应分别在超过5.0(Wu312)与10(478)mmol/L后,侧根密度显著下降.在10 mmol/LNO-3下,Wu312的侧根生长几乎完全被抑制.而478在20 mmol/L时,侧根密度仍可达到其最大值的30%(主根)～50%(胚根).植株地上部全氮及硝酸盐含量随NO-3供应的增加而升高,二者与侧根长度、侧根密度及冠根比的数学函数关系相同.     

Inhibition of NO3- and NO2- reduction by microbial Fe(III) reduction: evidence of a reaction between NO2- and cell surface-bound Fe2+

A recent study (D. C. Cooper, F. W. Picardal, A. Schimmelmann, and A. J. Coby, Appl. Environ. Microbiol. 69:3517-3525, 2003) has shown that NO(3)(-) and NO(2)(-) (NO(x)(-)) reduction by Shewanella putrefaciens 200 is inhibited in the presence of goethite. The hypothetical mechanism offered to explain this finding involved the formation of a Fe(III) (hydr)oxide coating on the cell via the surface-catalyzed, abiotic reaction between Fe(2+) and NO(2)(-). This coating could then inhibit reduction of NO(x)(-) by physically blocking transport into the cell. Although the data in the previous study were consistent with such an explanation, the hypothesis was largely speculative. In the current work, this hypothesis was tested and its environmental significance explored through a number of experiments. The inhibition of approximately 3 mM NO(3)(-) reduction was observed during reduction of a variety of Fe(III) (hydr)oxides, including goethite, hematite, and an iron-bearing, natural sediment. Inhibition of oxygen and fumarate reduction was observed following treatment of cells with Fe(2+) and NO(2)(-), demonstrating that utilization of other soluble electron acceptors could also be inhibited. Previous adsorption of Fe(2+) onto Paracoccus denitrificans inhibited NO(x)(-) reduction, showing that Fe(II) can reduce rates of soluble electron acceptor utilization by non-iron-reducing bacteria. NO(2)(-) was chemically reduced to N(2)O by goethite or cell-sorbed Fe(2+), but not at appreciable rates by aqueous Fe(2+). Transmission and scanning electron microscopy showed an electron-dense, Fe-enriched coating on cells treated with Fe(2+) and NO(2)(-). The formation and effects of such coatings underscore the complexity of the biogeochemical reactions that occur in the subsurface.     

Product formation and kinetic simulations in the pH range 1-14 account for a free-radical mechanism of peroxynitrite decomposition

The yields of nitrate and nitrite from decomposition of peroxynitrite in phosphate buffer at 37 degrees C were determined in the pH range 1-14. The NO(2)(-)/NO(3)(-) yields showed a stepwise variation with pH, with inflection points at approximately pH 3.1, 5.8, 6.8, 8.0, and 11.9. Nitrite formation increased strongly above pH 7 at the expense of nitrate, but above pH 12 nitrate again became the major product (80% at pH 14). At this pH, the Arrhenius parameters were E(a)=24.1+/-0.2kcal mol(-1) and A=(4.9+/-1.3)x10(12)s(-1). The yields of NO(2)(-), NO(3)(-), and O(2) measured at pH 5.8, 7.4, and 8.5 as a function of the initial peroxynitrite concentration (50-1000 microM) were linear only at pH 5.8. In the presence of carbon dioxide, oxygen production at pH 7.5 and pH 10 was found to be linear on the CO(2) concentration. The experimental observations were satisfactorily reproduced by kinetic simulations including principal component analyses. These data strongly suggest that the chemistry of peroxynitrite is exclusively mediated by z.rad;NO(2) and HO(z.rad;) radicals in the absence, and by z.rad;NO(2) and CO(3)(z.rad;-) radicals in the presence of CO(2).     

Effect of nitrite and nitrate on in situ sulfide production in an activated sludge immobilized agar gel film as determined by use of microelectrodes

Microelectrode, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE) analyses were used to investigate the effect of nitrite and nitrate on in situ sulfide production in an activated sludge immobilized agar gel film. Microelectrode measurements of O(2), H(2)S, NO(3)(-), NO(2)(-), and pH revealed that the addition of NO(2)(-) and NO(3)(-) forced sulfate reduction zones deeper in the agar gel and significantly reduced the in situ sulfide production levels. The sulfate reduction zone was consequently separated from O(2) and NO(2)(-) or NO(3)(-) respiration zones with increasing the concentrations of NO(2)(-) and NO(3)(-). These NO(2)(-) and NO(3)(-) treatments had only a transient effect on sulfide production. The in situ sulfide production quickly recovered to the previous levels when NO(2)(-) and NO(3)(-) were removed. The PCR-DGGE and FISH analyses revealed that 2-day-continuous addition of 500 microM NO(3)(-) did not change the metabolically active sulfate-reducing bacterial (SRB) community. On the basis of these data, it could be concluded that the addition of NO(2)(-) and NO(3)(-) did not kill SRB, but induced the interspecies competition for common carbon source (i.e., acetate) between nitrate-reducing heterotrophic bacteria and SRB and enhanced the oxidation of the produced sulfide, which were main possible causes of the suppression of in situ sulfide production in the agar gel.     

Modification of the cadmium reduction assay for detection of nitrite production using fluorescence indicator 2,3-diaminonaphthalene.

The measurement of nitric oxide (NO) is important for characterizing the regulatory roles of NO in various biological systems. In this communication we report that cadmium (Cd) reduction of nitrate (NO(-)(3)) to nitrite (NO(-)(2)) can be quantitated by using the fluorescence indicator, 2,3-diaminonaphthalene (DAN) to detect the sum of NO(-)(3) and NO(-)(2) (NO(-)(x)) from endothelial cells. This assay is at least 10-fold more sensitive than when Cd reduction is coupled with the spectrophotometric Greiss reaction and can be used to quantitate the small amounts of NO(-)(x) generated from the constitutive form of endothelial nitric oxide synthase (eNOS). In addition various P(2) purinoceptor agonists and antagonists do not interfere the Cd reduction/DAN assay. Thus the Cd reduction/DAN assay can be used not only to characterize P(2) purinoceptor release of NO(-)(x) from cultured endothelial cells but also to quantitate NO(-)(x) levels in serum.     

Is ceruloplasmin an important catalyst for S-nitrosothiol generation in hypercholesterolemia?

Nitric oxide (NO) reacts with thiol-containing biomolecules to form S-nitrosothiols (RSNOs). RSNOs are considered as NO reservoirs as they generate NO by homolytic cleavage. Ceruloplasmin has recently been suggested to have a potent catalytic activity towards RSNO production. Considering that NO activity is impaired in hypercholesterolemia and that RSNOs may act as important NO donors, we investigated the relation between concentrations of ceruloplasmin and RSNOs in plasma of hypercholesterolemic (HC) patients compared to normolipidemic (N) controls. Concentrations of ceruloplasmin (0.36 +/- 0.07 x 0.49 +/- 0.11 mg/dl, N x HC), nitrate (19.10 +/- 12.03 x 40.19 +/- 18.70 microM, N x HC), RSNOs (0.25 +/- 0.20 x 0.54 +/- 0.26 microM, N x HC), nitrated LDL (19.51 +/- 6.98 x 35.29 +/- 17.57 nM nitro-BSA equivalents, N x HC), and cholesteryl ester-derived hydroxy/hydroperoxides (CEOOH, 0.19 +/- 0.06 x 1.46 +/- 0.97 microM) were increased in plasma of HC as compared to N. No difference was found for nitrite levels between the two groups (1.01 +/- 0.53 x 1.02 +/- 0.33 microM, N x HC). The concentrations of RSNOs, nitrate, and nitrated LDL were positively correlated to those of total cholesterol, LDL cholesterol, and apoB. Ceruloplasmin levels were directly correlated to apoB and apoE concentrations. Data suggest that: (i) ceruloplasmin may have a role in the enhancement of RSNOs found in hypercholesterolemia; (ii) the lower NO bioactivity associated with hypercholesterolemia is not related to a RSNOs paucity or a defective NO release from RSNOs; and (iii) the increased nitrotyrosine levels found in hypercholesterolemia indicate that superoxide radicals contribute to inactivation of NO, directly generated by NO synthase or originated by RSNO decomposition.     

Cytochrome bo(3) from Escherichia coli: the binding and turnover of nitric oxide

The reaction of nitric oxide (NO) with fast and reduced cytochrome bo(3)(cyt bo(3)) from Escherichia coli has been investigated. The stoichiometry of NO binding to cyt bo(3) was determined using an NO electrode in the [NO] range 1-14 microM. Under reducing conditions, the initial decrease in [NO] following the addition of cyt bo(3) corresponded to binding of 1 NO molecule per cyt bo(3) functional unit. After this "rapid" NO binding phase, there was a slow, but significant rate of NO consumption ( approximately 0.3molNOmol bo(3)(-1)min(-1)), indicating that cyt bo(3) possesses a low level of NO reductase activity. The binding of NO to fast pulsed enzyme was also investigated. The results show that in the [NO] range used (1-14 microM) both fast and pulsed oxidised cyt bo(3) bind NO with a stoichiometry of 1:1 with an observed dissociation constant of K(d)=5.6+/-0.6 microM and that NO binding was inhibited by the presence of Cl(-). The binding of nitrite to the binuclear centre causes spectral changes similar to those observed upon NO binding to fast cyt bo(3). These results are discussed in relation to the model proposed by Wilson and co-workers [FEBS Lett. 414 (1997) 281] where the binding of NO to Cu(B)(II) results in the formation of the nitrosonium (Cu(B)(I)-NO(+)) complex. NO(+) then reacts with OH(-), a Cu(B) ligand, to form nitrite, which can bind at the binuclear centre. This work suggests for the first time that the binding of NO to oxidised cyt bo(3) does result in the reduction of Cu(B).     

Endothelial dysfunction is induced by proinflammatory oxidant hypochlorous acid

The myeloperoxidase (MPO)-derived oxidant hypochlorous acid (HOCl) plays a role in tissue injury under inflammatory conditions. The present study tests the hypothesis that HOCl decreases nitric oxide (NO) bioavailability in the vasculature of Sprague-Dawley rats. Aortic ring segments were pretreated with HOCl (1-50 microM) followed by extensive washing. Endothelium-dependent relaxation was then assessed by cumulative addition of acetylcholine (ACh) or the calcium ionophore A23187. HOCl treatment significantly impaired both ACh- and A23187-mediated relaxation. In contrast, endothelium-independent relaxation induced by sodium nitroprusside was unaffected. The inhibitory effect of HOCl on ACh-induced relaxation was reversed by exposure of ring segments to L-arginine but not D-arginine. In cellular studies, HOCl did not alter endothelial NO synthase (NOS III) protein or activity, but inhibited formation of the NO metabolites nitrate (NO3(-) and nitrite (NO2(-). The reduction in total NO metabolite production in bovine aortic endothelial cells was also reversed by addition of L-arginine. These data suggest that HOCl induces endothelial dysfunction via modification of L-arginine.     

Oxygen dependence of mitochondrial nitric oxide synthase activity

The effect of O(2) concentration on mitochondrial nitric oxide synthase (mtNOS) activity and on O(2)(-) production was determined in rat liver, brain, and kidney submitochondrial membranes. The K(mO(2)) for mtNOS were 40, 73, and 37 microM O(2) and the V(max) were 0.51, 0.49, and 0.42 nmol NO/minmg protein for liver, brain, and kidney mitochondria, respectively. The rates of O(2)(-) production, 0.5-12.8 nmol O(2)(-)/minmg protein, depended on O(2) concentration up to 1.1mM O(2). Intramitochondrial NO, O(2)(-), and ONOO(-) steady-state concentrations were calculated for the physiological level of 20 microM O(2); they were 20-39 nM NO, 0.17-0.33 pM O(2)(-), and 0.6-2.2 nM ONOO(-) for the three organs. These levels establish O(2)/NO ratios of 513-1000 that correspond to physiological inhibitions of cytochrome oxidase by intramitochondrial NO of 16-25%. The production of NO by mtNOS appears as a regulatory process that modulates mitochondrial oxygen uptake and cellular energy production.     

The [Ru(Hedta)NO](0.1-) system: structure, chemical reactivity and biological assays

The [Ru(II)(Hedta)NO(+)] complex is a diamagnetic species crystallizing in a distorted octahedral geometry, with the Ru-N(O) length 1.756(4) A and the RuNO angle 172.3(4) degrees . The complex contains one protonated carboxylate (pK(a)=2.7+/-0.1). The [Ru(II)(Hedta)NO(+)] complex undergoes a nitrosyl-centered one-electron reduction (chemical or electrochemical), with E(NO+/NO)=-0.31 V vs SCE (I=0.2 M, pH 1), yielding [Ru(II)(Hedta)NO](-), which aquates slowly: k(-NO)=2.1+/-0.4x10(-3) s(-1) (pH 1.0, I=0.2 M, CF(3)COOH/NaCF(3)COO, 25 degrees C). At pHs>12, the predominant species, [Ru(II)(edta)NO](-), reacts according to [Ru(II)(edta)NO](-)+2OH(-)-->[Ru(II)(edta)NO(2)](3-), with K(eq)=1.0+/-0.4 x 10(3) M(-2) (I=1.0 M, NaCl; T=25.0+/-0.1 degrees C). The rate-law is first order in each of the reactants for most reaction conditions, with k(OH(-))=4.35+/-0.02 M(-1)s(-1) (25.0 degrees C), assignable mechanistically to the elementary step comprising the attack of one OH(-) on [Ru(II)(edta)NO](-), with subsequent fast deprotonation of the [Ru(II)(edta)NO(2)H](2-) intermediate. The activation parameters were DeltaH(#)=60+/-1 kJ/mol, DeltaS(#)=-31+/-3 J/Kmol, consistent with a nucleophilic addition process between likely charged ions. In the toxicity up-and-down tests performed with Swiss mice, no death was observed in all the doses administered (3-9.08 x 10(-5) mol/kg). The biodistribution tests performed with Wistar male rats showed metal in the liver, kidney, urine and plasma. Eight hours after the injection no metal was detected in the samples. The vasodilator effect of [Ru(II)(edta)NO](-) was studied in aortic rings without endothelium, and was compared with sodium nitroprusside (SNP). The times of maximal effects of [Ru(II)(edta)NO](-) and SNP were 2 h and 12 min, respectively, suggesting that [Ru(II)(edta)NO](-) releases NO slowly to the medium in comparison with SNP.     

Uptake and Metabolism of Serotonin by Ependymal Primary Cultures

Serotonin uptake and metabolism was studied in ependymal primary cultures. Serotonin uptake was facilitated by two different systems, one of which was the neuronal serotonin transporter SERT, exhibiting a Vmax value of 3.8+/-0.1 pmol x min(-1) x (mg protein)(-1) and an apparent Michaelis-Menten constant of 0.41+/-0.03 microM. The main product of metabolism was 5-hydroxyindole acetic acid, which resulted from the action of monoamine oxidase A. This enzyme showed a maximal rate of 0.85+/-0.02 nmol x min(-1) x (mg protein)(-1) and a Michaelis-Menten constant of 78+/-5 microM. Ependymal cells were able to dispose of extracellular serotonin with initial rates of approximately 600 pmol x min(-1) x (mg protein)(-1) and of 4 pmol x min(-1) x (mg protein)(-1) when challenged with 500 microM and 1 microM extracellular serotonin, respectively. Ependymal cells are concluded to facilitate the "sink" action of the CSF by removing waste compounds upon passing of the fluid from the parenchymal extracellular space into the ventricular system.     

Effects of nitrite and ammonium on methane-dependent denitrification

For effective application of methane-dependent denitrification (MDD) in the treatment of wastewater containing NO(2)(-) or NH(4)(+), the effect of these inorganic nitrogen compounds on MDD activity needs to be clarified. The MDD activity of sludge acclimatized with CH(4) and O(2) was determined with mineral media of different nitrogen-compound compositions in the presence of 0.21 atm CH(4) and 0.20 atm O(2). Incubations with media containing only NO(2)(-) or two of the three inorganic nitrogen compounds (NO(3)(-)+NO(2)(-), NO(2)(-)+NH(4)(+) or NH(4)(+)+NO(3)(-)) resulted in MDD activity equal to or higher than that with media containing only NO(3)(-). However, there was no MDD activity in media containing NO(2)(-) at 10 degrees C, probably because of serious inhibition of NO(2)(-) on methane oxidation. MDD occurred in media containing only NH(4)(+), although the total nitrogen removal efficiency was very low. These results show that NO(2)(-) and NH(4)(+), in the presence of NO(x)(-), do not inhibit but rather promote MDD. Consequently, NH(4)(+) does not need to be completely oxidized to NO(3)(-) in the nitrification reactor before MDD. However, under psychrophilic conditions, NO(2)(-) seriously inhibited MDD. Therefore, the nitrification reactor must not discharge effluent containing NO(2)(-) under psychrophilic conditions.     

Total nitrogen oxide following exercise testing reflects endothelial function and discriminates health status

Nitric oxide (NO) bioavailability is important in vascular health, but unsuitable as a clinical measure due to biological oxidation. Total nitrogen oxides (NO(x)) are stable but background nitrate levels make it difficult to detect disease-based variation. We investigated the clinical discriminatory value of NO(x) as it relates to exercise capability (VO(2peak)) and brachial artery reactivity (BAR, an NO-dependent measure of endothelial health), in healthy (H), increased risk (RF), and known cardiovascular disease (CVD) subjects. BAR was measured using forearm occlusion/hyperemia stimulus. Subjects performed a maximal graded exercise test (GXT). Blood at rest, exercise termination, and 10 min into recovery was mixed equally with 0.1 M NaOH at 4 degrees C, filtered, and stored at -70 degrees C. NO(x) was measured by chemiluminescence. Seven of the RF group then exercise-trained for 6 months prior to retesting. The H group (n = 12) was younger, had higher VO(2peak), HDL levels, and baseline NO(x) values than the RF (n = 15) and CVD (n = 10) groups. NO(x) increased from baseline to recovery in the H group only (75.85 +/- 19.04 microM vs 97.76 +/- 31.93 microM; P 相似文献   

Hydrogen peroxide promotes endothelial dysfunction by stimulating multiple sources of superoxide anion radical production and decreasing nitric oxide bioavailability.

Hydrogen peroxide (H(2)O(2)) is an oxidant implicated in cell signalling and various pathologies, yet relatively little is known about its impact on endothelial cell function. Herein we studied the functional and biochemical changes in aortic vessels and cultured porcine aortic endothelial cells (PAEC) exposed to H(2)O(2). Exposure of aortic rings to 25 or 50 microM, but not 10 microM, H(2)O(2) for 60 min prior to constriction significantly decreased subsequent relaxation in response to acetylcholine (ACh), but not the nitric oxide ((.)NO) donor sodium nitroprusside. Treatment of PAEC with 50 microM H(2)O(2) significantly decreased ACh-induced accumulation of (.)NO, as measured with a (.)NO-selective electrode, yet such treatment increased nitric oxide synthase activity approximately 3-fold, as assessed by conversion of L-arginine to L-citrulline. Decreased (.)NO bioavailability was reflected in decreased cellular cGMP content, associated with increased superoxide anion radical (O(2)(-.)), and overcome by addition of polyethylene glycol superoxide dismutase. Increased cellular O(2)(-.) production was inhibited by allopurinol, diphenyliodonium and rotenone in an additive manner. The results show that exposure of endothelial cells to H(2)O(2) decreases the bioavailability of agonist-induced (.)NO as a result of increased production of O(2)(-.) likely derived from xanthine oxidase, NADPH-oxidase and mitochondria. These processes could contribute to H(2)O(2)-induced vascular dysfunction that may be relevant under conditions of oxidative stress such as inflammation.