FORMATION OF NITRIC OXIDE MYOGLOBIN BY NICOTINAMIDE ADENINE DINUCLEOTIDES AND FLAVINS

SUMMARY –Formation of nitric oxide myoglobin was studied under anaerobic conditions. NADH (reduced nicotinamide adenine dinucleotide) and NADPH (reduced nicotinamide adenine dinucleotide phosphate) as reducing agents alone did not produce nitric oxide myoglobin from metmyoglobin and nitrite. However, in the presence of FMN (flavin mononucleotide), either NADH or NADPH readily produced nitric oxide myoglobin. FAD (flavin adenine dinucleotide) and riboflavin also were effective for the formation of nitric oxide myoglobin by NADH. In the absence of myoglobin, the NADH-FMN system did not reduce nitrite to nitric oxide. Similarly, a diaphorasemethylene blue system readily produced nitric oxide myoglobin, but in the absence of myoglobin it did not reduce nitrite to nitric oxide. On the other hand, nitric oxide myoglobin was produced from deoxymyoglobin by the action of nitrite in the absence of reducing agents under anaerobic conditions. Deoxymyoglobin can directly reduce nitrite to nitric oxide; the NADH-FMN and diaphorase-methylene blue systems may participate in the formation of nitric oxide myoglobin by means of reduction of metmyoglobin to the deoxy form.     

Nitric oxide production during endotoxin-induced mastitis in the cow

Nitric oxide production was measured during endotoxin-induced mastitis. One hour after morning milking, the right hind quarters of 15 cows were infused with saline containing Escherichia coli endotoxin. Left hind control quarters were infused with saline only. At varying intervals before and after infusion, diagnostic markers of mastitis were recorded and nitric oxide production was evaluated by measuring nitrite plus nitrate levels in milk. In endotoxin-infused quarters, a significant increase in nitrite plus nitrate concentrations was observed 3 h postinfusion; concentrations decreased to preinfusion levels within 48 h. This change indicates that significant amounts of nitric oxide are released during endotoxin-induced mastitis. At 3 different time points, somatic cells were harvested from milk samples, plated, and maintained in culture for 24 h. The concentration of nitrite plus nitrate in medium from cells harvested 12 h postinfusion was increased, suggesting that nitric oxide is released, at least in part, by milk somatic cells. In a second set of experiments, we evaluated nitric oxide production when animals were infused with endotoxin and aminoguanidine, a specific inhibitor of the inducible form of nitric oxide synthase. In cows treated with aminoguanidine, the increase in nitrite plus nitrate observed after endotoxin infusion was prevented. These results suggest that nitric oxide production during endotoxin-induced mastitis resulted from the activity of the inducible form of nitric oxide synthase. They also support a possible involvement for nitric oxide in the inflammatory reaction observed during mastitis.     

A flow-injection spectrophotometric method for nitrate and nitrite determination through nitric oxide generation

A flow injection method with novel spectrophotometric detection for the determination of nitrite and nitrate in foodstuffs is presented. The method is based on the reduction of nitrite and nitrate to nitric oxide, with subsequent reaction with iron (II) and thiocyanate in an acid medium, forming FeSCNNO⁺. The absorbance of the complex, with a maximum at 460 nm, is proportional to the nitrite and nitrate concentrations. The NO is generated in two stages: (1) reduction of nitrate to nitrite in a cadmium copper reductor column and (2) reduction of the nitrite to NO in a sulfuric acid medium. The influence of reagent concentrations and manifold parameters were evaluated. Nitrite and nitrate can be determined in the range of 0.30–3.00 and 1.00–10.00 mg l⁻¹, respectively. The sampling rate of analyses was 30–40 h⁻¹ and, considering a sample of 5.0 g, the determination limit of the method was 20 and 13 mg kg⁻¹ of nitrate and nitrite, respectively. Nitrite and nitrate were determined in vegetables and meat products by the proposed method. The precision and accuracy of the proposed method were comparable to those of the reference spectrophotometric method (official AOAC reference method for the determination of nitrate in foodstuffs).     

Dissimilatory nitrate reduction metabolisms and their control in fungi

Most fungi grow under aerobic conditions by generating ATP through oxygen respiration. However, they alternatively express two pathways of dissimilatory nitrate reduction in response to environmental oxygen tension when the oxygen supply is insufficient. The fungus Fusarium oxysporum expressed the pathway of respiratory nitrate denitrification that is catalyzed by the sequential reactions of nitrate reductase and nitrite reductase. These enzymes are coupled with ATP generation through the respiratory chain and produce nitric oxide. Fungal nitric oxide reductase uses NADH as the direct electron donor in contrast to bacterial systems and thus might function in regeneration of NAD+ and detoxification of the toxic radical, nitric oxide. Another pathway of nitrate dissimilation by fungi reduces nitrate to ammonium and couples acetogenic reaction with substrate-level phosphorylation. This metabolic mechanism is also in feature of a variety of fungi and it is called ammonia fermentation. Thus, fungi adapt to various aerated conditions using these pathways of nitrate dissimilation in addition to conventional oxygen respiration.     

The breakdown into nitric oxide of compounds potentially derived from nitrite in a biological matrix

Summary N-Nitroso compounds can be determined using a chemiluminescence analyzer by means of the nitric oxide liberated as a volatile product of their denitrosation using hydrogen bromide [1, 2]. Much of the nitrite present in or added to a food matrix can be lost even during storage in a refrigerated condition and thus the possibility arises of its conversion into compounds other than nitrosamines and nitrosamides which could also give rise to nitric oxide under, for instance, thermal degradation. Among compounds potentially formed from nitrite that have been studied in relation to their breakdown to nitric oxide have been included an alkyl nitrate, an alkyl nitrite, an amine oxide, an azoxy compound, a nitramine, a nitro guanidine, a nitrolic acid, a nitrone, an oxime, a pseudonitrole, a pseudonitrosite, a thionitrite, a thionitrate as well as C-nitro and C-nitroso compounds. Only in the case of the nitrolic acid and the thionitrate was it likely that interference with the determination of N-nitroso compounds could occur to any considerable extent.

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Abspaltung von NO aus möglichen Folgeprodukten von Nitrit in einer biologischen Matrix

Zusammenfassung N-Nitrosoverbindungen können durch die Chemiluminiscenz-Methode über das Stickoxid bestimmt werden, das durch Denitrosierung mit HBr als flüchtiges Produkt entsteht. Ein großer Teil des Nitrits, das in Lebensmitteln enthalten ist oder zugesetzt wurde kann auch bei Kühllagerung bereits verloren gehen und in andere Produkte als Nitrosamine oder Nitrosamide umgewandelt werden, die bei der thermischen Zersetzung zur Bildung von NO Anlaß geben. Unter den mit Nitrit entstehenden Verbindungen, die zu einer Abspaltung von NO führen könnten, wurden näher untersucht ein Alkylnitrat, ein Alkylnitrit, ein Aminoxyd, eine Azoxy-Verbindung, ein Nitramin, ein Nitroguanidin, eine Nitrolsäure, ein Nitron, ein Oxim, ein Pseudonitrol, ein Pseudonitrosit, ein Thionitrit, sowie C-Nitro- und C-Nitroso-Verbindungen. Nur bei der Nitrolsäure und bei Thionitrat könnte eine Störung der Bestimmung von N-Nitrosoverbindungen über die NO-Abspaltung in beachtlichem Umfang eintreten.

     

Nitric oxide inhibits the formation of zinc protoporphyrin IX and protoporphyrin IX

The aim of this study was to elucidate the mechanism by which curing agents, especially nitrite, inhibit the formation of zinc protoporphyrin IX (ZPP) in dry-cured hams such as Parma ham. The oxidation–reduction potential of model solutions was increased by the addition of nitrite, but it was not clear whether the formation of ZPP is inhibited by the oxidizing property of nitrite. The effect of nitric oxide (NO) produced from nitrite on the formation of ZPP was examined. The amount of ZPP formed was decreased by the addition of NO donors. The amount of protoporphyrin IX (PPIX), which is the precursor of ZPP, was also decreased by the addition of NO donors. It is concluded that NO produced from nitrite inhibited the formation of PPIX and ZPP was therefore not formed in cured meat products with the addition of nitrite or nitrate.     

In-situ evidence for uranium immobilization and remobilization

The in-situ microbial reduction and immobilization of uranium was assessed as a means of preventing the migration of this element in the terrestrial subsurface. Uranium immobilization (putatively identified as reduction) and microbial respiratory activities were evaluated in the presence of exogenous electron donors and acceptors with field push-pull tests using wells installed in an anoxic aquifer contaminated with landfill leachate. Uranium(VI) amended at 1.5 microM was reduced to less than 1 nM in groundwater in less than 8 d during all field experiments. Amendments of 0.5 mM sulfate or 5 mM nitrate slowed U(VI) immobilization and allowed for the recovery of 10% and 54% of the injected element, respectively, as compared to 4% in the unamended treatment. Laboratory incubations confirmed the field tests and showed that the majority of the U(VI) immobilized was due to microbial reduction. In these tests, nitrate treatment (7.5 mM) inhibited U(VI) reduction, and nitrite was transiently produced. Further push-pull tests were performed in which either 1 or 5 mM nitrate was added with 1.0 uM U(VI) to sediments that already contained immobilized uranium. After an initial loss of the amendments, the concentration of soluble U(VI) increased and eventually exceeded the injected concentration, indicating that previously immobilized uranium was remobilized as nitrate was reduced. Laboratory experiments using heat-inactivated sediment slurries suggested that the intermediates of dissimilatory nitrate reduction (denitrification or dissimilatory nitrate reduction to ammonia), nitrite, nitrous oxide, and nitric oxide were all capable of oxidizing and mobilizing U(IV). These findings indicate that in-situ subsurface U(VI) immobilization can be expected to take place under anaerobic conditions, but the permanence of the approach can be impaired by disimilatory nitrate reduction intermediates that can mobilize previously reduced uranium.     

Relationship between nitrate/nitrite reductase activities in meat associated staphylococci and nitrosylmyoglobin formation in a cured meat model system

Quantitative determination of catalase, nitrate reductase, nitrite reductase and nitric oxide synthase activities (NOS) was performed on 11 different bacterial strains, mainly staphylococci, isolated from fermented sausages, bacon brine or cured meat products. All except one strain possessed catalase activity in the range from 1.0 to 6.1 μmol min^− 1 ml^− 1. Ten out of 11 bacteria strains showed nitrate reductase activity in the range between 50 and 796 nmol min^− 1 ml^− 1 and nine showed nitrite reductase activity in the range between 6 and 42 nmol min^− 1 ml^− 1. No evidence of NOS activity of the selected strains was detected. In a colour formation assay containing myoglobin all strains affected nitrosylmyoglobin (MbFe^IINO) formation in assays containing nitrite, whereas only strains having nitrate reductase activity generated MbFe^IINO in assays containing nitrate as the sole nitrosylating agent. The quantitative nitrate and nitrite reductase activity did not fully explain or correlate well with the observed rate of formation of MbFe^IINO, which seemed to be more affected by the growth rate of the different strains. The mechanism of the reduction of nitrite into NO of strains not having nitrite reductase activity remains to be fully elucidated, but could be due to a dual-mode action of nitrate reductase capable of acting on nitrate.     

Corn leaf nitrate reductase--a nontoxic alternative to cadmium for photometric nitrate determinations in water samples by air-segmented continuous-flow analysis

Development, characterization, and operational details of an enzymatic, air-segmented continuous-flow analytical method for colorimetric determination of nitrate + nitrite in natural-water samples is described. This method is similar to U.S. Environmental Protection Agency method 353.2 and U.S. Geological Survey method 1-2545-90 except that nitrate is reduced to nitrite by soluble nitrate reductase (NaR, EC 1.6.6.1) purified from corn leaves rather than a packed-bed cadmium reactor. A three-channel, air-segmented continuous-flow analyzer-configured for simultaneous determination of nitrite (0.020-1.000 mg-N/L) and nitrate + nitrite (0.05-5.00 mg-N/L) by the nitrate reductase and cadmium reduction methods-was used to characterize analytical performance of the enzymatic reduction method. At a sampling rate of 90 h(-1), sample interaction was less than 1% for all three methods. Method detection limits were 0.001 mg of NO2- -N/L for nitrite, 0.003 mg of NO3-+ NO2- -N/L for nitrate + nitrite by the cadmium-reduction method, and 0.006 mg of NO3- + NO2- -N/L for nitrate + nitrite bythe enzymatic-reduction method. Reduction of nitrate to nitrite by both methods was greater than 95% complete overthe entire calibration range. The difference between the means of nitrate + nitrite concentrations in 124 natural-water samples determined simultaneously bythe two methods was not significantly different from zero at the p = 0.05 level.     

替代亚硝酸盐生产安全腌肉制品的研究现状

硝酸盐和亚硝酸盐在腌肉制品加工中同时起着发色、抑菌、抗氧化和提高风味的作用,但硝酸盐和亚硝酸盐与肉中的二级胺反应会形成致癌物质N-亚硝胺,所以寻求一种更加安全有效的硝酸盐和亚硝酸盐替代方式,在实际生产中具有重要意义。本文就无硝腌肉制品在亚硝酸盐发色作用、抑菌作用和抗氧化作用的替代物方面开展的研究工作进行综述,提出了天然腌制的概念,重点介绍目前国内外利用蔬菜替代亚硝酸盐的方法,包括将蔬菜粉(或汁)中的硝酸盐用硝酸盐还原菌转化为亚硝酸盐后加入腌肉制品中(先发酵法)和将蔬菜粉(或汁)加入肉馅中再发酵(后发酵法)两种生产方式,并对目前的研究现状、进展、存在问题及今后发展方向进行了论述,以期为生产安全腌肉制品提供借鉴。     

Headspace concentration of selected dry-cured aroma compounds in model systems as affected by curing agents

The effect of curing agents (sodium chloride, nitrate, nitrite, glucose, and ascorbic acid) on the headspace concentration of six volatile compounds (2-methyl-butanal, 3-methyl-butanal, 2-pentanone, hexanal, methional, and octanal) has been studied. These volatile compounds were selected based on their presence and contribution to the flavour of typical Spanish dry-cured meat products. The release of volatile compounds to the headspace has been studied by using solid-phase microextraction (SPME) and gas chromatography analysis. The main effect was produced by sodium chloride, because it produced a salting-out effect on all the volatile compounds studied. Furthermore, the presence of nitrite, nitrate, and ascorbic acid in the solution also produced an increase in the headspace concentration of the volatile compounds while glucose decreased the headspace concentration of octanal and hexanal. The changes in the relative proportions of volatile compounds due to the effect of curing agents should be taken into account as they may change the flavour perception of dry-cured meat products.     

The effects of lactate on nitrosylmyoglobin formation from nitrite and metmyoglobin in a cured meat system

Two experiments were conducted to determine the effects of lactate on nitrite during meat curing. In the first experiment, using a model system, eight reaction components including nitrite and lactate, were used to assess the effect of each component on metmyoglobin reducing activity by excluding one component at a time. Excluding lactate, nicotinamide adenine dinucleotide (NAD), l-lactate dehydrogenase (LDH) or phenazine methosulfate (PMS) resulted in no reducing activity. A second experiment, utilising a meat mixture, investigated the effects of lactate (0%, 2%, 4% or 6%), nitrite (0 or 156 ppm), and packaging (oxygen-permeable or vacuum) on residual nitrite, meat colour and pH. Addition of lactate reduced residual nitrite in the meat mixtures. Both experiments support the hypothesis that lactate generates NADH which then reduces metmyoglobin to deoxymyoglobin. The resulting greater concentration of reduced myoglobin subsequently reacted with nitrite to produce more nitric oxide, reducing nitrite concentration and accelerating curing reactions.     

Photochemical loss of nitric acid on organic films: a possible recycling mechanism for NO(x)

The films coating urban impervious surfaces have been found to be comprised of about 7% inorganic nitrate and approximately 10% organic compounds (by mass). A simple steady-state analysis of the lifetime of the nitrate in the film suggests the existence of a loss process(es) in addition to washout by rainfall. We show here that gas-phase nitric acid can be taken up in organic films and lower the film pH. Photolysis of nitrated films using actinic illumination causes loss both of protons and of nitrate anion. We argue that this is possibly due to a combination of direct and indirect (photosensitized) photochemistry involving nitrate ions, yielding gas-phase HONO and/or NO2.     

Cured meat products without direct addition of nitrate or nitrite: what are the issues?

The growing popularity of food products marketed in the United States as "natural" and "organic" has resulted in a proliferation of marketing efforts to meet consumer demands for these foods. Because natural and organic foods are not permitted to use chemical preservatives, the traditional curing agents used for cured meats, nitrate and/or nitrite, cannot be added to natural and organic processed meat products. However, alternative processes that utilize ingredients with high nitrate content, such as vegetable-based ingredients, and a nitrate-reducing starter culture can produce processed meats with very typical cured meat properties. Because it is not possible to analytically measure the amount of nitrite produced by this process, several potential issues deserve consideration. Regulations, for example, should permit labeling that accurately reflects the process and products, manufacturing procedures must be standardized to achieve product consistency, marketing efforts should clearly communicate the nature of these products to consumers, product quality must be maintained, and microbiological safety must be assured.     

Subclinical mastitis in goats is associated with upregulation of nitric oxide-derived oxidative stress that causes reduction of milk antioxidative properties and impairment of its quality

The aim of this study was to verify the existence of a nitric oxide (NO) cycle in goat milk and to study how changes in it affect milk composition during subclinical mastitis. Fifteen lactating dairy goats in which one udder-half was free from bacterial infection and the contra-lateral one was naturally infected with various species of coagulase-negative staphylococci were used. In comparison to uninfected glands, subclinical mastitis was associated with a decrease in milk yield, lactose concentration, and curd yield and an increase in nitrite and nitrate concentrations and with measurements reflecting increased formation of NO-derived free-radical nitrogen dioxide. The occurrence of NO cycling in goat milk was largely confirmed. The increase in the NO-derived stress during subclinical infection was not associated with significant increase in oxidatively modified substances, 3-nitrotyrosine, and carbonyls on proteins, but with increased levels of peroxides on fat. However, the relatively modest nitrosative stress in subclinically infected glands was associated with significant reduction in total antioxidant capacity and vitamin C levels in milk. We concluded that subclinical mastitis in goats caused by coagulase-negative staphylococci imposes negative changes in milk yield, milk quality for cheese production, and negatively affects the nutritional value of milk as food. Thus, subclinical mastitis in goats should be considered as a serious economic burden both by farmers and by the dairy industry.     

离子色谱法同时测定烟草中的硝酸盐与亚硝酸盐

采用离子色谱建立一种烟草及烟草制品中硝酸盐和亚硝酸盐的同时测定方法。在超声条件下,用水萃取试样中的硝酸盐和亚硝酸盐离子,萃取液经阴离子交换色谱柱分离,用电导检测器检测,外标法定量。结果发现:该方法在测试范围内线性良好;硝酸盐的检出限为1.06 mg/kg,回收率在96.3%～99.0%之间,亚硝酸盐的检出限为1.23 mg/kg,回收率在96.1%～101.6%之间;相对标准偏差均小于5%;且操作简单、高效、重复性好、检出限低、样品回收率高,能满足当前烟草中硝酸盐和亚硝酸盐的同时快速检测要求。      

Membrane rejection of nitrogen compounds

Rejection characteristics of nitrogen compounds were examined for reverse osmosis, nanofiltration, and low-pressure reverse osmosis membranes. The rejection of nitrogen compounds is explained by integrating experimental results with calculations using the extended Nernst-Planck model coupled with a steric hindrance model. The molecular weight and chemical structure of nitrogen compounds appear to be less important in determining rejection than electrostatic properties. The rejection is greatest when the Donnan potential exceeds 0.05 V or when the ratio of the solute radius to the pore radius is greater than 0.8. The transport of solute in the pore is dominated by diffusion, although convective transport is significant for organic nitrogen compounds. Electromigration contributes negligibly to the overall solute transport in the membrane. Urea, a small organic compound, has lower rejection than ionic compounds such as ammonium, nitrate, and nitrite, indicating the critical role of electrostatic interaction in rejection. This suggests that better treatment efficiency for organic nitrogen compounds can be obtained after ammonification of urea.     

烤烟中硝酸盐和亚硝酸盐的测定

本文提出了一种使用紫外分光光度计测定硝酸盐和亚硝酸盐的方法。主要过程包括将烟样在弱碱性条件下热水中萃取,亚硝酸盐标准曲线的绘制,亚硝酸盐含量的测定,硝酸盐的还原,硝酸盐含量的测定,回收率的测定。该方法具有简单、高效、重复性好、不需专用仪器设备等特点,适用于各种烟草样品中硝酸盐和亚硝酸盐含量的测定。      

Determination of nitrate,nitrite and perchlorate anions in meat,milk and their products consumed in Hatay region in Turkey

Nitrates and nitrites added to food can cause formation of cancerous N-nitroso compounds, whereas exposure to perchlorate is especially emphasised as an important risk factor for newborns’ health. In this study, nitrate, nitrite and perchlorate concentrations in meat and milk products consumed in the Hatay region of Turkey were determined. Nitrate and nitrite were analysed with a spectrophotometric method, and perchlorate analysed via ion chromatography. The detected sodium nitrate and nitrite amounts in meat consumed in the Hatay region are less than the maximum levels as declared in the Turkish Food Codex. The amount of perchlorate was considered not to pose a threat as well. However, in 50% of the cheese samples, sodium nitrate amounts were found to be more than the maximum acceptable level in the Turkish Food Codex.     

Determination of nitrate and nitrite in dairy samples by sequential injection using an in-line cadmium-reducing column

A sequential injection analysis (SIA) system for the spectrophotometric determination of nitrate in dairy samples was developed. A test portion was aspirated into a carrier solution containing ethylendiaminetetracetic acid (EDTA) and ammonium buffer, which flowed into a copperized cadmium reduction column installed in-line for the determination of the nitrate plus nitrite contents of samples. For the nitrite determination, another test portion of sample was aspirated and directly sent to the detector without reduction. Nitrate content was calculated from the difference between nitrate plus nitrite (expressed as nitrite) and nitrite content. The spectrophotometric determination is based on the Griess reaction. The proposed method was used to test several dairy samples (ultrapasteurized milk with 1.7% milk fat, whey, raw bovine milk and several cheese varieties). Results were statistically in good agreement with those provided by the reference procedure, with a detection limit of 0.15 mg L⁻¹. A sampling rate of 21 determinations per hour can be achieved with this procedure.