Nitric oxide exchange in nitrosylmyoglobin

Summary The exchange of nitric oxide in nitrosylmyoglobin, the heme pigment of nitrite-cured meat, has been studied using nitrogen-15 labelling in aqueous solution under conditions (pH, concentration of ascorbate and nitrite) similar to those prevailing in meat during the curing process, and has been found to have a half-life of approximately 2 h at 40° C. One nitric oxide molecule is coordinated to the iron(II) centre of a myoglobin molecule and, in weakly acidic aqueous solution under anaerobic conditions, the exchange rate of the bound nitric oxide is proportional to the concentration of nitrosylmyoglobin, nitrite and hydrogen ion. The rate of exchange has a moderate temperature dependence, corresponding to an activation barrier ofH =47±3 kJ·mol^–1 at 25° C and pH 5.9, a value dramatically lower than that found for the enthalpy of activation for the oxidation of nitrosylmyoglobin by molecular oxygen,H =110 kJ·mol^–1. The difference in temperature dependence between the exchange and the autoxidation is discussed in relation to the function of nitrosylmyoglobin as antioxidant in cured meat products.

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Stickoxidaustausch in Stickoxidmyoglobin

Zusammenfassung Der Stickoxidaustausch im Häm-Farbstoff Stickoxidmyoglobin in Nitrit-gesalzenem Schinken wurde unter Verwendung einer Stickstoff-15-Markierung untersucht, und zwar in wäßriger Lösung und unter Verhältnissen (pH, Konzentration von Ascorbat und Nitrit), welche den Verhältnissen während des Einsalzens im Schinken ähnlich sind. Die Halbwertszeit des Austausches wurde bei 40 °C bei ungefähr 2 h festgestellt. Ein Stickoxid ist dem Eisen (II) des Myoglobins koordiniert; in schwach saurer Lösung und bei Sauerstofffreiheit ist die Austauschgeschwindigkeit des gebundenen Stickoxids proportional zur Konzentration von Stickoxidmyoglobin, Nitrit und Wasserstoffion. Die Austauschgeschwindigkeit ist etwas temperaturabhängig und entspricht einer Aktivierungsbarriere vonH =47±3 kJ·mol^–1 bei 25 °C und pH 5,9. Dieser Wert ist signifikant niedriger als der für die Aktivierungsenthalpie für Stickoxidmyoglobin-Oxydation durch molekularen Sauerstoff,H =110 kJ·mol^–1, festgestellte Wert. Der temperaturabhängige Unterschied zwischen Austausch und Autoxidation wird im Verhältnis zur Funktion des Stickoxidmyoglobins als Antioxidant in Schinken und ähnlichen Produkten diskutiert.

     

Nitric oxide synthases in pregnant rat uterus

The conversion of [14C]arginine into [14C]citrulline as an indicator of nitric oxide synthesis was studied in uteri isolated from rats on different days of gestation, after labour and during dioestrus. Nitric oxide synthesis was present in uterine tissues isolated at each stage of gestation and also in tissues collected during dioestrus and after labour. Expression of neuronal nitric oxide synthase was not detectable at any of the stages studied. Endothelial nitric oxide synthase was present at all the stages studied, but there was a significant increase on day 13 of gestation and a decrease thereafter, with the lowest expression recorded on the day after labour. Inducible nitric oxide synthase expression in rat uteri increased substantially during pregnancy, with the highest expression on day 13 of gestation; expression decreased at term and after labour. The changes in expression of inducible nitric oxide synthase were coincident with the changes in nitric oxide synthase activity in uteri treated with aminoguanidine. Thus, these findings indicate that an increase in expression of inducible nitric oxide synthase in the uterus may be important for maintenance of uterine quiescence during pregnancy and its decrease near the time of labour could have an effect on the start of uterine contractility.     

High-pressure effects on oxidation of nitrosylmyoglobin

The rate of oxidation of nitrosylmyoglobin by oxygen decreases with increasing hydrostatic pressure. At 15 °C in air-saturated solution with ionic strength 0.16 and pH 6.8 (tris-buffer), the first-order rate constant is smaller by a factor of 5 at 300 MPa compared to ambient conditions. The pressure-effect on rate is not primarily caused by protein denaturation, as the presence of urea (up to 4 M) at ambient pressure increases the rate of oxidation. From rate/pressure data a volume of activation of +8 ml·mol(-1) and a compressibility coefficient of activation of -3 × 10(-8) ml·mol(-1) Pa(-1) is estimated.     

Nitric oxide in blastocyst implantation in the rhesus monkey

Successful blastocyst implantation depends on the interaction between cells of maternal endometrium and conceptus, as well as adequate blood supply to the site of blastocyst implantation. Nitric oxide (NO) generally plays a significant role in the local regulation of vascular physiology in a variety of mammalian tissue systems, however, its role in blastocyst implantation and placentation in the primate is not known. The aim of the present study was to examine: (i) NADH-diaphorase activity and expression of three isoforms of nitric oxide synthase (NOS), namely endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) in pre-implantation stage monkey embryos, morula (n = 4) and blastocyst (n = 10), as well as, in different compartments of conceptus and maternal endometrium at primary implantation sites during lacunar (n = 6) and villous (n = 9) stages of placentation in the rhesus monkey, and (ii) the potential anti-nidatory effect of vaginal administration of NOS inhibitor during the peri-implantation period of conception cycles in rhesus monkeys. Pre-implantation stage blastocysts exhibited marked NADPH-diaphorase activity along with immunopositive iNOS mainly in the inner cell mass. During the lacunar stage, marked eNOS expression was observed in cytotrophoblast cells lining the embryonic cavity. However, cytotrophoblast cells lining villi, forming columns, and constituting anchoring villi expressed all the three isoforms of NOS in villous placenta stage tissue. During the lacunar stage, eNOS and iNOS protein expressions were observed in epithelial and decidual cells of endometrium. As gestation advanced, mRNAs for all three isoforms of NOS were observed to increase in epithelial and decidual cells, however, with no marked change in protein expression. Vaginal administration of a NOS inhibitor (N(G)-nitro-l-arginine methyl ester, L-NAME, 4, 6, and 8 mg/kg body weight or aminoguanidine, AG, 4 mg/kg body weight) during days 6 to 12 after ovulation resulted in pregnancy failure in a higher number of animals (L-NAME: 8 confirmed pregnancies in 25 animals; AG: 2 confirmed pregnancies in 8 animals) compared with control animals (5 pregnancies in 7 animals). It appears that NO may play an important role in the establishment of pregnancy in the rhesus monkey.     

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.     

The antioxidant activities of nitrite and nitrosylmyoglobin in cooked meats

Low concentrations of nitrite (20 mg/kg) caused significant (p < 0·001) inhibition of lipid oxidation, measured by the TBA test, in a cooked muscle system and 50 mg/kg nitrite resulted in a highly significant (p < 0·001) reduction in TBA values. Similar antioxidant effects of nitrite were observed in heated water-extracted pork muscle systems catalysed by 5 mg/g metmyoglobin (Mb) or 5 mg/kg Fe(2+), Cu(2+) or Co(2+). The cured meat pigment, nitrosylmyoglobin per se exhibited significant (p < 0·05) antioxidant effects in pork muscle systems catalysed by Mb or metal ions. Progressive depletion of nitrite occurred during refrigerated storage of heated and unheated nitrite-treated pork muscle, muscle aqueous extract and in systems containing Mb, Cu(2+) ot Co(2+). Nitrite depletion occurred much more rapidly in Fe(2+)-containing systems and nitrite concentration had decreased to 5% of the original concentration immediately after heating. In addition, nitrite caused a significant (p < 0·05) reduction in the concentration of non-haem iron in heated aqueous-extracts of beef muscle, whereas, in nitrite-free extracts, a highly significant (p < 0·001) increase in the concentration of non-haem iron, probably due to heat denaturation of the haem structure with release of iron, was observed. Based on the results of this study, three co-operative mechanisms for the antioxidative activities in meat are proposed: (a) by the formation of MbNO which has antioxidant properties per se, (b) on heating, MbNO forms a stable complex, nitrosylhaemochrome, which blocks the catalytic activity of haem iron and also prevents release of haem iron as non-haem iron, which is a highly effective catalyst and (c) nitrite appears to 'chelate' non-haem iron-and possibly copper and cobalt-forming a stable complex, thus inhibiting catalytic activity.     

Nitric oxide (NO) scavenging capacity of natural antioxidants

Nitric oxide (NO)-scavenging capacities of several hydrophilic antioxidants were determined by using the PTIO method, a competitive NO-scavenging method with 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). Relative NO-scavenging rates of antioxidants were measured with respect to PTIO and the scavenging rate constants were calculated based on PTIO’s rate constant. Results indicated that NO-scavenging rate constants of the antioxidants were: uric acid (2.5) > caffeic acid (1.2) > trolox (1.0) > genistein (0.19) > glutathione (0) ≈ N-acetylcysteine (0), where the numbers are expressed in trolox equivalent unit. The oxidation potentials of these antioxidants were measured and the order in the magnitude of oxidation potential was in good accordance with NO-scavenging capacity. Based on the results, we have suggested that the primary chemical process of the antioxidant reaction with NO can be characterised with the electron transfer from NO to the antioxidant.     

Nitric oxide reduction in coal combustion: role of char surface complexes in heterogeneous reactions

Nitrogen oxides are one of the major environmental problems arising from fossil fuel combustion. Coal char is relatively rich in nitrogen, and so this is an important source of nitrogen oxides during coal combustion. However, due to its carbonaceous nature, char can also reduce NO through heterogeneous reduction. The objectives of this work were on one hand to compare NO emissions from coal combustion in two different types of equipment and on the other hand to study the influence of char surface chemistry on NO reduction. A series of combustion tests were carried out in two different scale devices: a thermogravimetric analyzer coupled to a mass spectrometer and an FTIR (TG-MS-FTIR) and a fluidized bed reactor with an on line battery of analyzers. The TG-MS-FTIR system was also used to perform a specific study on NO heterogeneous reduction reactions using chars with different surface chemistry. According to the results obtained, it can be said that the TG-MS-FTIR system provides valuable information about NO heterogeneous reduction and it can give good trends of the behavior in other combustion equipments (i.e., fluidized bed combustors). It has been also pointed out that NO-char interaction depends to a large extent on temperature. In the low-temperature range (<800 degrees C), NO heterogeneous reduction seems to be controlled by the evolution of surface complexes. In the high-temperature range (>800 degrees C), a different mechanism is involved in NO heterogeneous reduction, the nature of the carbon matrix being a key factor.     

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.     

Photooxidation of nitrosylmyoglobin at low oxygen pressure. Quantum yields and reaction stoechiometries

Photooxidation of the nitrite-cured meat pigment, nitrosylmyoglobin, in aqueous solution saturated with a 20% CO₂/80% N₂ gas mixture with varying oxygen contents (0.1, 0.5 or 1.0%) was found to depend linearly on the oxygen content for both visible (436 nm) and UV-light (366 nm). Quantum yields were similar for the two wavelengths of excitation in agreement with previous findings at higher oxygen pressures. The reaction stoechiometry for photooxidation was different from that of thermal oxidation (investigated at the same oxygen pressures) with a unity MbFe(II)NO/O₂ ratio in the thermal reaction and a ratio larger than one for the photooxidation. For cured meat products packed in modified atmospheres light exposure may be even more harmful for the oxidative stability than expected from the concentration of residual oxygen.     

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.     

Nitric oxide fumigation stimulates flavonoid and phenolic accumulation and enhances antioxidant activity of mushroom

The effects of nitric oxide (NO) on antioxidant activity and contents of phenolics and flavonoids in mushroom Russula griseocarnosa were investigated. Freshly harvested mushrooms were fumigated with 0, 10, 20 and 30μLL(-1) NO at 20°C for 2h and then taken to examine the antioxidant activities using assays of reducing power, chelating effect on ferrous ions, scavenging effect on hydroxyl free radicals, and 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity. The results showed that the antioxidant activities of the mushrooms fumigated with NO were significantly increased when compared to the controls. Moreover, NO fumigation significantly enhanced phenolic and flavonoid contents and stimulated the activities of phenylalanine ammonia-lyase and chalcone synthase. The results indicated that NO fumigation might have potential application for enhancing the bioactive compounds and improving antioxidant activities in the mushrooms. Furthermore, the data suggested that the NO-induced phenolic and flavonoid accumulation was due to the activation of the biosynthetic pathways in the mushrooms.     

Nitric oxide prevents wound-induced browning and delays senescence through inhibition of hydrogen peroxide accumulation in fresh-cut lettuce

As a source of bioactive ingredients, lettuce is a preferable component of a healthy diet. In recent years the production of fresh-cut produce has become a fast growing business. However, the shreds are highly sensitive to wound-induced browning and premature senescence that substantially reduces the visual and sensory qualities and shortens the shelf life. To improve the fresh-cut quality, in this work, short pre-storage exposure of shreds from butterhead and iceberg lettuce to nitric oxide (NO) gas was applied. It was found that fumigation with 100 and 200 ppm NO for 1 or 2 h remarkably inhibited the browning of the cut surface and of other injured leaf areas; NO treatment delayed the senescence and substantially prolonged the shelf life upon storage at 4 °C and 12 °C. To obtain information on the physiological processes involved in the wound response, the generation of hydrogen peroxide (H₂O₂) and the occurrence of cell death were analyzed. The results revealed that the wounding stimulated the accumulation of H₂O₂ thus generating oxidative stress leading to cell death. A correlation between elevated H₂O₂ levels, cut surface browning, senescence and storability of the fresh-cuts was established. In comparison to mature leaves, younger leaves expressed a lesser susceptibility to wound-induced browning and the associated oxidative stress. Applied NO strongly inhibited the H₂O₂ accumulation which may explain its beneficial effects.Industrial relevanceWe demonstrate that short treatments with NO gas substantially inhibit wound-induced browning and largely improve the storability of fresh-cut lettuce. This offers an option for adopting NO treatments for optimization of the post-processing conditions. Implementation of reported findings into practice will offer innovative technological solutions for improvement of the post-harvest quality of fresh-cut lettuce applicable for the industry, distributors and retailers. Moreover, our findings indicate that the browning disorder is to a large extent dependent on the severity of the wound-induced oxidative stress and cell death occurrence. This discovery opens a possibility for the development of metabolic and morphological markers appropriate for the prediction of the quality and shelf life of fresh-cut lettuce with perspective for expanding the research and introducing the tests toward other perishable leafy vegetables.     

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.     

Nitric oxide stimulates human sperm motility via activation of the cyclic GMP/protein kinase G signaling pathway

Nitric oxide (NO), a modulator of several physiological processes, is involved in different human sperm functions. We have investigated whether NO may stimulate the motility of human spermatozoa via activation of the soluble guanylate cyclase (sGC)/cGMP pathway. Sperm samples obtained by masturbation from 70 normozoospermic patients were processed by the swim-up technique. The kinetic parameters of the motile sperm-rich fractions were assessed by computer-assisted sperm analysis. After a 30-90 min incubation, the NO donor S-nitrosoglutathione (GSNO) exerted a significant enhancing effect on progressive motility (77, 78, and 78% vs 66, 65, and 62% of the control at the corresponding time), straight linear velocity (44, 49, and 48?μm/s vs 34, 35, and 35.5?μm/s), curvilinear velocity (81, 83, and 84?μm/s vs 68?μm/s), and average path velocity (52, 57, and 54?μm/s vs 40, 42, and 42?μm/s) at 5?μM but not at lower concentrations, and in parallel increased the synthesis of cGMP. A similar effect was obtained with the NO donor spermine NONOate after 30 and 60 min. The GSNO-induced effects on sperm motility were abolished by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (a specific sGC inhibitor) and mimicked by 8-bromo-cGMP (8-Br-cGMP; a cell-permeating cGMP analog); the treatment with Rp-8-Br-cGMPS (an inhibitor of cGMP-dependent protein kinases) prevented both the GSNO- and the 8-Br-cGMP-induced responses. On the contrary, we did not observe any effect of the cGMP/PRKG1 (PKG) pathway modulators on the onset of hyperactivated sperm motility. Our results suggest that NO stimulates human sperm motility via the activation of sGC, the subsequent synthesis of cGMP, and the activation of cGMP-dependent protein kinases.     

Formation and identification of nitrosylmyoglobin by Staphylococcus xylosus in raw meat batters: A potential solution for nitrite substitution in meat products

Staphylococcus xylosus and Pediococcus pentosaceus isolated from Chinese dried sausage were assessed for their ability to convert metmyoglobin into nitrosylmyoglobin in Mann–Rogosa–Sharp broth model systems and raw pork meat batters without the addition of nitrite. The results showed that samples in model systems with S. xylosus cultures had an absorption spectra that is typical of nitrosylmyoglobin, an obvious pink colour (judged by visual inspection) and a significantly higher a*-value than the control samples or samples inoculated with P. pentosaceus. In raw meat batters, the a*-values of the S. xylosus samples were almost the same as those for the meat with nitrite added. The complementary analysis of meat batter samples by photochemical information from UV–vis, electron spin resonance and resonance Raman spectroscopy revealed that the existing status of the myoglobin in meat batters inoculated with S. xylosus was mainly pentacoordinate nitrosylmyoglobin. This study provides a potential solution for nitrite substitute in meat products.     

氧化氮合酶在骨关节炎中的作用

近年氧化氮对炎症性疾病的作用引起了广泛的重视,氧化氮合酶则对氧化氮的产生起着关键作用。现对诱导型氧化氮合酶在骨关节炎发病过程中的作用,以及氧化氮合酶抑制剂用于治疗骨关节炎的研究进展作一综述。