Cellular metabolism of proxyl nitroxides and hydroxylamines

Previous data from model systems indicated that the proxyl nitroxides should be especially resistant to bioreduction and therefore could be an effective solution to this often problematic characteristic of nitroxides. Therefore, we investigated the rate of reduction by cells and by the usual model system, ascorbate, of four proxyl nitroxides and three reference nitroxides. We found that, while the rate of reduction by ascorbate of the proxyl nitroxides was slower than the rate of a prototypic pyrrolidine nitroxide (PCA), the reverse was true for reduction by cells. We also studied the rate of oxidation of the corresponding hydroxylamines. The rate of oxidation by cells of the proxyl hydroxylamines was relatively fast, especially for the most lipophilic derivative. These results indicate that: (i) proxyl nitroxides may not be unusually resistant to bioreduction by functional biological systems; (ii) accurate knowledge of relative rates of metabolism of nitroxides and hydroxylamines in cells and tissues will require direct studies in these systems because the rates may not closely parallel those observed in model (chemical) systems; and (iii) proxyl nitroxides show potential value as agents to measure oxygen concentrations by the rates of oxidation of their corresponding hydroxylamines.     

Metabolism of aqueous soluble nitroxides in hepatocytes: effects of cell integrity, oxygen, and structure of nitroxides

The optimum use of nitroxides in viable biological systems, including live animals, requires knowledge of the metabolism of nitroxides by major organ systems, especially the liver. We report here details of the metabolism of several prototypic aqueous soluble nitroxides in suspensions of freshly isolated hepatocytes. The general patterns of metabolism were similar to those observed in other types of cells (previous studies have been done principally in cells from tissue culture, such as CHO cells) including the primary initial reaction being reduction to the hydroxylamine, an increased rate of metabolism of some nitroxides in hypoxic cells, faster rates of reduction of nitroxides on six-membered piperidine rings compared to five-membered pyrrolidine rings, and most metabolism being intracellular. Metabolism in hepatocytes differed from other cell lines in having (1) significant reduction in the extracellular medium due to ascorbate that was released from damaged hepatocytes; (2) decreased rates of metabolism in freeze-thawed cells due to damage to subcellular organelles. These results provide much of the data needed to understand the role of the liver in the metabolism of nitroxides by intact animals and explain some previously puzzling results which indicated an apparent unusually high rate of metabolism of a charged nitroxide (Cat1) by hepatocytes. Our results also indicate that the use of freshly isolated cells or tissue homogenates may introduce experimental artifacts in the study of the metabolism of nitroxides.     

Effect of shear rate on hybridoma cell metabolism

The effect of shear rate on cell growth and monoclonal antibody production of hybridoma cells was studied. The dependence of agitation rate on antibody production is discussed by measuring the amount of monoclonal antibody in cells cultured by a spinner vessel. The effect of shear rate is also studied by exposing a homogeneous shear flow to hybridoma cells in a cone-and-plate viscometer. The dependence of shear rate on hybridoma cells was observed and the increase of antibody production was arised from the increase of secretion from cells.     

Effects of oxygen on the metabolism of nitroxide spin labels in cells

The products of the reduction of nitroxides in cells are the corresponding hydroxylamines, which cells can oxidize back to the nitroxides in the presence of oxygen. Both the reduction of nitroxides and the oxidation of hydroxylamines are enzyme-mediated processes. For lipid-soluble nitroxides, the rates of reduction are strongly dependent on the intracellular concentration of oxygen; severely hypoxic cells reduce nitroxides more rapidly than cells supplied with oxygen. In contrast, the rates of oxidation of hydroxylamines increase smoothly with increasing intracellular oxygen concentration up to 150 microM. In order to separate the effects on the rates of metabolism of nitroxides due directly to oxygen from effects due to the redox state of enzymes, we studied the cells under conditions in which each of these variables could be changed independently. Oxygen affects the metabolism of these nitroxides primarily by interacting with cytochrome c oxidase to change the redox state of the enzymes in the respiratory chain. Our results are consistent with the conclusions that in these cells reduction of lipophilic nitroxides occurs at the level of ubiquinone in the respiratory chain in mitochondria, and oxidation of the corresponding hydroxylamines occurs at the level of cytochrome c oxidase.     

Effect of oxygen tension and rate of pressure reduction during decompression on central gas bubbles

Reinertsen, R. E., V. Flook, S. Koteng, and A. O. Brubakk.Effect of oxygen tension and rate of pressure reduction duringdecompression on central gas bubbles. J. Appl.Physiol. 84(1): 351-356, 1998.Reduction inascent speed and an increase in theO₂ tension in the inspired airhave been used to reduce the risk for decompression sickness. It haspreviously been reported that decompression speed andO₂ partial pressure are linearly related for human decompressions from saturation hyperbaric exposures. The constant of proportionality K(K = rate/partial pressure of inspiredO₂) indicates the incidence ofdecompression sickness. The present study investigated the relationshipamong decompression rate, partial pressure of inspiredO₂, and the number of central gasbubbles after a 3-h dive to 500 kPa while breathing nitrox with an O₂ content of 35 kPa. Weused transesophageal ultrasonic scanning to determine the number ofbubbles in the pulmonary artery of pigs. The results show that, for agiven level of decompression stress, decompression rate andO₂ tension in the inspired air canbe traded off against each other by using pulmonary artery bubbles asan end point. The results also seem to confirm that decompressions thathave a high K value are morestressful.

     

Effect of oxygen transfer rate on levels of key enzymes of xylose metabolism in Debaryomyces hansenii

The titers of key enzymes of xylose metabolism were measured and correlated with the kinetics of xylitol production by Debaryomyces hansenii under different oxygen transfer rates (OTR) in a batch reactor. An OTR change from 2.72 to 4.22 mmol O₂ l⁻¹ min⁻¹ resulted in a decrease in NADPH-dependent xylose reductase (XR) and NAD ± -dependent xylitol dehydrogenase (XDH) activities. For higher values of OTR (12.93 mmol O₂ l⁻¹ min⁻¹, the XDH titer increased twofold whereas the XR titer did not show a significant change. At the lowest OTR (2.72 mmol O₂ l⁻¹ min⁻¹), xylitol (and ethanol) production rates showed the highest values. However, xylitol specific productivity was twice as high as ethanol specific productivity. The titer of the NADPH-forming enzyme, glucose-6-phosphate dehydrogenase (GPDH), increased from 333 to 412 mU mg⁻¹ when the OTR was increased. However, 6-phosphogluconate dehydrogenase (PGDH) activity remained unchanged and at a lower level, which indicates that this enzyme is responsible for the carbon flux control of the oxidative branch of the pentose phosphate pathway. The activity of the alcohol-forming enzyme was repressed at the higher amount of oxygen, decreasing its activity more than 50%. The changes in ADH suggested that two different metabolic regions under oxygen-limited conditions can be hypothesized for xylose metabolism by D. hansenii. For low OTR values (up to 4.22 mmol O₂ l⁻¹ min⁻¹), a fermentative-type activity is displayed. At higher OTR values (above 4.22 mmol O₂ l⁻¹ min⁻¹), no significant fermentative activity is reported.     

干旱胁迫对小麦旗叶活性氧代谢及灌浆速率的影响

在防雨池栽条件下,研究了干旱胁迫对小麦旗叶活性氧代谢的影响.结果表明,小麦旗叶内H2O2和O-·2水平随干旱胁迫的加剧和衰老进程的加快而逐渐升高,活性氧清除系统中的SOD和CAT活性逐渐下降,膜脂过氧化产物MDA含量升高,导致叶片内可溶性蛋白质和叶绿素含量下降,是干旱胁迫造成对籽粒产量贡献较大的旗叶伤害的主要原因之一,从而导致穗粒重下降.     

Effect of water-soluble vitamins on rat diaphragm carbohydrate metabolism

Oxygen and glucose uptakes and glycogen content of isolated rat diaphragm were studied in phosphate-saline medium containing 120 mg% glucose in the presence of moderate up to very high levels of various water-soluble vitamins. Of the significant findings, calcium pantothenate, methyl-methionine-sulfonium chloride, and ascorbic acid, each at a level of 0.75 mg, depressed oxygen uptake. At the latter concentration, inositol and pyridoxine hydrochloride caused a small but definite increase in the mean glycogen level, and PABA depressed both oxygen and glucose uptake by the muscle. More physiologically significant results were obtained with α-lipoic acid, which markedly decreased the glycogen content even at a level of 5 μg, and citrovorum factor (leucovorin; 10 μg), which strongly depressed both the glucose uptake and glycogen. The effect with citrovorum factor did not extend to folic or tetrahydrofolic acids, the three being compared at a level of 1.1 μM.     

Effect of agitation rate on cell release rate and metabolism during continuous fermentation with entrapped growing

The effects of agitation rate (50 to 150 rpm) on cell metabolism and cell release rates were studied during continuous fermentation of de Man, Rogosa and Sharpe medium (MRS) by immobilizedLactobacillus casei subsp.casei in κ-carrageenan/locust bean gum gel beads. Biomass concentration in the outflow was significantly higher at high agitation rates. Shear forces promoted cell loss from the beads by disrupting cell-filled cavities in the gel, near the particle surface. Moreover, high agitation rates enhanced fluid-to-particle mass transfer.     

Effects of oxygen on the membrane structure and the metabolism of lipophilic nitroxide in rat liver microsomes

The effects of oxygen on the metabolism of lipophilic nitroxide and the membrane structure of microsomes were investigated with an ESR spectrometer equipped with a hand-made gas-permeable sampling tube. The half life of gas change in the membrane was confirmed to be 48 s by the line-width of the ESR signal. The signal intensity of lipophilic nitroxide in microsomal membranes decreased with NADPH under N2 and increased under O2. The change of signal intensity was reversible, suggesting that lipophilic nitroxide can be used as an indicator of the redox state in membranes for in vivo ESR imaging.     

Effect of specific oxygen uptake rate on Enterobacter aerogenes energetics: carbon and reduction degree balances in batch cultivations

The effect of oxygen availability on the metabolism of Enterobacter aerogenes NCIMB 10102 was studied through batch fermentations of glucose performed increasing the specific oxygen uptake rate up to 72.7 mmol(O2) C-mol(DW) (-1) x h(-1). The final concentrations of fermentation products of this biosystem (2,3-butanediol, hydrogen, acetoin, formate, acetate, carbon dioxide, ethanol, lactate, succinate, and biomass) were utilized to check the use of simple carbon mass and reduction degree balances for the study of microbial energetics even in batch cultivations.     

Hypoxia/reoxygenation increases the permeability of endothelial cell monolayers: role of oxygen radicals

We assessed the effect of hypoxia/reoxygenation on 14C-albumin flux across endothelial monolayers. Cultured bovine pulmonary artery endothelial cells were grown to confluence on nitrocellulose filters (pore size 12 microns). The endothelialized filters were mounted in Ussing-type chambers which were filled with cell culture medium (M 199). Equimolar amounts (33 nM) of 14C-labeled and unlabeled albumin were added to the "hot" and "cold" chambers, respectively. The monolayers were then exposed to successive periods (90 min) of normoxia (pO2 145 mmHg), hypoxia (pO2 20 mmHg), and reoxygenation (pO2 145 mmHg). A gas bubbling system was used to control media pO2 and to ensure adequate mixing. Four aliquots of culture media were taken during each period in order to calculate the 14C-albumin permeability across the endothelialized filter. In some experiments, either the xanthine oxidase inhibitor, oxypurinol (10 microM), or superoxide dismutase (600 U/mL), was added to the media immediately prior to the experiments. As compared to the normoxic control period, albumin permeability was 1.5 times higher during hypoxia (p less than 0.01) and 2.3 times higher during reoxygenation (p less than 0.01). The reoxygenation-induced increase in albumin permeability was prevented by either oxypurinol or superoxide dismutase. These data indicate that xanthine oxidase-derived oxygen radicals contribute to the hypoxia/reoxygenation-induced endothelial cell dysfunction. The altered endothelial barrier function induced by hypoxia/reoxygenation is consistent with the microvascular dysfunction observed following reperfusion of ischemic tissues.     

Reversible reduction of nitroxides to hydroxylamines: roles for ascorbate and glutathione

Biological applications of stable nitroxyl radicals, NR, include their use as contrast agents for magnetic resonance imaging, spin labels, superoxide dismutase mimics, and antioxidants. The rapid reduction of NR in biological samples into hydroxylamines (HA) significantly limits their application. In turn, reoxidation of HA back to the NR has been used for detection of reactive oxygen species (ROS). In this work comparative studies of the reduction of pyrrolidine, imidazoline, and imidazolidine NR by ascorbate were performed taking advantage of recently synthesized tetraethyl-substituted NR with much higher stability toward reduction both in vitro and in vivo. Surprisingly, these NR kept 10-50% of initial intensity of electron paramagnetic resonance signal for about 1 h in the presence of 100-fold excess of ascorbate. To explain these data, reoxidation of the corresponding HA by ascorbate radical and dehydroascorbic acid back to the NR was proposed. This hypothesis was supported by direct measurement of the NR appearance from the HA on ascorbate radical generation by ascorbate oxidase, or in the presence of the dehydroascorbic acid. The reversible reaction between NR and ascorbate was observed for the various types of NR, and the rate constants for direct and reverse reactions were determined. The equilibrium constants for one-electron reduction of the tetraethyl-substituted NR by ascorbate were found to be in the range from 2.65x10(-6) to 10(-5) which is significantly lower than corresponding values for the tetramethyl-substituted NR (more or about 10(-4)). This explains the establishment of an EPR-detectable quasi-equilibrium level of tetraethyl-substituted NR in the presence of an excess of ascorbate. The redox reactions of the NR-HA couple in ascorbate-containing media were found to be significantly affected by glutathione (GSH). This effect was attributed to the reduction of ascorbate radicals by GSH, and the rate constant of this reaction was found to be equal to 10 M-1 s-1. In summary, the data provide new insight into the redox chemistry of NR and HA, and significantly affect interpretation and strategy of their use as redox- and ROS-sensitive probes, or as antioxidants.     

Effect of phosphate and oxygen concentrations on alginate production and stoichiometry of metabolism of Azotobacter vinelandii under microaerobic conditions

Alginate production by Azotobacter vinelandii was studied in batch and continuous cultures under microaerobic conditions. In batch culture at a pO₂ of 2–3% (air saturation) alginate production was enhanced by decreasing the PO³⁻ ₄ level in the medium. Alginate yield from biomass (Y _P/X) reached the highest value of 0.66 g/g at the lowest phosphate level (100 mg/l), compared to 0.40 g/g and 0.25 g/g at higher phosphate levels (200 mg/l and 400 mg/l, respectively). In contrast, biomass formation behaved differently and the growth yield (Y _X/S) decreased with decreasing PO₄ ³⁻ concentrations. Moreover, the respiratory quotient (RQ) of the culture was dependent on the initial phosphate concentration, especially in the phosphate-limited phase of growth. As the initial phosphate level decreased from 400 mg/l to 100 mg/l, the average RQ value of the culture declined from 1.46 to 0.89. The low RQ value is very close to the theoretical optimum RQ, calculated to be 0.8 on the basis of the stoichiometry of the metabolic pathways for alginate formation from sucrose. This optimum RQ was also confirmed in continuous culture at different dilution rates. Independent of the dilution rate, a pO₂ value of 2–5% (air saturation) was found to be optimal for alginate production, the corresponding RQ values being 0.80–0.84. In addition, the molecular mass and composition of alginate were also found to be affected by both phosphate and oxygen concentrations. In conclusion, the RQ appears to be a useful parameter for optimum control of alginate production with this microorganism. Received: 31 March 1999 / Received revision: 2 July 1999 / Accepted: 5 July 1999     

Substrate reduction and oxygen activation during microsomal metabolism of quinones

2-Dimethylamino-3-chloro-1,4-naphthaquinone (DCNQ) was used to study oxygen and substrate activation in microsomal system. DCNQ was shown to be bound to microsomal cytochrome P-450 as a type I substrate; its N-demethylation was catalyzed by cytochrome P-450. Cytochrome P-450 and NADPH-cytochrome P-450 reductase are capable of DCNQ reduction to semi- and hydroquinones. The OH-radical formed in the presence of DCNQ, NADPH and reductase was detected, using a spin trap (5,5-dimethylpyrroline-N-oxide). The OH-radical formation was shown to be stimulated by the Fe-EDTA complex. Using the OH-radical scavengers (mannitol, N-butanol, alpha-naphthol) and the catalase inhibitor sodium azide, it was shown that the OH-radical participates in microsomal oxidation of DCNQ and aminopyrine. It was assumed that in the course of microsomal oxidation the reduced DCNQ is responsible for: i) stimulation of molecular oxygen reduction to H2O2; ii) reduction of Fe ions (Fe3+----Fe2+) which cause the decomposition of H2O2 in the Fenton reaction resulting in the formation of a strong oxidizing agent--a hydroxyl radical.     

Effect of silver ion, carbon dioxide, and oxygen on ethylene action and metabolism

The relationship between ethylene action and metabolism was investigated in the etiolated pea seedling (Pisum sativum L. cv. Alaska) by inhibiting ethylene action with Ag⁺, high CO₂, and low O₂ and then determining if ethylene metabolism was inhibited in a similar manner. Ag⁺ (100 milligrams per liter) was clearly the most potent antiethylene treatment. Ag⁺ pretreatment inhibited the growth retarding action of 0.2 microliters per liter ethylene by 48% and it also inhibited the incorporation of 0.2 microliters per liter ¹⁴C₂H₄ into pea tips by the same amount. As the ethylene concentration was increased from 0.2 to 30 microliters per liter, the effectiveness of Ag⁺ in reducing ethylene action and metabolism declined in a similar fashion. Although Ag⁺ significantly inhibited the incorporation of ¹⁴C₂H₄ into tissue metabolites, the oxidation of ¹⁴C₂H₄ to ¹⁴CO₂ was unaffected in the same tissue.     

Effect of nickel on oxygen free radical metabolism

The effect of nickel on superoxide dismutase activity (SOD), as well as on rate of hydroxydopamine oxidation, was studied in vitro since lipid peroxidation has been implicated in cell damage by nickel, whose toxicity and carcinogenicity are well established. Nickel strongly inhibits SOD activity. The degree of inhibition is directly proportion to the nickel concentration (tested range 0.066 to 0.33 microgram/mL in the reaction mixture); to the substrate concentration (tested range 0.4 x 10 4M to 1.1 x 10 4M 6-hydroxydopamine); and to reaction mixture. Autoxidation of 6-hydroxydopamine was increased by nickel concentrations higher than 15 micrograms/mL. The combination of excessive oxygen free radical production and inhibition of their elimination by inhibition of SOD activity may contribute to the nickel toxicity that has been reported in industrial accidents, as well as to the high incidence of cancer occurring in nickel workers. It may also contribute to many complications in uremic patients, in whom increased serum nickel levels were reported to be in a similar range to those inhibiting SOD.