Determination of the inorganic degradation products sulfate and sulfamate in the antiepileptic drug topiramate by capillary electrophoresis

A capillary electrophoresis (CE) method has been developed as an alternative method for the determination of the inorganic degradation products sulfate and sulfamate in topiramate drug product and drug substance, currently performed by ion chromatography. The anions are separated in a background electrolyte containing potassium chromate and boric acid, followed by indirect UV detection. By adding tetradecyltrimethylammonium bromide to the electrolyte, analysis is performed under co-electroosmotic flow conditions. Variations in injection volumes and migration times are compensated for by use of an internal standard. The validation of the method, which was performed according to ICH guidelines (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use) [1], comprises specificity, accuracy, linearity, precision, sensitivity and robustness. In addition, the results of an actual tablet sample analysis obtained by this CE method are statistically shown to be in close agreement with those obtained by an ion chromatographic method.     

Energetics of syntrophic ethanol oxidation in defined chemostat cocultures

The ethanol-oxidizing, proton-reducing Pelobacter acetylenicus was grown in chemostat cocultures with either Acetobacterium woodii, Methanobacterium bryantii, or Desulfovibrio desulfuricans. Stable steady state conditions with tightly coupled growth were reached at various dilution rates between 0.02 and 0.14 h^-1. Both ethanol and H₂ steady state concentrations increased with growth rate and were lower in cocultures with the sulfate reducer < methanogen < homoacetogen. Due to the higher affinity for H₂, D. desulfuricans outcompeted M. bryantii, and this one A. woodii when inoculated in cocultures with P. acetylenicus. Cocultures with A. woodii had lower H₂ steady state concentrations when bicarbonate reduction was replaced by the energetically more favourable caffeate reduction. Similarly, cocultures with D. desulfuricans had lower H₂ concentrations with nitrate than with sulfate as electron acceptor. The Gibbs free energy (G) available to the H₂-producing P. acetylenicus was independent of growth rate and the H₂-utilizing partner, whereas the G available to the latter increased with growth rate and the energy yielding potential of the H₂ oxidation reaction. The critical Gibbs free energy (G_c), i.e. the minimum energy required for H₂ production and H₂ oxidation, was-5.5 to-8.0 kJ mol^-1 H₂ for P. acetylenicus,-5.1 to-6.3 kJ mol^-1 H₂ for A. woodii,-7.5 to-9.1 kJ mol^-1 H₂ for M. bryantii, and-10.3 to-12.3 kJ mol^-1 H₂ for D. desulfuricans. Obviously, the potentially available energy was used more efficiently by homoacetogens > methanogens > sulfate reducers.     

Protonmotive force in freshwater sulfate-reducing bacteria,and its role in sulfate accumulation in Desulfobulbus propionicus

The protonmotive force in several sulfate-reducing bacteria has been determined by means of radiolabelled membrane-permeant probes (tetraphenyl-phosphonium cation, TPP⁺, for , and benzoate for pH). In six of ten freshwater strains tested only the pH gradient could be determine, while the membrane potential was not accessible due to nonspecific binding of TPP⁺. The protonmotive force of the other four strains was between –110 and –155 mV, composed of a membrane potential of –80 to –140 mV and a pH gradient between 0.25 and 0.8 (inside alkaline) at pH_out=7. In Desulfobulbus propionicus the pH gradient decreased with rising external pH values. This decrease, however, was compensated by an increasing membrane potential. Sulfate, which can be highly accumulated by the cells, did not affect the protonmotive force, if added in concentrations of up to 4 mM. The highest sulfate accumulation observed (2500-fold), which occurred at external sulfate concentrations below 5 M, could be explained by a symport of three protons per sulfate, if equilibrium with the protonmotive force was assumed. At higher sulfate concentrations the accumulation decreased and suggested an electroneutral symport of two protons per sulfate. At sulfate concentrations above 500 M, the cells stopped sulfate uptake before reaching an equilibrium with the protonmotive force.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - MOPS morpholinopropanesulfonic acid - TPP⁺ tetraphenylphosphonium cation - EDTA ethylenediaminetetraacetic acid - pH transmembrane pH gradient (pH_in-pH_out) - transmembrane electrical potential difference     

Electron transport across the plasmalemma of Lemna gibba G1

Lemna gibba L., grown in the presence or absence of Fe, reduced extracellular ferricyanide with a V _max of 3.09 mol · g^-1 fresh weight · h^-1 and a K _m of 115 M. However, Fe³⁺-ethylenediaminetetraacetic acid (EDTA) was reduced only after Fe-starvation. External electron acceptors such as ferricyanide, Fe³⁺-EDTA, 2,6-dichlorophenol indophenol or methylene blue induced a membrane depolarization of up to 100 mV, but electron donors such as ferrocyanide or NADH had no effect. Light or glucose enhanced ferricyanide reduction while the concomitant membrane depolarization was much smaller. Under anaerobic conditions, ferricyanide had no effect on electrical membrane potential difference (E_m). Ferricyanide reduction induced H⁺ and K⁺ release in a ratio of 1.16 H⁺+1 K⁺/2 e^- (in +Fe plants) and 1.28 H⁺+0.8 K⁺/2 e^- (in -Fe plants). Anion uptake was inhibited by ferricyanide reduction. It is concluded that the steady-state transfer of electrons and protons proceeds by separate mechanisms, by a redox system and by a H⁺-ATPase.Abbreviations E _m electrical membrane potential difference - EDTA ethylenediaminetetraacetic acid - DCPIP dichlorophenol indophenol - +Fe control plant - -Fe iron-deficient plant - FW fresh weight - H⁺ electrochemical proton gradient     

The effect of sulfate reduction on the thermophilic (55°C) methane fermentation process

Summary The continuously operated suspended growth anaerobic contact system was utilized to estimate the effect of sulfate reduction on the thermophilic (55°C) methane fermentation process. Results indicated that reduction in methanogenesis in the presence of sulfate was due to two separate, but related, processes;i.e. competitive and sulfide inhibition. Although prevention of competitive inhibition would be difficult under normal fermenter operation, sulfide inhibition could be minimized by environmental selection of sulfide tolerant microbial populations through biomass recycle and pH control. Stable fermenter operation was achieved at soluble sulfide concentrations as high as 330 mg/l soluble sulfide. Using batch fermenters, a maximum thermophilic sulfate reduction rate of 3.7 mg SO₄ ^2––S/g volatile solids (VS)-day was estimated. The importance of reporting sulfate reduction rates on a biomass basis is demonstrated by a simple population adjustment kinetic model.This research study was conducted at the Department of Agricultural Engineering, Cornell University, Riley Robb Hall, Ithaca, NY 14853, U.S.A.     

Fate of elemental sulfur in an intertidal sediment

Abstract: Sediment from a tidal flat at Wedderwarden, near the mouth of the Weser estuary, northern Germany, was amended with elemental sulfur, and concentrations of metabolic end products were monitored. The production of both sulfate and sulfide was consistent with disproportionation as the most important fate of the added elemental sulfur. A population of bacteria conducting active elemental sulfur disproportionation was also enriched from the sediment. In the enrichments, containing both elemental sulfur and Fe oxides as a sulfide 'scrub', sulfide and sulfate were produced in a ratio of     , somewhat lower than the predicted ratio of     . The mismatch between predicted and observed production ratios is explained by the channelling of electrons into autotrophic or mixotrophic CO₂ fixation rather than sulfide formation. The production of organic carbon, in the correct amount to explain the observed sulfide to sulfate production ratio, was verified by organic carbon analysis. Finally, rates of sulfate reduction were identical in the elemental sulfur amended sediment, and in control sediment with no added sulfur. Hence, the heterotrophic bacterial community was completely unaffected by an active metabolism conducting elemental sulfur disproportionation.     

Glutathione synthesis in maize genotypes with different sensitivities to chilling

The effect of chilling on enzymes, substrates and products of sulfate reduction, gultathione synthesis and metabolism was studied in shoots and roots of maize (Zea mays L.) genotypes with different chilling sensitivity. At full expansion of the second leaf, chilling at 12 °C inhibited dry weight increase in shoots and roots compared to controls at 25 °C and induced an increase in adenosine 5-phosphosulfate sulfotransferase and -glutamylcysteine synthetase (EC 6.3.2.2) activity in the second leaf of all genotypes tested. Glutathione synthetase (EC 6.3.2.3) activity was about one order of magnitude higher than -glutamylcysteine synthetase activity, but remained unchanged during chilling except for one genotype. During chilling, cysteine and glutathione content of second leaves increased to significantly higher levels in the two most chilling-tolerant genotypes. Comparing the most tolerant and most sensitive genotype showed that chilling induced a greater incorporation of³⁵S from [³⁵S]sulfate into cysteine and glutathione in the chilling-tolerant than in the sensitive genotype. Chilling decreased the amount of³⁵S-label incorporated into proteins in shoots of both genotypes, but had no effect on this incorporation in the roots. Glutathione reductase (EC 1.6.4.2) and nitrate reductase (EC 1.6.6.1) activity were constitutively higher in the chilling-tolerant genotypes, but showed no changes in most examined genotypes during 3 d at 12 °C. Our results indicate that in maize glutathione is involved in protection against chilling damage.Abbreviations APSSTase adenosine 5-phosphosulfate sulfotransferase - EC -glutamylcysteine - GR glutathione reductase - OSH glutathione - NR nitrate reductase We thank M. Suter for preparing [³⁵S]adenosine 5-phosphosulfate, Dr. A. Fleming (both our Institute) for correcting the English and M. Soldati (Eschlikon, Switzerland) for his help with the plant material. This work was supported by COST 814 Crop development for the wet and cool regions of Europe.     

Purification and characterization of ferredoxin-sulfite reductases from leek (Allium tuberosum) leaves

Ferredoxin-sulfite reductases (Fd-SiRs) [hydrogen-sulfide: ferredoxin oxidoreductase, EC 1.8.7.1] from leek leaves have been purified to homogeneity. The enzymes (SiR 1, SiR 2 and SiR 3) were separated by Mono Q chromatography. The collective molecular mass of the enzymes was estimated to be 65 kDa by gel filtration. In all three cases, subunit analysis by SDS-PAGE yielded a single protein band corresponding to a molecular mass of 64 kDa, indicating that the enzymes each exist as a monomer. In the oxidized forms, SiR 1, SiR 2 and SiR 3 all exhibited nearly identical absorption maxima at 279∼280, 389∼390, 588 and 714 nm, indicating that siroheme is involved in the catalysis of sulfite reduction. On enzymatic properties, SiR 1, SiR 2 and SiR 3 could only react with the physiological electron donor, feriedoxin. The enzymes exhibited different heat stabilities. The pH active curve obtained from SiR 2 was different from the others. Moreover, SiR 1 exhibited a lower Km value for ferredoxin than SiR 2. Although the N-terminal sequence was the same, the results of some enzymatic properties, amino acid analysis, and peptide mapping suggested the presence of the Fd-SiR isozymes in leek leaves.     

Net Uptake of Sulfate and its Transport to the Shoot in Spinach Plants Fumigated with H2S or SO2: Does Atmospheric Sulfur Affect the “Inter-Organ” Regulation of Sulfur Nutrition?*

Spinach plants (Spinacea oleracea L. cv. Estivato) were grown on nutrient solutions under deficient, normal and excess sulfate supply. In both young and mature plants net uptake of sulfate and its transport to the shoot increased with increasing sulfate supply, but both processes proceeded at a higher rate in young as compared to mature plants. The relative sulfate transport, i.e. the relative amount of the sulfate taken up that is transported to the shoot, decreased with increasing sulfate supply. Apparently, net uptake of sulfate is not strictly controlled by the sulfur demand of the shoot, but xylem loading appears to counteract excess transport of sulfate to the shoot. Fumigation with H₂S or SO₂ reduced net uptake of sulfate by the roots in sulfur-deficient plants and absolute as well as relative sulfate transport to the shoot independent of the three sulfate levels supplied to the plant. At the same time thiol contents of the shoot and the root were enhanced by fumigation with H₂S and SO₂. These findings are consistent with the idea that thiols produced in the leaves can mediate demand-driven control of sulfate uptake by the roots and its transport to the shoot.