Development of a sensitive and quantitative HPLC-FLD method for the determination of obestatin in human plasma

Obestatin is a gastrointestinal system peptide. The quantification of this peptide is conventionally performed using immunological techniques. In this study, a selective and sensitive HPLC method coupled with fluorescence detection for the quantitation of obestatin in human plasma was developed and validated. The separation was obtained on a C₁₈ (4.6 × 100 mm, 3.5-μm particles) column using a mobile phase composed of acetonitrile and water, both including 0.1% trifluoroacetic acid. The developed method was found to be linear in the concentration range of 20 to 1000 ng/mL, with a coefficient of determination of 0.9982. The precision results were less than 10%, and the accuracy results were between 92% and 107%. The detection and quantification limit values were obtained as 2.8 and 9.4 ng/mL, respectively. Analyte solutions were found stable for 24 h at room temperature, three freeze–thaw cycles, and 2 weeks at −20°C. The developed method was successfully used for the quantification of obestatin in human plasma samples. In conclusion, the developed method is sensitive and specific for measuring the plasma concentrations of obestatin.     

Extension of the yeast metabolic model to include iron metabolism and its use to estimate global levels of iron-recruiting enzyme abundance from cofactor requirements

Metabolic networks adapt to changes in their environment by modulating the activity of their enzymes and transporters; often by changing their abundance. Understanding such quantitative changes can shed light onto how metabolic adaptation works, or how it can fail and lead to a metabolically dysfunctional state. We propose a strategy to quantify metabolic protein requirements for cofactor-utilising enzymes and transporters through constraint-based modelling. The first eukaryotic genome-scale metabolic model to comprehensively represent iron metabolism was constructed, extending the most recent community model of the Saccharomyces cerevisiae metabolic network. Partial functional impairment of the genes involved in the maturation of iron-sulphur (Fe-S) proteins was investigated employing the model and the in silico analysis revealed extensive rewiring of the fluxes in response to this functional impairment, despite its marginal phenotypic effect. The optimal turnover rate of enzymes bearing ion cofactors can be determined via this novel approach; yeast metabolism, at steady state, was determined to employ a constant turnover of its iron-recruiting enzyme at a rate of 3.02 × 10 ⁻¹¹ mmol·(g biomass) ⁻¹·h ⁻¹.     

Pharmacological preconditioning with erythropoietin reduces ischemia–reperfusion injury in the small intestine of rats

AimsConsidering the implications that arose from several recent experimental studies using recombinant human erythropoietin in rodents, erythropoietin has been regarded as a pharmacological preconditioning agent. The purpose of the present study was to evaluate whether erythropoietin has a preconditioning effect against ischemia and reperfusion injury in the small intestine of the rat.Main methodsIntestinal ischemia was induced in male Wistar rats by clamping the superior mesenteric artery for 30 min, followed by reperfusion for 180 min. Recombinant human erythropoietin (1000 or 3000 U/kg) or vehicle was administered intraperitoneally 24 h prior to ischemia. After collection of ileal tissue, evaluation of damage was based on measurements of the accumulation of polymorphonuclear neutrophils by technetium-99m-labeled leukocyte uptake, content of malondialdehyde, reduced glutathione, contractile responses to agonists, and an evaluation of histopathological features in intestinal tissue.Key findingsTreatment with erythropoietin 24 h before ischemia significantly reduced the tissue content of malondialdehyde and increased that of reduced glutathione. Pretreatment also significantly suppressed leukocyte infiltration into the postischemic tissue, as evidenced by the lower content of myeloperoxidase and technetium-99m-labeled leukocytes. Physiological and histopathological improvements were also significant with the rHuEpo treatment.SignificanceResults of the present study indicate that rHuEpo is an effective preconditioning agent in ischemic injury of the small intestine. Protection provided by recombinant human erythropoietin is closely related to the inhibition of oxidative stress and leukocyte infiltration, which might be among the possible protective mechanisms of erythropoietin in intestinal ischemia and reperfusion.