26 research outputs found

    Characterization of Polymer Membranes by MALDI Mass-Spectrometric Imaging Techniques

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    For physical and chemical characterization of polymers, a wide range of analytical methods is available. Techniques like NMR and X-ray are often combined for a detailed characterization of polymers used in medical applications. Over the past few years, MALDI mass-spectrometry has been developed as a powerful tool for space-resolved analysis, not least because of its mass accuracy and high sensitivity. MALDI imaging techniques combine the potential of mass-spectrometric analysis with imaging as additional spatial information. MALDI imaging enables the visualization of localization and distribution of biomolecules, chemical compounds, and other molecules on different surfaces. In this study, surfaces of polymeric dialyzer membranes, consisting of polysulfone (PS) and polyvinylpyrrolidone (PVP), were investigated, regarding chemical structure and the compound’s distribution. Flat membranes as well as hollow fiber membranes were analyzed by MALDI imaging. First, analysis parameters like laser intensity and laser raster step size (spatial resolution in resulting image) were established in accordance with polymer’s characteristics. According to the manufacturing process, luminal and abluminal membrane surfaces are characterized by differences in chemical composition and physical characteristics. The MALDI imaging demonstrated that the abluminal membrane surface consists more of polysulfone than polyvinylpyrrolidone, and the luminal membrane surface displayed more PVP than PS. The addition of PVP as hydrophilic modifier to polysulfone-based membranes increases the biocompatibility of the dialysis membranes. The analysis of polymer distribution is a relevant feature for characterization of dialysis membranes. In conclusion, MALDI imaging is a powerful technique for polymer membrane analysis, regarding not only detection and identification of polymers but also localization and distribution in membrane surfaces

    Representative multi-capillary column/ion mobility spectra (MCC/IMS) of breath samples.

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    <p>Breath sample of (A) a healthy adult, (B) an end-stage renal disease proband before and (C) after hemodialysis treatment. Areas of interest are marked and labeled by numbers. Substances corresponding to these numbers are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046258#pone-0046258-t002" target="_blank">Table 2</a>. Signal intensity is coded by colours (yellow: very high; red high, blue: moderate, white: no signal).</p

    Harmful Effects of the Azathioprine Metabolite 6-Mercaptopurine in Vascular Cells: Induction of Mineralization

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    <div><p>Vascular mineralization contributes to the high cardiovascular morbidity and mortality in patients who suffer from chronic kidney disease and in individuals who have undergone solid organ transplantation. The immunosuppressive regimen used to treat these patients appears to have an impact on vascular alterations. The effect of 6-mercaptopurine (6-MP) on vascular calcification has not yet been determined. This study investigates the effect of 6-MP on vascular mineralization by the induction of trans-differentiation of rat vascular smooth muscle cells <i>in vitro</i>. 6-MP not only induces the expression of osteo-chondrocyte-like transcription factors and proteins but also activates alkaline phosphatase enzyme activity and produces calcium deposition in <i>in vitro</i> and <i>ex vivo</i> models. These processes are dependent on 6-MP-induced production of reactive oxygen species, intracellular activation of mitogen-activated kinases and phosphorylation of the transcription factor Cbfa1. Furthermore, the metabolic products of 6-MP, 6-thioguanine nucleotides and 6-methyl-thio-inosine monophosphate have major impacts on cellular calcification. These data provide evidence for a possible harmful effect of the immunosuppressive drug 6-MP in vascular diseases, such as arteriosclerosis.</p></div

    Signal intensities of exemplary analytes that accumulate during hemodialysis.

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    <p>Signal intensities of analytes P9 and P11 in 28 healthy controls, 26 patients with chronic renal failure (CRF) stage 2–4 according to K/DOQI-criteria, 28 patients with end-stage renal disease (ESRD, CRF stage 5D) prior to and 22 after hemodialysis. Signal intensities were tested for statistical significance by two-tailed t-tests; *p<0.05, **p<0.01, ***p<0.001.</p

    Signal intensities of exemplary analytes that accumulate with decreasing renal function and are eliminated by dialysis.

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    <p>Figures A–C present signal intensities of exemplary analytes P1–P3 and Figure D the sum of the signal intensities of the five analytes that accumulate with decreasing function and are eliminated by dialysis (P1–P5) in 28 healthy controls, 26 patients with chronic renal failure (CRF) stage 2–4 according to K/DOQI-criteria, 28 patients with end-stage renal disease (ESRD, CRF stage 5D) prior to and 22 after hemodialysis. Signal intensities were tested for statistical significance by two-tailed t-tests; *p<0.05, **p<0.01, ***p<0.001.</p

    Scheme of an ion mobility spectrometer (MCC/IMS).

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    <p>The multi-capillary column (MCC) provides a preseparation of the molecules in the gas phase. In the ionization chamber proton transfer from the reactant ions to the analyte molecules takes place, thus forming protonated analyte ions. The drift time of the ions in the electric field depends on size and shape of the analytes. The retention time in the MCC and mobility in the IMS characterize the identity of the analyte. The intensity of the signal is a measure of the analyte's concentration.</p

    Receiver-operating-characteristic (ROC) curves to distinguish different stages of renal failure.

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    <p>ROC curves for the sum of the signal intensities of hydroxyacetone, hydroxy-2-butanone, ammonia, 0.5468–17.0, and 0.5985–55.6 in differentiating (A) healthy subjects and patients with chronic renal failure (CRF) corresponding to an eGFR of 10–59 ml/min per 1.73 m<sup>2</sup> (AUC 0.76), (B) healthy subjects and patients with endstage renal disease (ESRD, AUC 0.83), and (C) healthy subjects and all patients with impaired renal function (CRF and ESRD; AUC 0.80).</p

    mRNA expression of osteogenic proteins.

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    <p>(A–C) VSMCs were stimulated with 6-MP as indicated and mRNA expression was detected after 48 h. Data represent means±SEM, n≥6,*p<0.05 vs. control. (D) VSMCs were stimulated with 6-MP for 48 h. Nuclear proteins were extracted. Cbfa1, Cbfa1-phospho and TATA-bp were detected via Western blot. Representative images and relative band intensities of 3 independent blots of Cbfa1-phospho are shown. (E) MEK1 and ERK1/2 activation was detected via Bio-Plex (n≥6). Values are given as % of control and are normalized to total kinase. (F) mRNA expression of cbfa1 after 48 h treatment with 6-MP (100 µmol/L) ± U0126 (1 µmol/L) (n>6). Data represent means±SEM, *p<0.05 vs. control. ALP: alkaline phosphatase, cbfa1: core binding factor alpha-1, 6-MP: 6-mercaptopurine, OCN: osteocalcin.</p

    Involvement of ROS.

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    <p>(A,B,E,F) VSMCs were stimulated as indicated for 30 min before labeling cells with DHE. Superoxide production was (A) visualized via fluorescence microscopy (representative images from 3 independent experiments) or (B,E,F) quantified in a fluorescence plate reader (n≥6). (C,D) Hydrogen peroxide production is measured in H<sub>2</sub>DCFDA-labeled cells via flow cytometry. (C) Representative histograms of flow data (grey: control, white: 6-MP). (D) Quantification of fluorescence intensity by % of labeled control (n≥6). (E) Stimulation with 6-MP (100 µmol/L) or its metabolites (each 10 µmol/L). (F) Stimulation with 6-MP (100 µmol/L) alone or costimulation with inhibitors (tiron [1 mmol/L] and allopurinol [1 µmol/L]). DHE: dihydroethidium, 6-MP: 6-mercaptopurine, 6-MTIMP: 6-methylthioinosine monophosphate, 6-TGDP: 6-thioguanosine diphosphate, 6-TGMP: 6-thioguanosine monophosphate, 6-TGN: 6-thioguanine nucleotide, 6-TGTP: 6-thioguanosine triphosphate, 6-TU: 6-thiouric acid.</p
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