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  • 1
    Keywords: evaluation ; Germany ; human ; QUANTIFICATION ; TOOL ; DISEASE ; PATIENT ; RAT ; SUSCEPTIBILITY ; smoking ; SPECTROMETRY ; PATHOGENESIS ; MASS-SPECTROMETRY ; HELICOBACTER-PYLORI ; INFLAMMATORY-BOWEL-DISEASE ; INJURY ; MASSES ; LIGHT ; BILE ; COLITIS ; GASTRIC-MUCOSA ; GASTRODUODENAL MUCOSAL PROTECTION ; inflammatory bowel disease ; mucosa barrier ; NanoESI MS/MS ; PANCREATITIS ; phosphatidylcholine ; PHOSPHOLIPIDS ; STANDARDS ; ulcerative colitis ; WEIGHT
    Abstract: Background: A defective mucus composition represents a key pathogenetic factor for intestinal injury. Phosphatidylcholine (PC) is an essential component contributing to formation of a hydrophobic mucus layer. For evaluation of PC in the pathogenesis of inflammatory bowel disease, the concentration and composition of PC in the rectal mucus Of patients with ulcerative colitis was determined. Electrospray ionization (ESI) tandem mass spectrometry (MS/MS) allows quantification of PC species and enables analysis of crude extracts. Methods: Lipid extracts of material obtained by light scrapings of the intestinal lumen were analysed quantitatively by nanoESI MS/MS with synthetic internal PC and lysophosphatidylcholine (LPC) standards. PC and LPC species from rectoscopically acquired Mucus aliquots of patients with ulcerative colitis were compared to Crohn disease and control subjects. Results: Patients with inactive ulcerative colitis showed significantly less PC and LPC (median 346 [IQR: 2304051 pmol total PC/mg dry weight) in rectal mucus compared to Crohn disease (median 1126 [IQR: 4651-9411 pmol total PC/mg dry weight) and control subjects (median 1285 [IQR: 850-1639] pmol total PC/mg dry weight) (P 〈 0.05). The molecular species of PC and LPC were not significantly different between the groups. The most abundant species were PC 16:0/18:1; PC 16:0/18:2;, PC 18:0/18:1; PC 18:0/18:2 LPC 16:0: and LPC 18:0. Conclusion: NanoESI MS/MS is a suitable tool for analysing and quantifying small amounts of PC in human mucus. Patients with ulcerative colitis have significant less PC in their intestinal mucus despite a comparable PC molecular species composition pattern. This suggests that a low amount of protective mucus PC is a characteristic feature in ulcerative colitis and explains an increased susceptibility to luminal contents
    Type of Publication: Journal article published
    PubMed ID: 15513358
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  • 2
    Keywords: CELLS ; IN-VITRO ; PROTECTION ; CELL ; evaluation ; Germany ; PERFUSION ; VITRO ; PROTEIN ; MICE ; RAT ; animals ; colon ; INJECTION ; SPECTROSCOPY ; FORM ; NO ; IN-SITU ; DIFFERENCE ; MEMBRANE ; RATES ; electrospray ionization ; TANDEM MASS-SPECTROMETRY ; IONIZATION ; EPITHELIAL-CELLS ; Jun ; ORIGIN ; MASSES ; BILE ; phosphatidylcholine ; bile acid ; intestinal mucus layer ; LAMELLAR BODIES ; MDR3 P-GLYCOPROTEIN ; MUCOSAL BARRIER ; perfusion study ; phosphatidylcholine transport ; SURFACTANT-LIKE PARTICLES
    Abstract: Background: Intestinal mucus not only facilitates substrate absorption, but also forms a hydrophobic, phosphatidylcholine (PC) enriched, barrier against luminal gut contents. Methods: For evaluation of the origin of PC in intestinal mucus, we first analyzed the mucus PC in mice with absent biliary phospholipid secretion (mdr2 ( - / -) mice) using electrospray ionization (ESI) tandem mass spectroscopy (MS/MS). Second, in situ perfused rat jejunum, ileum and colon were analyzed after i.v. bolus injections of 155 pmol [H-3]-PC. Additional in vitro experiments were performed with isolated mucosal cells after incubation with the PC precursor [H-3]-choline. Results: In mdr2 ( - / -) mice and control animals no significant quantitative difference in mucus PC was found, indicating that mucus PC is of intestinal and not biliary origin. In situ perfusion studies detected intestinal secretion of [H-3]-PC, which was stimulated in presence of 2 mM taurocholate (TC). Secretion rates of [H-3]-PC were highest in ileum (9.0 +/- 0.8 fmol h(-1) x cm(-1)), lower in jejunum (4.3 +/- 0.5) and minimal in colon (0.8 +/- 0.2). It compares to an intestinal secretion of native PC originating to 64% from bile, 9% from jejunum, 28% from ileum, and 1% from colon. Complementary in vitro studies showed 30-min secretion rates for [H-3]-PC to be highest in enterocytes from ileum (26.5 +/- 5.3% of intracellular [H-3]-PC) and jejunum (19.8 +/- 2.9%), and significantly lower in colonocytes (8.4 +/- 1.3%). Conclusion: PC in the intestinal mucus originates from secretion by ileal and jejunal enterocytes. (C) 2004 Elsevier B.V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 15158757
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  • 3
    Abstract: OBJECTIVE: Cancer cells in the body release soluble and membranous factors that manipulate the tumor environment to facilitate growth and survival. Recent years have provided evidence that small microvesicles that are termed exosomes may play a pivotal role in this process. Exosomes are membrane vesicles with a size of 40-100nm that are released by both tumor and normal cells and can be found in various body fluids. Tumor-derived exosomes carry functional proteins, mRNAs, and miRNAs and could serve as novel platform for tumor diagnosis and prognosis. However, marker proteins that allow enrichment of tumor-derived exosomes over normal exosomes are less well defined. METHODS: We used Western blot analysis and antibody coupled magnetic beads to characterize CD24 and EpCAM as markers for exosomes. We investigated ovarian carcinoma ascites, pleural effusions and serum of breast carcinoma patients. As non-tumor derived control we used exosomes from ascites of liver cirrhosis patients. RESULTS: Exosomes could be isolated from all body fluids and contained marker proteins as well as miRNAs. We observed that CD24 and EpCAM were selectively present on ascites exosomes of tumor patients and copurified together on anti-EpCAM or anti-CD24 magnetic beads. In breast cancer patients CD24 was present but EpCAM was absent from serum exosomes. Instead, the intact EpCAM ectodomain was recovered in a soluble form. We provide evidence that EpCAM can be cleaved from exosomes via serum metalloproteinase(s). CONCLUSION: Loss of EpCAM on serum exosomes may hamper enrichment by immune-affinity isolation. We suggest that CD24 could be an additional marker for the enrichment of tumor-derived exosomes from blood.
    Type of Publication: Journal article published
    PubMed ID: 21601258
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  • 4
    Keywords: Germany ; human ; DISEASE ; DISEASES ; PROTEIN ; MICE ; PATIENT ; ACID ; HUMANS ; mass spectrometry ; tandem mass spectrometry ; TANDEM MASS-SPECTROMETRY ; PATHOGENESIS ; MASS-SPECTROMETRY ; STRATEGIES ; RANDOMIZED-TRIAL ; ULCERATIVE-COLITIS ; CROHNS-DISEASE ; rodent ; INFLAMMATORY-BOWEL-DISEASE ; COLITIS ; GASTRIC-MUCOSA ; GASTRODUODENAL MUCOSAL PROTECTION ; inflammatory bowel disease ; phosphatidylcholine ; SPHINGOMYELIN ; USA ; ACID-RESIDUES ; NOV ; lipid ; PULMONARY SURFACTANT ; therapeutic ; STRATEGY ; INVESTIGATE ; CD ; COLONIC-MUCOSA ; EXTRACTS ; G-Protein ; GASTROINTESTINAL MUCUS ; lysophosphatidylcholine ; nano ESI mass spectrometry
    Abstract: Background: Phospholipids are essential for the normal function of the intestinal mucus barrier. The objective of this study was to systematically investigate phospholipids in the intestinal mucus of humans Suffering from inflammatory bowel diseases, where it barrier defect is strongly supposed to be pathogenetic. Methods: Optimal mucus recovery was first validated in healthy mice and the method was then transferred to the endoscopic acquisition of ileal and colonic mucus from 21 patients with ulcerative colitis (UC), 10 patients with Crohn's disease (CD), and 29 healthy controls. Nano-electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to determine phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and sphingomyelin (SM) in lipid extracts of mucus specimens. Results: Human and rodent mucus contained very similar phospholipid species. In the ileal and colonic mucus from patients suffering from UC, the concentration of PC was highly significantly lower (607 +/- 147 pmol/100 mu g protein and 745 +/- 148 pmol/100 mu g protein) compared to that of patients with CD (3223 +/- 1519 pmol/100 mu g protein and 2450 +/- 431 pmol/100 mu g protein) and to controls (3870 +/- 760 pmol/100 mu g protein and 2790 +/- 354 pmol/100 mu g protein), overall, P = 0.0002 for ileal specimens and P 〈 0.0001 tor colonic specimens. Independent of disease activity, patients suffering from UC showed an increased Saturation grade of PC fatty acid residues and it higher LPC-to-PC ratio. Conclusions: The intestinal mucus barrier of patients with UC is significantly altered concerning its phospholipid concentration and species composition. These alterations may be very important for the pathogenesis of this disease and underline new therapeutic strategies
    Type of Publication: Journal article published
    PubMed ID: 19504612
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  • 5
    Keywords: DISEASE ; ASSAY ; TANDEM MASS-SPECTROMETRY ; PATHOGENESIS ; cholesterol ; SERUM ; COLITIS ; PHOSPHOLIPIDS ; BILE-ACIDS ; ABCB4
    Abstract: AIM: To analyze phospholipid profiles in intrahepatic bile from patients with primary sclerosing cholangitis (PSC) and secondary sclerosing cholangitis (SSC). METHODS: Intrahepatic bile specimens collected via endoscopic retrograde cholangiography from 41 patients were analyzed. Fourteen of these patients were diagnosed with PSC, 10 with SSC, 11 with choledocholithiasis or no identifiable biliary disease, and 6 with cholangiocellular carcinoma (CCC). Bile acid, cholesterol, protein, and bilirubin contents as well as pancreas lipase activity in bile were determined by biochemical methods. Phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) species were quantified using nano-electrospray ionization tandem mass spectrometry. RESULTS: Bile from all the examined patient groups showed a remarkably similar PC and LPC species composition, with only minor statistical differences. Total biliary PC concentrations were highest in controls (8030 +/- 1843 mumol/L) and lowest in patients with CCC (1969 +/- 981 mumol/L) (P = 0.005, controls vs SSC and CCC, respectively, P 〈 0.05). LPC contents in bile were overall low (4.2% +/- 1.8%). Biliary LPC/PC ratios and ratios of biliary PC to bilirubin, PC to cholesterol, PC to protein, and PC to bile acids showed no intergroup differences. CONCLUSION: PC and LPC profiles being similar in patients with or without sclerosing cholangitis, these phospholipids are likely not of major pathogenetic importance in this disease group.
    Type of Publication: Journal article published
    PubMed ID: 24023488
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