An on-line solid phase extraction procedure for the routine quantification of urinary methylmalonic acid by liquid chromatography-tandem mass spectrometry

Background: The goal of this study was to develop and to validate an improved isotope-dilution-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of methylmalonic acid (MMA) in urine. Methods: A previously described sample preparation protocol requires two solvent extraction steps, including evaporation. The first extraction is to extract the analyte from the sample, and second occurs following derivatization of the extract. In the method described here, the second evaporation step was substituted by on-line solid phase extraction employing column-switching and a permanent co-polymer based extraction cartridge. A standard validation protocol was applied to investigate the performance of the method. Results: The method was found to be linear in the clinically relevant range of concentrations (6-100 mu mol/L). Total coefficients of variation were below 10% and inaccuracy was <10% for quality control samples at three concentrations. Conclusions: By omitting one evaporation step, the semi-automated method described in this article enables for more convenient work-flow in the quantification of urinary MMA compared to the previous protocol. This is of relevance for MMA measurement in the routine clinical laboratory setting. Validation demonstrated acceptable analytical performance. Clin Chem Lab Med 2010;48:1647-50

External quality assessment of urinary methylmalonic acid quantification - results of a pilot study

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Overexpression and altered glycosylation of MUC1 in malignant mesothelioma

Current interest in the MUC1/EMA mucin relates to its role in malignancy, and its potential as a therapeutic target. MUC1/EMA expression has been observed in the majority of epithelioid mesotheliomas. However, little is known of the characteristics of MUC1/EMA in mesothelioma. Herein, we studied the cell surface and soluble expression of the MUC1/EMA glycoprotein, and determined the mRNA and genomic expression profiles in mesothelioma. We found that the anti-MUC1 antibody, E29, was the most diagnostically useful of seven antibody clones examined with a sensitivity of 84% (16 out of 19 cases) and no false positive results. MUC1 mRNA expression was significantly higher in mesothelioma samples than in benign mesothelial cells. No amplification of the MUC1 gene was observed by FISH. Seven of 9 mesothelioma samples expressed MUC1-secreted mRNA isoform in addition to the archetypal MUC1/transmembrane form. CA15.3 (soluble MUC1) levels were significantly higher in the serum of mesothelioma patients than in healthy controls but were not significantly different to levels in patients with benign asbestos-related disease. CA15-3 in effusions could differentiate malignant from benign effusions but were not specific for mesothelioma. Thus, as in other cancers, alterations in MUC1 biology occur in mesothelioma and these results suggest that specific MUC1 characteristics may be useful for mesothelioma diagnosis and should also be investigated as a potential therapeutic target

Kinetic regulation of multi-ligand binding proteins

Background: Second messengers, such as calcium, regulate the activity of multisite binding proteins in a concentration-dependent manner. For example, calcium binding has been shown to induce conformational transitions in the calcium-dependent protein calmodulin, under steady state conditions. However, intracellular concentrations of these second messengers are often subject to rapid change. The mechanisms underlying dynamic ligand-dependent regulation of multisite proteins require further elucidation. Results: In this study, a computational analysis of multisite protein kinetics in response to rapid changes in ligand concentrations is presented. Two major physiological scenarios are investigated: i) Ligand concentration is abundant and the ligand-multisite protein binding does not affect free ligand concentration, ii) Ligand concentration is of the same order of magnitude as the interacting multisite protein concentration and does not change. Therefore, buffering effects significantly influence the amounts of free ligands. For each of these scenarios the influence of the number of binding sites, the temporal effects on intermediate apo- and fully saturated conformations and the multisite regulatory effects on target proteins are investigated. Conclusions: The developed models allow for a novel and accurate interpretation of concentration and pressure jump-dependent kinetic experiments. The presented model makes predictions for the temporal distribution of multisite protein conformations in complex with variable numbers of ligands. Furthermore, it derives the characteristic time and the dynamics for the kinetic responses elicited by a ligand concentration change as a function of ligand concentration and the number of ligand binding sites. Effector proteins regulated by multisite ligand binding are shown to depend on ligand concentration in a highly nonlinear fashion