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Steven Pauwels, Ivo Jans, Jaak Billen, Annemieke Heijboer, Annemieke Verstuyf, Geert Carmeliet, Chantal Mathieu, Miguel Maestro, Etienne Waelkens, Pieter Evenepoel, Roger Bouillon, Dirk Vanderschueren, Pieter Vermeersch
BACKGROUND: The measurement of 1α,25(OH)2D3 in human serum poses a true challenge as concentrations are very low and structurally similar metabolites can interfere. MATERIALS AND METHODS: During optimization of our in-house LC-MSMS method for serum 1α,25(OH)2D3 a previously co-eluting isobaric interference was separated. The isobar was identified as 1β,25(OH)2D3 by comparing retention time and fragmentation spectra to standards (other isobaric dihydroxylated vitamin D3 analogs)...
February 10, 2017: Journal of Steroid Biochemistry and Molecular Biology
Hilke Jungen, Hilke Andresen-Streichert, Alexander Müller, Stefanie Iwersen-Bergmann
The detection of disaccharides in urine is under investigation to act as a marker for intravenous abuse of disaccharide formulations, like liquid methadone with syrup (sucrose), methadone tablets (lactose and sucrose), or buprenorphine tablets (lactose). As the detection time in urine has not yet been investigated and a routine method for detecting disaccharides is still lacking, a study was performed to estimate the window of detection in urine after intravenous consumption of disaccharides. Furthermore, an analytical LC-MSMS method for the quantification of sucrose and lactose in urine was validated...
January 2017: Journal of Analytical Toxicology
Sandra den Hoedt, Carola I F Janssen, Guiseppe Astarita, Daniele Piomelli, Frank P J Leijten, Simone M Crivelli, Adrie J M Verhoeven, Helga E de Vries, Jochen Walter, Pilar Martinez-Martinez, Eric J G Sijbrands, Amanda J Kiliaan, Monique T Mulder
BACKGROUND: Apolipoprotein E (ApoE) is known for its role in lipid trafficking and the ɛ4 allele is a risk factor for late onset Alzheimer's disease (AD). Recently, aberrant ceramide and fatty acid (FA) levels have been implicated in AD. OBJECTIVE: To determine the specific effects of human ApoE4 (hE4) on cerebral ceramide and FA content during chow or a high fat/high cholesterol (HFHC) diet. METHODS: Cerebral ceramide and FA profiles were determined by LC-MSMS in 15-month-old female wild-type (WT), ApoE-knockout (E0), and hE4-knockin mice fed chow or a HFHC diet for 3 months...
December 30, 2016: Journal of Alzheimer's Disease: JAD
Turan Demircan, Ilknur Keskin, Seda Nilgün Dumlu, Nilüfer Aytürk, Mahmut Erhan Avşaroğlu, Emel Akgün, Gürkan Öztürk, Ahmet Tarık Baykal
Salamander axolotl has been emerging as an important model for stem cell research due to its powerful regenerative capacity. Several advantages, such as the high capability of advanced tissue, organ, and appendages regeneration, promote axolotl as an ideal model system to extend our current understanding on the mechanisms of regeneration. Acknowledging the common molecular pathways between amphibians and mammals, there is a great potential to translate the messages from axolotl research to mammalian studies...
January 2017: Proteomics
Kerry J Welsh, Brian R Stolze, Xiaolin Yu, Trisha R Podsiadlo, Lisa S Kim, Steven J Soldin
OBJECTIVES: Patients with non-thyroidal illness syndrome have many abnormalities in thyroid hormone tests. Such patients have medical comorbidities associated with low serum proteins and are on multiple medications that interfere with thyroid hormone measurement by immunoassay platforms. It is unknown if these thyroid hormone measurements reflect physiologic conditions or if they are artifacts of testing methodology. METHODS: Fifty patients were selected from the intensive care unit (ICU) from our institution...
November 25, 2016: Clinical Biochemistry
Zhou Meng, Sylvain Le Marchand, Deep Agnani, Matt Szapacs, Harma Ellens, Joe Bentz
From fits of drug transport kinetics across confluent MDCKII-hMDR1-NKI and Caco-2 cell monolayers we estimated the levels of efflux active P-glycoprotein (P-gp) in these two cell lines (companion paper, Meng at el., 2016a). In the present work, we compared the efflux active P-gp number to the total P-gp level, using LC/MSMS, and showed that in Caco-2 cells total P-gp is about 10-fold greater than efflux active P-gp, while in MDCKII-hMDR1-NKI cells these values are within 2-fold. We further visualized the microvilli in MDCKII-hMDR1-NKI and Caco-2 cells using three-dimensional structured illumination super-resolution microscopy and found that the microvilli in Caco-2 cells are taller and more densely packed than those in MDCK-NKI-hMDR1 cells...
November 16, 2016: Drug Metabolism and Disposition: the Biological Fate of Chemicals
Ashley M Hopkins, Mahin Moghaddami, David J R Foster, Susanna M Proudman, Richard N Upton, Michael D Wiese
Leflunomide's active metabolite teriflunomide inhibits dihydro-oroate dehydrogenase, an enzyme essential to proliferation of T lymphocytes. As teriflunomide must reach the target site to have this effect, this study assessed the distribution of teriflunomide into T lymphocytes, as intracellular concentrations may be a superior response biomarker to plasma concentrations. CD3 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were used to extract CD3(+) T cells from the peripheral blood of patients with rheumatoid arthritis who were taking a stable dose of leflunomide...
January 2017: Drug Metabolism and Disposition: the Biological Fate of Chemicals
Carol Evans, Sarah Tennant, Petros Perros
Patients with differentiated thyroid cancer (DTC) usually have an excellent prognosis. Following surgical and radioiodine treatment to remove the cancer cells and suppressive doses of levothyroxine, long-term follow-up, including measurement of serum thyroglobulin (Tg) using a sensitive assay is required to detect recurrence. To interpret Tg results clinicians need to know the corresponding serum TSH concentration, have an appreciation of the clearance of Tg from patient serum following various interventions and the limitations of its measurement...
2016: Scandinavian Journal of Clinical and Laboratory Investigation. Supplementum
Marie Tré-Hardy, Arnaud Capron, Marina Venzon Antunes, Rafael Linden, Pierre Wallemacq
The purpose of this study was to develop and validate a new liquid chromatography-tandem mass spectrometric (LC-MSMS) assay for the simultaneous quantification of tamoxifen (TAM) and its main therapeutically active metabolites, N-desmethyltamoxifen (NDT), 4-hydroxytamoxifen (4HT) and endoxifen (END) in dried blood spots. Ultrasound assisted methanolic extraction was used for TAM and metabolites extraction from dried blood spot. After evaporation and methanol reconstitution, the extract was injected into a LC-MSMS system...
November 2016: Clinical Biochemistry
Caroline Noyon, Cédric Delporte, Damien Dufour, Melissa Cortese, Alexandre Rousseau, Philippe Poelvoorde, Jean Nève, Luc Vanhamme, Karim Zouaoui Boudjeltia, Thierry Roumeguère, Pierre Van Antwerpen
Myeloperoxidase promotes several kinds of damage and is involved in the development of various diseases (as atherosclerosis and cancers). An example of these damage is the chlorination of nucleic acids, which is considered as a specific marker of the MPO activity on those acids. This study aimed to develop and validate a method to analyze oxidized and MPO-specific chlorinated nucleosides in biological matrixes (cells, tissues and plasma). Although a lot of methods to quantify oxidized or chlorinated nucleosides have already been established, none of them took into account all these derivatives together...
July 1, 2016: Talanta
Dahai Shao, Shusheng Tang, Rosanne A Healy, Paula M Imerman, Dwayne E Schrunk, Wilson K Rumbeiha
Orellanine (3,3',4,4'-tetrahydroxy-2,2'-bipyridine-1,1'-dioxide) is a tetrahydroxylated di-N-oxidized bipyridine compound. The toxin, present in certain species of Cortinarius mushrooms, is structurally similar to herbicides Paraquat and Diquat. Cortinarius orellanus and Cortinarius rubellus are the major orellanine-containing mushrooms. Cortinarius mushrooms are widely reported in Europe where they have caused human poisoning and deaths through accidental ingestion of the poisonous species mistaken for the edible ones...
May 2016: Toxicon: Official Journal of the International Society on Toxinology
Likhona Siphe Masika, Zhen Zhao, Steven John Soldin
OBJECTIVES: Thyroid dysfunction is a common medical condition affecting an estimated 30 million people in the US alone. Employing gold standard Liquid chromatography-tandem mass spectrometry (LC-MSMS) methods we have examined the extent of inaccuracy of immunoassay (IA) measurement for total T3 (TT3) at low, normal and high concentrations. DESIGN AND METHODS: 268 TT3 Roche Cobas 6000 immunoassay TT3 values (covering the low, normal, and high ranges) were compared with LC-MSMS results...
August 2016: Clinical Biochemistry
Konstantina Fragaki, Annabelle Chaussenot, Jean-François Benoist, Samira Ait-El-Mkadem, Sylvie Bannwarth, Cécile Rouzier, Charlotte Cochaud, Véronique Paquis-Flucklinger
BACKGROUND: Coenzyme Q10 (CoQ10 or ubiquinone) deficiency can be due either to mutations in genes involved in CoQ10 biosynthesis pathway, or to mutations in genes unrelated to CoQ10 biosynthesis. CoQ10 defect is the only oxidative phosphorylation disorder that can be clinically improved after oral CoQ10 supplementation. Thus, early diagnosis, first evoked by mitochondrial respiratory chain (MRC) spectrophotometric analysis, then confirmed by direct measurement of CoQ10 levels, is of critical importance to prevent irreversible damage in organs such as the kidney and the central nervous system...
January 8, 2016: Biological Research
Ivan Verrastro, Karina Tveen-Jensen, Rudiger Woscholski, Corinne M Spickett, Andrew R Pitt
Phosphatase and tensin homolog (PTEN) is involved in a number of different cellular processes including metabolism, apoptosis, cell proliferation and survival. It is a redox-sensitive dual-specificity protein phosphatase that acts as a tumor suppressor by negatively regulating the PI3K/Akt pathway. While direct evidence of redox regulation of PTEN downstream signaling has been reported, the effect of PTEN redox status on its protein-protein interactions is poorly understood. PTEN-GST in its reduced and a DTT-reversible H2O2-oxidized form was immobilized on a glutathione-sepharose support and incubated with cell lysate to capture interacting proteins...
January 2016: Free Radical Biology & Medicine
Juraj Lenčo, Olga Lenčová-Popelová, Marek Link, Anna Jirkovská, Vojtěch Tambor, Eliška Potůčková, Jiří Stulík, Tomáš Šimůnek, Martin Štěrba
Due to their cardiac origin, H9c2 cells rank among the most popular cell lines in current cardiovascular research, yet molecular phenotype remains elusive. Hence, in this study we used proteomic approach to describe molecular phenotype of H9c2 cells in their undifferentiated (i.e., most frequently used) state, and its functional response to cardiotoxic drug doxorubicin. Of 1671 proteins identified by iTRAQ IEF/LC-MSMS analysis, only 12 proteins were characteristic for striated muscle cells and none was cardiac phenotype-specific...
December 10, 2015: Experimental Cell Research
Karina Tveen-Jensen, Ana Reis, Corinne M Spickett, Andrew R Pitt
Oxidative post-translational modifications (oxPTMs) can alter the function of proteins, and are important in the redox regulation of cell behaviour. The most informative technique to detect and locate oxPTMs within proteins is mass spectrometry (MS). However, proteomic MS data are usually searched against theoretical databases using statistical search engines, and the occurrence of unspecified or multiple modifications, or other unexpected features, can lead to failure to detect the modifications and erroneous identifications of oxPTMs...
October 2014: Free Radical Biology & Medicine
Keiryn L Bennett, Xia Wang, Cory E Bystrom, Matthew C Chambers, Tracy M Andacht, Larry J Dangott, Félix Elortza, John Leszyk, Henrik Molina, Robert L Moritz, Brett S Phinney, J Will Thompson, Maureen K Bunger, David L Tabb
Questions concerning longitudinal data quality and reproducibility of proteomic laboratories spurred the Protein Research Group of the Association of Biomolecular Resource Facilities (ABRF-PRG) to design a study to systematically assess the reproducibility of proteomic laboratories over an extended period of time. Developed as an open study, initially 64 participants were recruited from the broader mass spectrometry community to analyze provided aliquots of a six bovine protein tryptic digest mixture every month for a period of nine months...
December 2015: Molecular & Cellular Proteomics: MCP
Lauren A Kristofco, Luis Colon Cruz, Samuel P Haddad, Martine L Behra, C Kevin Chambliss, Bryan W Brooks
Because basic toxicological data is unavailable for the majority of industrial compounds, High Throughput Screening (HTS) assays using the embryonic and larval zebrafish provide promising approaches to define bioactivity profiles and identify potential adverse outcome pathways for previously understudied chemicals. Unfortunately, standardized approaches, including HTS experimental designs, for examining fish behavioral responses to contaminants are rarely available. In the present study, we examined movement behavior of larval zebrafish over 7 days (4-10 days post fertilization or dpf) during typical daylight workday hours to determine whether intrinsic activity differed with age and time of day...
January 2016: Aquatic Toxicology
P G J Ter Horst, J H Proost, J P Smit, M T Vries, L T W de Jong-van de Berg, B Wilffert
PURPOSE: Clomipramine is one of the drugs for depression during pregnancy; however, pharmacokinetic data of clomipramine and its active metabolite desmethylclomipramine in this vulnerable period are lacking. In this study, we describe clomipramine and desmethylclomipramine concentrations including their ratios during pregnancy. Second, we describe Center for Epidemiologic Studies Depression scale (CES-D) scores during pregnancy. METHODS: During 13 pregnancies, every trimester and 3 months after pregnancy, the clomipramine and desmethylclomipramine concentrations were measured with LC-MSMS and the severity of depression was assessed by taking the CES-D score...
December 2015: European Journal of Clinical Pharmacology
Antje Grosche, Alexandra Hauser, Marlen Franziska Lepper, Rebecca Mayo, Christine von Toerne, Juliane Merl-Pham, Stefanie M Hauck
To date, the proteomic profiling of Müller cells, the dominant macroglia of the retina, has been hampered because of the absence of suitable enrichment methods. We established a novel protocol to isolate native, intact Müller cells from adult murine retinae at excellent purity which retain in situ morphology and are well suited for proteomic analyses. Two different strategies of sample preparation - an in StageTips (iST) and a subcellular fractionation approach including cell surface protein profiling were used for quantitative liquid chromatography-mass spectrometry (LC-MSMS) comparing Müller cell-enriched to depleted neuronal fractions...
February 2016: Molecular & Cellular Proteomics: MCP
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