Increased plasma cholesterol levels are listed between the major atherosclerosis risk factors. The final plasma cholesterol levels result from the interplay between the genetic and environmental (diet, physical activity) factors. Little is known, how dietary factor influence epigenetics. We have analyzed, if an over-generation feeding of rat with cholesterol influences total liver-DNA methylation, and if total liver-DNA methylation differ between the different rat strains (Prague hereditary hypercholesterolemic rats, Prague hereditary hypertriglyceridemic rats and Wistar Kyoto rats). The animals were feed with high fat (additional 5 % over normal capacity) high cholesterol (2 %) diet for 14 days. DNA methylation in the liver tissue in different generations was analyzed using the liquid chromatography coupled with tandem mass spectrometry. We have not observed any significant changes in total liver-DNA methylation over the 9 generations of animals feed by fat/cholesterol enriched diet. Additionally, there were no differences in DNA methylation between different rat strains. In animal model, the dietary changes (hypercholesterolemic diet) not significantly influence the total DNA methylation status within the liver.
- MeSH
- cholesterol dietní aplikace a dávkování škodlivé účinky MeSH
- dieta s vysokým obsahem tuků * škodlivé účinky MeSH
- hypercholesterolemie chemicky indukované genetika metabolismus MeSH
- játra metabolismus MeSH
- krysa rodu rattus MeSH
- metylace DNA genetika MeSH
- potkani Wistar MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
BACKGROUND: Tauopathies represent heterogeneous groups of neurodegenerative diseases that are characterised by abnormal deposition of the microtubule-associated protein tau. Alzheimer's disease is the most prevalent tauopathy, affecting more than 35 million people worldwide. In this study we investigated changes in metabolic pathways associated with tau-induced neurodegeneration. METHODS: Cerebrospinal fluid (CSF), plasma and brain tissue were collected from a transgenic rat model for tauopathies and from age-matched control animals. The samples were analysed by targeted and untargeted metabolomic methods using high-performance liquid chromatography coupled to mass spectrometry. Unsupervised and supervised statistical analysis revealed biochemical changes associated with the tauopathy process. RESULTS: Energy deprivation and potentially neural apoptosis were reflected in increased purine nucleotide catabolism and decreased levels of citric acid cycle intermediates and glucose. However, in CSF, increased levels of citrate and aconitate that can be attributed to glial activation were observed. Other significant changes were found in arginine and phosphatidylcholine metabolism. CONCLUSIONS: Despite an enormous effort invested in development of biomarkers for tauopathies during the last 20 years, there is no clinically used biomarker or assay on the market. One of the most promising strategies is to create a panel of markers (e.g., small molecules, proteins) that will be continuously monitored and correlated with patients' clinical outcome. In this study, we identified several metabolic changes that are affected during the tauopathy process and may be considered as potential markers of tauopathies in humans.
- MeSH
- apoptóza genetika MeSH
- biologické markery metabolismus MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- metabolomika MeSH
- modely nemocí na zvířatech MeSH
- mozek metabolismus patologie MeSH
- mutace genetika MeSH
- potkani inbrední SHR MeSH
- potkani transgenní MeSH
- proteiny tau genetika metabolismus MeSH
- tauopatie diagnóza genetika MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Untargeted metabolite profiling using high-resolution mass spectrometry coupled with liquid chromatography (LC-HRMS), followed by data analysis with the Compound Discoverer 2.0™ software, was used to study the metabolism of imatinib in humans with chronic myeloid leukemia. Plasma samples from control (drug-free) and patient (treated with imatinib) groups were analyzed in full-scan mode and the unknown ions occurring only in the patient group were then, as potential imatinib metabolites, subjected to multi-stage fragmentation in order to elucidate their structure. The application of an untargeted approach, as described in this study, enabled the detection of 24 novel structurally unexpected metabolites. Several sulphur-containing compounds, probably originating after the reaction of reactive intermediates of imatinib with endogenous glutathione, were found and annotated as cysteine and cystine adducts. In the proposed mechanism, the cysteine adducts were formed after the rearrangement of piperazine moiety to imidazoline. On the contrary, in vivo S-N exchange occurred in the case of the cystine adducts. In addition, N-O exchange was observed in the collision cell in the course of the fragmentation of the cystine adducts. The presence of sulphur in the cysteine and cystine conjugates was proved by means of ultra-high resolution measurements using Orbitrap Elite. The detection of metabolites derived from glutathione might improve knowledge about the disposition of imatinib towards bioactivation and help to improve understanding of the mechanism of its hepatotoxicity or nephrotoxicity in humans.
- MeSH
- antitumorózní látky krev metabolismus moč MeSH
- chromatografie kapalinová MeSH
- cystein metabolismus MeSH
- cystin metabolismus MeSH
- imatinib mesylát krev metabolismus moč MeSH
- inhibitory proteinkinas krev metabolismus moč MeSH
- lidé MeSH
- síra krev metabolismus moč MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The discovery of tyrosine kinase inhibitors (TKIs) brought a major breakthrough in the treatment of patients with chronic myeloid leukemia (CML). Pathogenetic CML events are closely linked with the Bcr-Abl protein with tyrosine kinase activity. TKIs block the ATP-binding site; therefore, the signal pathways leading to malignant transformation are no longer active. However, there is limited information about the impact of TKI treatment on the metabolome of CML patients. Using liquid chromatography mass spectrometric metabolite profiling and multivariate statistical methods, we analyzed plasma and leukocyte samples of patients newly diagnosed with CML, patients treated with hydroxyurea and TKIs (imatinib, dasatinib, nilotinib), and healthy controls. The global metabolic profiles clearly distinguished the newly diagnosed CML patients and the patients treated with hydroxyurea from those treated with TKIs and the healthy controls. The major changes were found in glycolysis, the citric acid cycle, and amino acid metabolism. We observed differences in the levels of amino acids and acylcarnitines between those patients responding to imatinib treatment and those who were resistant to it. According to our findings, the metabolic profiling may be potentially used as an additional tool for the assessment of response/resistance to imatinib.
- MeSH
- aminokyseliny metabolismus MeSH
- chronická myeloidní leukemie krev metabolismus MeSH
- citrátový cyklus účinky léků MeSH
- glykolýza účinky léků MeSH
- hydroxymočovina farmakologie terapeutické užití MeSH
- imatinib mesylát farmakologie terapeutické užití MeSH
- inhibitory proteinkinas farmakologie MeSH
- krevní plazma chemie metabolismus MeSH
- leukocyty chemie metabolismus MeSH
- lidé MeSH
- metabolom * MeSH
- metabolomika metody MeSH
- monitorování léčiv metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: With an increasing number of cancer patients receiving tyrosine kinase inhibitors (TKIs), therapeutic drug monitoring of these molecules is becoming more widespread today. It is mainly based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) methods with typical run times of several minutes. In an online solid phase extraction-MS/MS (SPE-MS/MS) system, the chromatography column is replaced with a reusable solid phase extraction (SPE) cartridge and the analysis time is shortened to less than half a minute. The aim of this study was to develop such a method and test the performance of this high-throughput system in the analysis of imatinib (IMA), nilotinib (NIL), and lapatinib (LAP) in human plasma. METHODS: Samples were prepared by simple protein precipitation with methanol containing deuterated internal standards. After centrifugation, the supernatant was diluted 10 fold with a mixture of methanol and water (1:1). A C4 cartridge was used for SPE and the analytes were eluted by acetonitrile. All the analytes were measured within a wide calibration range (50-5000 ng/mL for nilotinib and imatinib, 100-10,000 ng/mL for lapatinib). The method was compared with the LC-MS/MS method by the analysis of 176 clinical samples. RESULTS: Intraday and interday inaccuracies within 15% and a coefficient of variation less than 15% were achieved for all the TKIs that were measured. Even though the matrix effects were higher in comparison with LC-MS/MS methods, their effect on the performance of the method was eliminated by the usage of deuterated internal standards. The total run time of the new method was 29 seconds for one analysis and the results were fully comparable with LC-MS/MS. CONCLUSIONS: Routine clinical practice requiring high-throughput methods for therapeutic drug monitoring of TKIs may benefit from the online SPE-MS/MS method that provides fast, low-cost analysis, and results that are comparable with conventional methods.
- MeSH
- chinazoliny chemie MeSH
- chromatografie kapalinová metody MeSH
- extrakce na pevné fázi metody MeSH
- imatinib mesylát krev MeSH
- inhibitory proteinkinas krev MeSH
- kalibrace MeSH
- krevní plazma chemie MeSH
- lidé MeSH
- monitorování léčiv metody MeSH
- pyrimidiny krev MeSH
- reprodukovatelnost výsledků MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- tyrosinkinasy metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
5-Ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytidine (EdC) are mainly used as markers of cellular replicational activity. Although EdU is employed as a replicational marker more frequently than EdC, its cytotoxicity is commonly much higher than the toxicity of EdC. To reveal the reason of the lower cytotoxicity of EdC, we performed a DNA analysis of five EdC-treated human cell lines. Surprisingly, not a single one of the tested cell lines contained a detectable amount of EdC in their DNA. Instead, the DNA of all the cell lines contained EdU. The content of incorporated EdU differed in particular cells and EdC-related cytotoxicity was directly proportional to the content of EdU. The results of experiments with the targeted inhibition of the cytidine deaminase (CDD) and dCMP deaminase activities indicated that the dominant role in the conversion pathway of EdC to EdUTP is played by CDD in HeLa cells. Our results also showed that the deamination itself was not able to effectively prevent the conversion of EdC to EdCTP, the conversion of EdC to EdCTP occurs with much lesser effectivity than the conversion of EdU to EdUTP and the EdCTP is not effectively recognized by the replication complex as a substrate for the synthesis of nuclear DNA.
- MeSH
- bromodeoxyuridin metabolismus MeSH
- buněčná smrt MeSH
- buněčné jádro metabolismus MeSH
- cytidindeaminasa metabolismus MeSH
- deoxycytidin analogy a deriváty metabolismus MeSH
- deoxyuridin analogy a deriváty metabolismus MeSH
- DNA metabolismus MeSH
- lidé MeSH
- malá interferující RNA metabolismus MeSH
- metabolom MeSH
- nádorové buněčné linie MeSH
- protilátky metabolismus MeSH
- replikace DNA MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
