A new CZE method was developed for the determination of 12 purine and pyrimidine nucleotides, two adenine coenzymes and their reduced forms, and acetyl coenzyme A in various cell extracts. As the concentration levels of these metabolites in living cells are low; CZE was combined with field-enhanced sample stacking. As a result, the separation conditions were optimised to achieve a suitable resolution at the relatively high sample volume provided by this on-line pre-concentration technique. The optimum BGE was 150 mM glycine buffer (pH 9.5). Samples were introduced hydrodynamically using a pressure of 35 mbar (3.5 kPa) for 25 s, and data were collected at a detection wavelength of 260 nm. An applied voltage of 30 kV (positive polarity) and capillary temperature of 25°C gave the best separation of these compounds. The optimised method was validated by determining the linearity, sensitivity and repeatability and it was successfully applied for the analysis of extracts from Paracoccus denitrificans bacteria and from stem cells.
- MeSH
- acetylkoenzym A analýza MeSH
- adenosintrifosfát analýza MeSH
- chemické techniky analytické metody normy MeSH
- cytidintrifosfát analýza MeSH
- embryonální kmenové buňky chemie MeSH
- guanosintrifosfát analýza MeSH
- lidé MeSH
- limita detekce MeSH
- Paracoccus denitrificans chemie MeSH
- reprodukovatelnost výsledků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Recent evidence suggests that energy metabolism contributes to molecular mechanisms controlling stem cell identity. For example, human embryonic stem cells (hESCs) receive their metabolic energy mostly via glycolysis rather than mitochondrial oxidative phosphorylation. This suggests a connection of metabolic homeostasis to stemness. Nicotinamide adenine dinucleotide (NAD) is an important cellular redox carrier and a cofactor for various metabolic pathways, including glycolysis. Therefore, accurate determination of NAD cellular levels and dynamics is of growing importance for understanding the physiology of stem cells. Conventional analytic methods for the determination of metabolite levels rely on linear calibration curves. However, in actual practice many two-enzyme cycling assays, such as the assay systems used in this work, display prominently nonlinear behavior. Here we present a diaphorase/lactate dehydrogenase NAD cycling assay optimized for hESCs, together with a mechanism-based, nonlinear regression models for the determination of NAD(+), NADH, and total NAD. We also present experimental data on metabolic homeostasis of hESC under various physiological conditions. We show that NAD(+)/NADH ratio varies considerably with time in culture after routine change of medium, while the total NAD content undergoes relatively minor changes. In addition, we show that the NAD(+)/NADH ratio, as well as the total NAD levels, vary between stem cells and their differentiated counterparts. Importantly, the NAD(+)/NADH ratio was found to be substantially higher in hESC-derived fibroblasts versus hESCs. Overall, our nonlinear mathematical model is applicable to other enzymatic amplification systems.
- MeSH
- buněčné extrakty MeSH
- elektroforéza kapilární MeSH
- embryonální kmenové buňky metabolismus MeSH
- kalibrace MeSH
- lidé MeSH
- NAD metabolismus MeSH
- nelineární dynamika * MeSH
- oxaziny metabolismus MeSH
- regresní analýza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cíl studie: Uvést stručný přehled současných metod pro analýzu metabolomu embrya, vlastních zkušeností v této oblasti a perspektivy dalšího vývoje. Typ studie: Literární přehled. Název a sídlo pracoviště: Gynekologicko-porodnická klinika, Lékařská fakulta Masarykovy univerzity a Fakultní nemocnice Brno; Ústav biochemie, Přírodovědecká fakulta a CEITEC, Masarykova univerzita, Brno. Předmět a metoda studie: V současné době jsou k dispozici vysoce citlivé analytické metody umožňující přesné stanovení řady molekul, které souvisí s látkovou přeměnou – metabolismem – embrya v prvních dnech jeho vývoje. Jednou z perspektivních metod je kapilární elektroforéza. V uplynulých letech byly analyzovány parametry metabolismu embryí kultivovaných in vitro a byl hodnocen jejich vztah k morfologii a dosažení těhotenství. Pozornost se soustředila nejčastěji na metabolismus aminokyselin, glukózy a pyruvátu. Výsledky studií však mají zatím nejednoznačné výsledky. Závěr: Analýza kultivačního média kapilární elektroforézou patří ke slibným metodám stanovení metabolomu. Rozsáhlý výzkum přináší nové poznatky, které by ve svém výsledku měly vést k vytvoření klinicky použitelné metody výběru embrya a zvýšení efektivity transferu jednoho embrya. Klíčová slova: metabolomika, analýza metabolomu, kapilární elektroforéza, embryo, asistovaná reprodukce.
Objective: To review current technologies for the analytical examination of the embryonic metabolome and its perspectives. Design: Review article. Setting: Department of Gynecology and Obstetrics, Faculty of Medicine, Masaryk University, and University Hospital, Brno, Department of Biochemistry, Faculty of Science and CEITEC, Masaryk University, Brno. Methods and results: Nowadays, very sensitive analytical technologies are available. They enable exact measurement of various molecules – products of embryo metabolism during first days of cultivation. The capillary electrophoresis is one of promising method. Recent studies analysed metabolic differences between embryos that result in a pregnancy and those that do not. Amino acid levels, glucose or pyruvate in the embryo culture media were analysed most frequently. However, results of these studies are ambiguous. Conclusions: The capillary electrophoresis with contactless conductivity detection may provide a useful data of the embryonic metabolome. A comprehensive analysis of the used culture medium may represent a valuable adjunct to morphological criteria for enhanced rates of implantation and delivery. Key words: metabolomics, metabolome analysis, capillary electrophoresis, embryo, assisted reproductive techniques.
- MeSH
- asistovaná reprodukce MeSH
- chemické techniky analytické * metody MeSH
- elektroforéza kapilární MeSH
- kultivace embrya * MeSH
- kultivační média MeSH
- lidé MeSH
- metabolom * MeSH
- metabolomika metody MeSH
- terminologie jako téma MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
This review defines metabolomics and clarifies its history and significance. Various strategies of metabolomics research are described together with their main application fields. The review focuses on the methods of sample preparation and on the analytical methodologies that are most frequently used in metabolomic studies NMR, MS, GC/MS, HPLC/MS and CE/MS. Practical importance of metabolomics and its role in system biology are mentioned as well.
The main aim of this work was to demonstrate the applicability of capillary zone electrophoresis in combination with field enhanced sample stacking in targeted metabolome analyses of adenine nucleotides--AMP, ADP, ATP, coenzymes NAD(+), NADP(+) and their reduced forms in Paracoccus denitrificans. Sodium carbonate/hydrogencarbonate buffer (100 mM, pH 9.6) with the addition of beta-CD at a concentration of 10 mM was found to be an effective BGE for their separation within 20 min. Besides this, special attention was paid to the development of the procedure for the extraction of specific metabolites from the bacterium P. denitrificans. This procedure was not only optimised to achieve the highest metabolite yields but also to obtain a sample that was fully compatible with the online preconcetration strategy used. The developed methodology was finally applied in a study of the bacterium P. denitrificans at various stages of the active respiratory chain.
