Nonlinear Regression Models for Determination of Nicotinamide Adenine Dinucleotide Content in Human Embryonic Stem Cells
| Autor: | Zdenek Glatz, Petr Dvorak, Olga Kyrylenko, Anton Salykin, Petr Kuzmic, Jindra Musilova, Sergiy Kyrylenko |
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| Rok vydání: | 2013 |
| Předmět: |
Cell Extracts
Cancer Research Oxidative phosphorylation Biology Nicotinamide adenine dinucleotide Cofactor 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Lactate dehydrogenase Oxazines Humans Glycolysis Embryonic Stem Cells 030304 developmental biology 0303 health sciences Electrophoresis Capillary Cell Biology NAD Metabolic pathway Nonlinear Dynamics Biochemistry chemistry Calibration biology.protein Regression Analysis NAD+ kinase Stem cell 030217 neurology & neurosurgery |
| Zdroj: | Stem Cell Reviews and Reports. 9:786-793 |
| ISSN: | 1558-6804 1550-8943 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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