naa a22 4500 510739067 CHVBK 20180411083021.0 cr unu---uuuuu 180411e20131201xx s 000 0 eng 10.1007/s12015-013-9454-3 doi (NATIONALLICENCE)springer-10.1007/s12015-013-9454-3 Nonlinear Regression Models for Determination of Nicotinamide Adenine Dinucleotide Content in Human Embryonic Stem Cells [Elektronische Daten] [Anton Salykin, Petr Kuzmic, Olga Kyrylenko, Jindra Musilova, Zdenek Glatz, Petr Dvorak, Sergiy Kyrylenko] 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. Springer Science+Business Media New York, 2013 NAD nationallicence Human embryonic stem cells nationallicence hESC-derived fibroblasts nationallicence Enzymatic cycling nationallicence Nonlinear regression nationallicence Biochemical assay nationallicence ADH : Alcohol dehydrogenase nationallicence BSA : Bovine serum albumin nationallicence CE : Capillary electrophoresis nationallicence hESCs : Human embryonic stem cells nationallicence NAD : Nicotinamide adenine dinucleotide nationallicence PBS : Phosphate buffer saline nationallicence POM1 : Sodium polyoxotungstate nationallicence RFU : Relative fluorescence units nationallicence Salykin Anton Department of Biology, Faculty of Medicine, Masaryk University, Building A3, Kamenice 735/5, 625 00, Brno-Bohunice, Czech Republic aut Kuzmic Petr BioKin Ltd, Watertown, MA, USA aut Kyrylenko Olga A. I. Virtanen Institute, University of Eastern Finland, Kuopio, Finland aut Musilova Jindra Department of Biochemistry, Faculty of Science and Central-European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic aut Glatz Zdenek Department of Biochemistry, Faculty of Science and Central-European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic aut Dvorak Petr Department of Biology, Faculty of Medicine, Masaryk University, Building A3, Kamenice 735/5, 625 00, Brno-Bohunice, Czech Republic aut Kyrylenko Sergiy Department of Biology, Faculty of Medicine, Masaryk University, Building A3, Kamenice 735/5, 625 00, Brno-Bohunice, Czech Republic aut Stem Cell Reviews and Reports Springer US; http://www.springer-ny.com 9/6(2013-12-01), 786-793 1550-8943 9:6<786 2013 9 12015 https://doi.org/10.1007/s12015-013-9454-3 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s12015-013-9454-3 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Salykin Anton Department of Biology, Faculty of Medicine, Masaryk University, Building A3, Kamenice 735/5, 625 00, Brno-Bohunice, Czech Republic aut NATIONALLICENCE 700 1- Kuzmic Petr BioKin Ltd, Watertown, MA, USA aut NATIONALLICENCE 700 1- Kyrylenko Olga A. I. Virtanen Institute, University of Eastern Finland, Kuopio, Finland aut NATIONALLICENCE 700 1- Musilova Jindra Department of Biochemistry, Faculty of Science and Central-European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic aut NATIONALLICENCE 700 1- Glatz Zdenek Department of Biochemistry, Faculty of Science and Central-European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic aut NATIONALLICENCE 700 1- Dvorak Petr Department of Biology, Faculty of Medicine, Masaryk University, Building A3, Kamenice 735/5, 625 00, Brno-Bohunice, Czech Republic aut NATIONALLICENCE 700 1- Kyrylenko Sergiy Department of Biology, Faculty of Medicine, Masaryk University, Building A3, Kamenice 735/5, 625 00, Brno-Bohunice, Czech Republic aut NATIONALLICENCE 773 0- Stem Cell Reviews and Reports Springer US; http://www.springer-ny.com 9/6(2013-12-01), 786-793 1550-8943 9:6<786 2013 9 12015 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer