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Metabolites 2016, 6(4), 34; doi:10.3390/metabo6040034

Analysis of Mammalian Cell Proliferation and Macromolecule Synthesis Using Deuterated Water and Gas Chromatography-Mass Spectrometry

1
Institute for Physical Activity and Nutrition Research, School of Exercise and Nutrition Sciences, Deakin University, Geelong 3216, Victoria, Australia
2
Systems Biology and Personalised Medicine Division, The Walter and Eliza Hall Institute of Medical Research, Parkville 3052, Victoria, Australia
3
Department of Medical Biology, University of Melbourne, Parkville 3052, Victoria, Australia
4
Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville 3052, Victoria, Australia
5
MRL, Merck & Co. Inc., Kenilworth, NJ 07033, USA
6
Department of Surgery, University of Melbourne, Parkville 3052, Victoria, Australia
7
School of Biomedical Sciences, Monash University, Clayton 3168, Victoria, Australia
*
Author to whom correspondence should be addressed.
Academic Editors: Devin Benheim, Farhana R. Pinu, Konstantinos A. Kouremenos, Damien L. Callahan and David P. De Souza
Received: 2 September 2016 / Revised: 10 October 2016 / Accepted: 10 October 2016 / Published: 13 October 2016
View Full-Text   |   Download PDF [2397 KB, uploaded 13 October 2016]   |  

Abstract

Deuterated water (2H2O), a stable isotopic tracer, provides a convenient and reliable way to label multiple cellular biomass components (macromolecules), thus permitting the calculation of their synthesis rates. Here, we have combined 2H2O labelling, GC-MS analysis and a novel cell fractionation method to extract multiple biomass components (DNA, protein and lipids) from the one biological sample, thus permitting the simultaneous measurement of DNA (cell proliferation), protein and lipid synthesis rates. We have used this approach to characterize the turnover rates and metabolism of a panel of mammalian cells in vitro (muscle C2C12 and colon cancer cell lines). Our data show that in actively-proliferating cells, biomass synthesis rates are strongly linked to the rate of cell division. Furthermore, in both proliferating and non-proliferating cells, it is the lipid pool that undergoes the most rapid turnover when compared to DNA and protein. Finally, our data in human colon cancer cell lines reveal a marked heterogeneity in the reliance on the de novo lipogenic pathway, with the cells being dependent on both ‘self-made’ and exogenously-derived fatty acid. View Full-Text
Keywords: deuterated water; biomass; GC-MS; stable isotopes; C2C12; colon cancer; protein synthesis; lipogenesis; DNA synthesis deuterated water; biomass; GC-MS; stable isotopes; C2C12; colon cancer; protein synthesis; lipogenesis; DNA synthesis
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Foletta, V.C.; Palmieri, M.; Kloehn, J.; Mason, S.; Previs, S.F.; McConville, M.J.; Sieber, O.M.; Bruce, C.R.; Kowalski, G.M. Analysis of Mammalian Cell Proliferation and Macromolecule Synthesis Using Deuterated Water and Gas Chromatography-Mass Spectrometry. Metabolites 2016, 6, 34.

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