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

Deuterated water ((2)H(2)O), 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 (2)H(2)O 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.