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Determination of Rate of [3H-methyl]-choline Incorporation into Cellular Lipids and Non-lipid Metabolites   

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Original research article

A brief version of this protocol appeared in:
PLOS ONE
Mar 2016

Abstract

The choline-containing phospholipid, phosphatidylcholine (PtdCho) is the most common mammalian phospholipid found in cell membrane (Ide et al., 2013). It is also a component of intracellular signalling pathways (Cui and Houweling, 2002). Herein is described a measure of the rate of accumulation of choline by lipid soluble PtdCho and lyso-Ptdcho which can further be discriminated by chromatographic analysis (Smith and Phyu, 2016). Determination of the accumulation of [3H-methyl]-choline into water-soluble components is also described. The procedure could be used to measure the effect of drugs and other factors on choline incorporation into phospholipids. After exposure of cells to test conditions (e.g., drugs) adherent cells in tissue culture flasks are incubated with radiolabelled [3H-methyl]-choline in medium for 15 min (pulse). The [3H-methyl]-choline is then rapidly removed and incubation continued in the presence of non-radioactive medium (chase). Cellular distribution of [3H-methyl] is then determined by cell fractionation and measurement of radioactivity in the lipid and non-lipid cellular components.

Background

Phospholipid metabolism is essential in formation of cell membranes (Ide et al., 2013) and cell signalling (Cui and Houweling, 2002). Both the formation of choline-containing metabolites and the accumulation of choline into lipids are pivotal processes during cellular proliferation. Perturbations in phospholipid metabolisms are associated with cancer and other disorders (Gibellini and Smith, 2010). Measurement of these processes is central to understanding medical imaging modalities that detect choline incorporation by tumour tissue using [11C]-choline-PET (positron emission tomography) (Podo et al., 2007) and choline metabolite content using 31P or 1H (proton) magnetic resonance spectroscopy (Saeedi et al., 2005).

Here is described a method of quantitating the incorporation of choline into aqueous and lipid components. The method is straightforward, relatively inexpensive and easy to set up. It is also quantitative. Alternative methods include NMR spectroscopy (Mori et al., 2015) which can be used to measure the content of phospholipids in tissues and tissue extracts but requires very expensive equipment and specialist knowledge and thin layer chromatography which is inexpensive but is considered to be a qualitative technique.

Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite:  Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Smith, T. A. and Phyu, S. M. (2016). Determination of Rate of [3H-methyl]-choline Incorporation into Cellular Lipids and Non-lipid Metabolites. Bio-protocol 6(22): e2019. DOI: 10.21769/BioProtoc.2019.
  2. Smith, T. A. and Phyu, S. M. (2016). Metformin decouples phospholipid metabolism in breast cancer cells. PLoS One 11(3): e0151179.
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