Last Month's Most Accessed Feature: Choose your solvent: For detecting metabolites in colon cancer cells

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  • Published: Oct 3, 2017
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thumbnail image: Last Month's Most Accessed Feature: Choose your solvent: For detecting metabolites in colon cancer cells

Choose your solvent: For detecting metabolites in colon cancer cellsInconsistent metabolites

As metabolic processes in cancer cells and healthy cells can differ substantially, metabolomics offers a good way for scientists to explore the unique characteristics of cancer cells, as well as to identify potential biomarkers for diagnosing cancer and novel drug targets. When comparing how metabolites differ between healthy and cancerous cells, however, different studies often identify different sets of metabolites, which simply adds to the confusion.

Now, a team of researchers from Spain and the US has discovered one reason for this inconsistency, by showing that the type of metabolites that are detected is strongly influenced by the method used to extract them from cancer cells.

They made this discovery by extracting metabolites from colon cancer cells using four different methods and then analyzing the extracted metabolites by gas chromatography-mass spectrometry (GC-MS). The four methods all adopted the same basic approach, which involved first snap-freezing the cells to stop all metabolism at a specific point in time. Next, the researchers, led by Alejandro Cifuentes at the Laboratory of Foodomics in Madrid, added glass beads and an extraction solvent to the cells, before shaking this mixture rapidly to disrupt the cell walls and extract the metabolites into the solvent.

Organic and aqueous solvents

The four methods only differed in the extraction solvent, with Cifuentes and his team testing two organic solvents acetonitrile and a mixture of acetonitrile, isopropanol and water and two aqueous solvents water and water with formic acid. Using these four extraction solvents, they were able to detect a total of 290 metabolites by GC-MS, of which they could identify 150. In terms of number of identified metabolites, plain water turned out to be the most effective extraction solvent, producing 100 identified metabolites, followed by acetonitrile-isopropanol-water with 96, acidified water with 94 and acetonitrile with 82.

More interesting, though, was comparing the metabolites identified with the four extraction solvents. Of the 150 identified metabolites, just 49 were identified with all four extraction solvents; 72 metabolites were identified with both organic solvents, of which 18 were unique to the organic solvents, while 80 metabolites were found with both aqueous solvents, of which 14 were unique. This general variation in the metabolites extracted by the four solvents was confirmed by statistical analysis, with principal component analysis grouping the two aqueous solvents together and the two organic solvents together based on the identified metabolites.

From fatty acids to amino acids

Next, Cifuentes and his colleagues investigated what type of metabolites were identified by the four extraction solvents and found some clear patterns. Perhaps unsurprisingly, the metabolites extracted by the two organic solvents included a lot of fatty acids and lipids, whereas the two aqueous solvents tended to extract more amino acids, peptides and carbohydrates. However, both organic and aqueous solvents were equally as effective at extracting nucleic acids. There were even differences within the organic solvents and aqueous solvents, with acetonitrile-isopropanol-water extracting a broader range of metabolites than acetonitrile on its own, which extracted nucleic acids almost exclusively.

Finally, using the information on the type of identified metabolites, Cifuentes and his colleagues determined the specific metabolic pathways that were most effectively sampled by the four extraction methods. The found that organic solvents were best at sampling metabolites from pathways involved in RNA transcription and fatty acid oxidation, while aqueous solvents were best for pathways involved in the synthesis and metabolism of amino acids, proteins and sugars.

This clearly shows that in order to get a full understanding of all the metabolites in a cancer cell scientists need to utilize both aqueous and organic solvents. But that’s not all, because previous studies, including some by Cifuentes, have shown that different analytical techniques, including capillary electrophoresis, liquid chromatography and GC, are better at detecting some metabolites than others. So what this study ultimately shows is that resolving the confusion of cancer cell metabolomics is going to require a lot more analytical studies.

Analytica Chimica Acta (Article in Press): "GC-MS based metabolomics of colon cancer cells using different extraction solvents"


Article by Jon Evans

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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