Metabolic turnover and sampling the metabolome
Metabolism is in constant flux and the turnover of metabolites is fast compared to the transcriptome and proteome.
Samples that are metabolically active such as cells, or tissues contain enzymes and should be quenched at the point of sampling to inactivate the enzymes and stop the turnover of metabolites. Sampling of the intracellular metabolome provides a snap-shot of metabolism at a given point in time. The sample should be preserved to ensure that it is representative of the metabolic state at the point of sampling. Many metabolites have turnover rates less than 1 second and so the quenching procedure must stop metabolism quicker than the metabolic turnover rate to preserve the metabolic profile at the point of sampling.
To stop the turnover of metabolites the samples are quenched. The quenching step should halt metabolism faster than the turnover of metabolites and without the loss of metabolites from the sample. The loss of metabolites is termed metabolite leakage and is problematic and often unavoidable during the quenching of certain sample types.
The quenching step is performed by either a rapid change in the temperature or pH of the sample to inhibit enzymatic activity and metabolic turnover. The rapid cooling of samples is the most popular method to quench metabolism and is usually performed by plunging the samples into liquid nitrogen, a dry-ice ethanol bath or a pre-cooled quenching solution such as 60% aqueous methanol solution. The use of liquid nitrogen is appropriate for samples such as plant material or tissue samples. Small samples such as leaves will freeze in 10-30 seconds whereas larger samples such as fruits will take longer and may take several minutes to fully freeze. With the direct freezing of samples it is important to consider any contaminating metabolites before the freezing process. Tissue samples are surrounded by blood and will require washing with water prior to quenching to remove the blood metabolites before the tissue is quenched. A chilled solution such as saline solution at 4°C may be used. The wash step will result in a slight delay to the quenching procedure but is necessary to prevent contamination of the sample.
Cell culture samples are often quenched in organic solvents that have been pre-chilled to -48°C. The cultures are sprayed into the quenching solution and the biomass is rapidly separated from the culture media and quenching solution by centrifugation or fast-filtration methods. The use of organic solvents often resulting in the leakage of metabolites from mammalian and microbial cells and so the contact time between the cells and quenching solution should be minimised and suitable controls should be included to assess the loss of metabolites during the quenching step. This often involves the analysis of the exometabolome or a combined samples of the intra- and exo-metabolome to determine if metabolites have leaked from the cells.
Various studies have investigated different quenching solutions to prevent the leakage of metabolites during the quenching procedure. These include chilled glycerol-saline solution, chilled saline solution and the addition of the buffers tricine , N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid (HEPES) or ammonium hydrogen carbonate to the quenching solution. There are advantages and disadvantages to the majority of the quenching solutions currently used to stop metabolism of cell cultures. This may be that the solution does not reduce the temperature of the culture sufficiently or the inclusion of a component in the quenching solution may not be desirable for the analytical procedure to be applied. The chosen method should be optimised for the particular sample type prior to the collection of samples.
© University of Birmingham and Birmingham Metabolomics Training Centre.