You are here - Home ::

Sample Extraction

The point of sample extraction is to dissolve all the metabolites in which you are interested and kill all the enzymes that could metabolise them. This must be done quickly so that the levels you measure reflect the levels in vivo. Metabolites can be unstable for biological reasons or chemical; for instance, triose phosphates are unstable in alkaline solution (and not terribly stable in any solution) chemically. On the other hand ATP and ADP are not especially unstable chemicals, but can be unstable in real tissues because myokinase, which interconverts 2ADP to ATP + AMP, is an enzyme that is hard to kill.

Typical methods include:

Chloroform-Methanol extraction
Extraction in hot ethanol
TCA-ether extraction
Perchloric acid extraction

When you have made your extract you will have to filter it. The particles in an HPLC column may be as small as 3 microns, and the frit that holds them in place will be even smaller. Suspended material that you cannot even see will block this. If you are doing mass spectroscopy without a column there is still a problem, because the sample must pass through a filter in the mass detector, and also through a very fine needle. There is a wide choice of filters. On a purely selfish level, dirty, particulate samples yield spiky, messy spectra. This is especially relevant for samples that automatically contain fine suspended particulates, for instance eluted tlc-plate bands.

Chloroform-Methanol extraction

Suitable for both water-soluble and organic-soluble metabolites.

The Planrt Metobolite Group in Golm publish a good method, which they use routinely for their broad-based metabolite profiling.

Extraction in hot ethanol

Very easy to do, and suited to polar or mildly non-polar metabolites. Not, however, the best method. Plant tissue is heated at 80°C in 80% ethanol. After a while the tissue is removed, and heated again in fresh ethanol. This may be repeated any number of times, often with gradually decreasing concentrations of ethanol. The number of extractions you need to do depends on the bulkiness of the tissue you are extracting. For instance, 1cm disks of potato tuber might be extracted once in 80% ethanol, twice in 50%, and maybe finally in water. To find out how many extractions you need to do on your tissue, you should ideally measure the metabolite-of-interest in all of the ethanol fractions, and keep on extracting until you get a fraction that no longer contains a relevant amount of metabolite. If you know that (for instance) three extractions are enough, subsequently you would pool all three ethanol extracts and measure this, summed, extract.

The main weakness of this method is that the enzymes will remain active until they meet hot ethanol. Deep in a chunk of plant tissue that may take some time, and during this time the tissue will be warming up. Warm conditions are just what an enzyme needs to mess up your results. Note also that commercial sources of Phosphoglucoisomerase sometimes contain traces of alcohol dehydrogenase, so this method may not be suitable for enzyme linked assays of fructose, depending on how you do it.

TCA-ether extraction

An excellent method for acid-stable, water-soluble metabolites, but hard work. The method is described in Jelitto et. al. (1992), Planta 188, 238-244, and we include a summary on this site.

Perchloric acid extraction

Not as good as TCA-ether but suitable for similar metabolites. I would rather not process perchloric acid extracts by LC-MS or HPLC because they are basically saturated solutions of potassium perchlorate (which has a very low solubility). Thus they form precipitate at the drop of a hat (for instance on application of a gradient in HPLC), which gums up columns something chronic. Therefore I am not including any summary here or any references, but please ask if you must use this method.

Reference

Jelitto T., Sonnewald U., Willmitzer L., Hajirezeai M., and Stitt M. (1992) Inorganic pyrophosphate content and metabolites in potato and tobacco plants expressing E. coli pyrophosphatase in their cytosol. Planta 188, 238-244