I'm developing a metabolite extraction protocol for Clostridium thermocellum, a gram positive anaerobic thermophilic bacterium. The extraction solvent I'm using is 2:2:1 acetonitrile:methanol:water. I was having problems detecting pyruvate in my samples and discovered that my stock solution of sodium pyruvate, when diluted in methanol, caused the pyruvate to "disappear." I can observe the disappearance of pyruvate over time by measuring pyruvate with lactate dehydrogenase and NADH. I've sent some samples to a collaborator with an LC/MS and he has confirmed that the pyruvate seems to have disappeared, but we can't figure out what it might have been converted to. The cuvette only contains sodium pyruvate, water and methanol.
How long time the solutions were stored and at what temperature?
You may check for the formation of pyruvate methyl ester (the acidic environment can catalyze this reaction), this substance has a low boiling point and can easily evaporate, in this manner the ester is subtracted to the reaction equilibrium of ester formation so displacing the reaction toward its formation .
If it is so in a little time you will lose all your pyruvate, since methanol, being a solvent, is abundantly in excess respect to the acid .
How long time the solutions were stored and at what temperature?
You may check for the formation of pyruvate methyl ester (the acidic environment can catalyze this reaction), this substance has a low boiling point and can easily evaporate, in this manner the ester is subtracted to the reaction equilibrium of ester formation so displacing the reaction toward its formation .
If it is so in a little time you will lose all your pyruvate, since methanol, being a solvent, is abundantly in excess respect to the acid .
Pyruvate can easily polymerize as well. I suggest you keep your stock solutions in water. 100mM NaPyruvate is stable in sterile water at 4 deg for a decently long time (years). In my experience, frequent freeze/thaw cycles led to degradation of pyruvate as well.
As for your extracts: Do not store your metabolite extracts, but use them immediately. If you have on LC/MS system with autosampler, make sure you don't load so many samples that they will stand in the autosamples for hours, before they are actually loaded. I have been able to extract and detect pyruvate in perchloric acid extracts (with ~70% recovery, so not brilliant, but decent), but this extract will clog up your column due to its high salt content.
Agilent has an extraction technique that calls for isopropanol:CH3CN:H2O. You might try it. The reactivity of the isopropanol might be a bit different than the MeOH if you are getting ester formation. Here is the extraction I use:
Sample prep details: Add 30ul of biological matrix to a glass vial. Extract each of the 30ul aliquots with 0.2ml isopropanol:acetonitrile:water (3:3:2v/v), centrifuge and take to dryness under nitrogen and freeze till analysis.
Perhaps, you should search for the potential products of pyruvate decarboxylation (acetaldehyde? acetoin? ethanol?). I have not seen reports of this particular reaction. However, it is well known that alcoholic fermentation in the presence of Brewers' yeast causes decarboxylation of pyruvic acid and the irreversible formation of acetaldehyde, a process that ultimately transform pyruvic acid into ethanol. Almost three decades ago, Chao and Jordan reported the presence of acetaldehyde and acetoin using Brewers yeast and pyruvic acid (Biochemistry, 1984, 23:3576-3582). It is also good to remember that the providers of sodium pyruvate recommed keeping this product in water (where it can be stable for 24 months) rather than methanol.
The solution to my problem turned out to be a bad stock of methanol. I purchased a new bottle and the problem went away. I never figured out what exactly was the cause of the pyruvate disappearance in the bad bottle of methanol though. Thanks to everyone for your suggestions.
- Badges
- Science method