TNO Prins Maurits Laboratory, P.O. Box 45, 2280, AA Rijswijk, The Netherlands

may be they can help you.

If you simply want to introduce 13C, why don't you use 13C-glucose?I think it is a far more stable way to introduce your isotopes into the TCA cycle.

I guess that the aim of your studies is describing metabolism of cyanobacteria you're working on.

At the short sampling times you're working, you should assess dynamic label incorporation to metabolic intermediaries. And the rate at which label incorporates to intermediaries is a function of the total flux through the pathway, which explains the huge differences in labelling efficiency observed.

Since you're working on a photosynthetic microorganism, labelled bicarbonate incorporates through the Calvin Cycle, entering glycolysis and other pathways of carbohydrate metabolism.

In the presence of light, photosynthesis is the major source of energy (ATP) and reducing power (NADPH). Under these conditions, the cell has no need to use the TCA cycle for these purposes, and flux through this pathway should be very low. Remember that another important function of the TCA cycle is the synthesis of certain amino acids, such as glutamate and aspartate, which carbon skeleton derives from TCA cycle intermediaries. 

According to this, I think that there are two ways you can increase labelling of TCA cycle intermediaries:

a) Making sure that your cultures are actively growing (i.e. synthesizing proteins). For that, perform experiments during exponential growth phase of cultures, ideally in the phase of more active (fast) growth, with fresh medium not limited in nitrogen source. And, most of all, increase the sampling times to the range of hours (think a bit first about growth rates in your biological system).

b) Culturing cyanobacteria under heterotrophic or mixotrophic conditions. See a nice work by the group of Prof. Shimizu in Japan http://www.sciencedirect.com/science/article/pii/S1096717602902260 

I hope this answer helps. 

Best regards, V.

I forgot to mention that the rate of label incorporation is also a function of the concentration of the metabolite in the cell. Under equal "flux conditions" for two metabolites, the "relative rate" of label incorporation would be faster in a metabolite which concentration in the cell is lower. 

V.

As far as you are using labeled NaHCO3 I believe you want to monitor the C1 dynamic reaching the TCA cycle?

As Vincente Bernal mention, the entrance will be the Calvin Cycle, therefore if you want to get an idea of how much could reach the TCA cycle, you could monitor the isotopologues of PEP (M+1,2,3) and Acetyl-CoA (M+1,2).

On the technical point of view, the poor labeling may be also link to the incubation/medium conditions.

For example, if you are not using 13C-CO2 atmosphere for store and incubation, you may have natural CO2 that can dissolved in the medium and enter the Calvin cycle as unlabeled source.

@ Fabrizio Donnarumma: Firstly, I want to study the labelling pattern under photoautotrophic conditions and hence has to use NaHCO3 as my labelled substrate so that I can give a step change from unlabelled CO2 to labelled NaHCO3. Second, I am also planning to do mixotrophic studies but my culture does not have a preference for organic substrates like glucose and glycerol. I have even tried acclimating these cultures to the substrate, but still the uptake of label is poor.

@Vicente Bernal: I make sure that I do my sampling from exponentially growing cultures. I generally take my samples at 0.4 and 0.8 OD as after 1.0OD, there can be shading due to light limitation.

I am also planning to do mixotrophic studies like you suggested, but my culture has poor utilization of organic substrates like glucose and glycerol.

You mentioned that the rate of label incorporation is also a function of the concentration of the metabolite in the cell. Since I am growing the cultures optimally in seed cultures, I expect that the cultures may have optimal amounts of metabolites inside the cell. So, in that case, do you think that growing the seed culture in sub-optimal conditions and then transfer to optimal conditions containing labelled substrate will help in increasing its uptake?

@Giuseppe Martano: I do my experiments in a fermentor with a continuous inlet of 12C-CO2. But before labelling, I put off the inlet of 12C-CO2 and then add 13C-NaHCO3. As you rightly mentioned, I want to monitor the isotopologues of PEP, Acetyl-CoA etc. Since I want to see a step change, I have to initially grow the cultures in 12C-CO2 and then introduce my labelled NaHCO3

I really thank you all for giving me wonderful insights. Please do write back if you have any more suggestions. Since I am very new to this field, all your suggestions and advice will really help me out. Thanks once again

Also, do you think the reason for the poor detection of the labeled metabolites could be attributed to their shorter shelf-life than their corresponding unlabeled fractions? So, is it possible that the labelled metabolites are more easily degraded than unlabeled ones and hence I see good intensities with Mo (unlabeled) at 0 secs, but do not see good intensities with labeled fractions

A lot of possible reasons for this low 13C incorporation (dilution by CO2 at natural abundance, experiment not long enough, very low turn-over of these metabolites, etc).

I think this paper may help you: http://www.ncbi.nlm.nih.gov/pubmed/22688667

And no, the labelled metabolites aren't more easily degraded than unlabeled ones. Reaction rates can slightly change depending on the isotopic content of the substrates, but not significant enough -

Thank you so much for clearing my doubts regarding the stability of labelled metabolites. I will definitely go through the article you have suggested. 

may i ask you:which column did you choose for your LC-MS analysis.