Unfortunately, your question does not provide enough detail to give a helpful answer. Do you plan to trace oleic acid or its metabolites? Which organism? In-vivo or in-vitro? Quantitative measurment or qualitative detection?

Basically, you need to work out the dilution space of the system you wish to study to calculate the tracer recovery in the sample(s) you are going to collect for analysis.

Dear sir,

I want to do in-vivo 13C oleic acid to get the amino acids profile so i can back track and find from where it is coming.

OK. In-vivo study in animal or human subjects? If human, which age group? You do realise acetylCoA generated by beta-oxidation of LCFAs is a very versatile and widely used biochemical key molecule? A huge number of processes will consume acetylCoA derived from LCFA breakdown of which de-novo synthesis of some non-essential AA is just one? Furthermore, there are only 7 (9) AA in total that could potentially have a 13C-labelled C2 unit in their carbon skeleton, and synthesis of 3 (4) of those precedes (biochemically) synthesis of the other 4 (5). In other words, while the dilution space for your tracer is huge, recovery of your tracer in your target products will be quite low.

Dear Augenstein

I am working with the bacterial system. I want to label oleate to trace AA through GC-MS. I agree about the recovery. Can you suggest any lab which works on this so I can collaborate with them and get my procedure done.

Hello Shikha,

For GC-MS based labelled studies, I would try to read all the papers from Dougal Allen here: https://www.danforthcenter.org/scientists-research/principal-investigators/douglas-allen and ask him questions where you do not understand stuffs.

For general metabolic labelling studies, I would highly recommend consulting Dr. Teresa Fan: https://toxicology.med.uky.edu/users/wtfa224 and read her papers and tutorials on YouTube.

But then, without having a "clear experimental route", "challenges in data analysis and interpretation" I WOULD NOT start the experiment at all!

So better do lot of trials after reading all / most of their papers and then establishing the protocols, and see

Another serious limitation what I see is that without having access to a Mass-spec with resolution >1000,000 (a million) or so, if you can discern/ differentiate between labelled C-positions, which is what you are looking for. Make sure you have the right platform before doing such fancy experiments. Nonetheless, it is challenging, but WORTH looking at her core facility if you are willing to shell $$ amounts to get the work done there or start a collaboration: http://bioinformatics.cesb.uky.edu/RCSIRM/WebHome

Can discuss more too.

All the best wishes and thanks,

Biswa

If you are interested in seeing how many 13C-AcCoA units are incoporated into which non-essential AA one would expect to show 13C incorporation, you could run two experiments at two different levels of enrichment.

(A) Incubate your culture with [U-13C]-oleic acid diluted with natural abundance oleic acid to a level of 10 APE.

(B) Incubate your culture with [U-13C]-oleic acid diluted with natural abundance oleic acid to a level of 20 APE.

Either level of enrichment should be ample to be detected in your target AAs by GC/MS. Comparing the results from the two experiments should provide you with interesting quantitative information.

Thank you so much Wolfram Meier-Augenstein and Biswapriya Biswavas Misra