Hi,
I am trying to measure which metabolic pathways are active in my cells and how they change when I modify the conditions. I would like to perform a metabolic flux analysis but I would like to avoid tracers as much as possible. Is there any other alternative method? Many thanks in advance.
That will depend on wat exactly you want to see. Form some major pathways respirometry is a suitable approach. You measure respiration of your cells in standard conditions (e.g. in cell culture medium) and then add specific inhibitors. Doing so you can discern total energy turnover (or metabolic activity) and protein synthesis-related turnover (by using e.g. cycloheximide as inhibitor) and the turnover related to the main ion pumping activity (e.g. ouabain as inhibitor of the sodium pump)...
Buttgereit, F. and Brand, M. D. (1995) A hierarchy of ATP-consuming processes in mammalian cells. Biochem. J. 312, 163-167.
Siems, W., Dubiel, W., Dumdey, R., Muller, M. and Rapoport, S. M. (1984) Accounting for the ATP-consuming processes in rabbit reticulocytes. Eur. J. Biochem.139, 101-107.
Wieser W, Krumschnabel G. Biochem J. Hierarchies of ATP-consuming processes: direct compared with indirect measurements, and comparative aspects. 2001 Apr 15;355(Pt 2):389-95.
Dear Javier,
if you do not have access to tracer substrates, you may use stoichiometric metabolic flux analysis or flux balance analysis instead. Successful examples for various organisms are given below:
sMFA:
https://www.ncbi.nlm.nih.gov/pubmed/18613101
https://www.ncbi.nlm.nih.gov/pubmed/18609599
https://www.ncbi.nlm.nih.gov/pubmed/17931730
https://www.ncbi.nlm.nih.gov/pubmed/15758216
FBA:
https://www.ncbi.nlm.nih.gov/pubmed/19128495
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4855977/
It is solely based on stoichiometric balance equations around metabolite pools in central carbon metabolism. However, the quality of your outcome pretty much depends on your assumptions to constrain your biochemical network (e.g. pathway definition, biomass composition, respiration efficiency, cofactor balancing etc.).
Nevertheless, I would always recommend to apply 13C-MFA (if possible) as the sensitive labeling information adds to the stoichiometric information (overdetermined equation system) thus reducing your solution space and making your flux data much more confident.
Good luck.
Michael
Dear Javier,
for exchange fluxes (intake or secretion of nutrients), an approximation that might work is to measure the difference in the concentration of a metabolite in the medium in a given interval of time. You could use these exchange fluxes to constrain your FBA model and then estimate internal fluxes.
However, if you need the flux of a single cell you should probably divide the flux per the integral of the growth curve in the considered interval of time. I have some concerns on this way to proceed and I would appreciate some comments on this.
Chiara
Thanks a lot for all the answers. I will explore the different possibilities.
- Badges
- Science topic