Metabolic flux analysis
Metabolic flux analysis is an experimental fluxomics technique used to examine production and consumption rates of metabolites in a biological system. At an intracellular level, it allows for the quantification of metabolic fluxes, thereby elucidating the central metabolism of the cell. Various methods of MFA, including isotopically stationary metabolic flux analysis, isotopically non-stationary metabolic flux analysis, and thermodynamics-based metabolic flux analysis, can be coupled with stoichiometric models of metabolism and mass spectrometry methods with isotopic mass resolution to elucidate the transfer of moieties containing isotopic tracers from one metabolite into another and derive information about the metabolic network. Metabolic flux analysis has many applications such as determining the limits on the ability of a biological system to produce a biochemical such as ethanol, predicting the response to gene knockout, and guiding the identification of bottleneck enzymes in metabolic networks for metabolic engineering efforts.
Metabolic flux analysis may use 13C-labeled isotope tracers for isotopic labeling experiments. Nuclear magnetic resonance techniques and mass spectrometry may then be used to measure metabolite labeling patterns to provide information for determination of pathway fluxes. Because MFA typically requires rigorous flux calculation of complex metabolic networks, publicly available software tools have been developed to automate MFA and reduce its computational burden.
Experimental method
Although using a stoichiometric balance and constraints of the metabolites comprising the metabolic network can elucidate fluxes, this approach has limitations including difficulty in stimulating fluxes through parallel, cyclic, and reversible pathways. Moreover, there is limited insight on how metabolites interconvert in a metabolic network without the use of isotope tracers. Thus, the use of isotopes has become the dominant technique for MFA.Isotope labeling experiments
Isotope labeling experiments are optimal for gathering experimental data necessary for MFA. Because fluxes determine the isotopic labeling patterns of intracellular metabolites, measuring these patterns allows for inference of fluxes. The first step in the workflow of isotope labeling experiments is cell culture on labeled substrates. A substrate such as glucose is labeled by isotope, most often 13C, and is introduced into the culture medium. The medium also typically contains vitamins and essential amino acids to facilitate cells' growth. The labeled substrate is then metabolized by the cells, leading to the incorporation of the 13C tracer in other intracellular metabolites. After the cells reach steady-state physiology, cells are then lysed to extract metabolites. For mammalian cells, extraction involves quenching of cells using methanol to stop their cellular metabolism and subsequent extraction of metabolites using methanol and water extraction. Concentrations of metabolites and labeled isotope in metabolites of the extracts are measured by instruments like liquid chromatography-mass spectrometry or NMR, which also provide information on the position and number of labeled atoms on the metabolites. This data are necessary for gaining insight into the dynamics of intracellular metabolism and metabolite turnover rates to infer metabolic flux.Methodologies
Isotopically stationary
A predominant method for metabolic flux analysis is isotopically stationary MFA. This technique for flux quantitation is applicable under metabolic and isotopic steady-state, two conditions that assume that metabolite concentrations and isotopomer distributions are not changing over time, respectively. Knowledge of the stoichiometric matrix comprising the consumption and production of metabolites within biochemical reactions is needed to balance fluxes around the assumed metabolic network model. Assuming metabolic steady-state, metabolic fluxes can thus be quantitated by solving the inverse of the following simple linear algebra equation:To reduce the possible solution space for flux distributions, isotopically stationary MFA requires additional stoichiometric constraints such as growth rates, substrate secretion and uptake, and product accumulation rates as well as upper and lower bounds for fluxes. Although isotopically stationary MFA allows precise deduction of metabolic fluxes through mathematical modeling, the analysis is limited to batch cultures during the exponential phase. Moreover, after addition of a labeled substrate, the time-point for when metabolic and isotopic steady-state may be accurately assumed can be difficult to determine.