Dynamic metabolic flux analysis

Towards dynamic metabolic flux analysis in CHO cell cultures. Biotechnol. /., 7, 61-74. 

The -based metabolic flux analysis is a more advanced technique that calculates the metabolic flux vector r by additionally using the -labelled pattern of the stable and abundant protein-bound amino acids determined by either gas chromatography coupled with mass spectroscopy (GC/MS) and/or nuclear magnetic resonance (NMR) (Christensen et al. 2002 Sauer 2006 Wiechert et al. 2(X)1 Zamboni et al. 2005, 2009). The most sophisticated technique known as kinetic flux profiling has recently been developed to calculate the metabolic flux vector r by measuring the dynamic incorporation of labeled substrates (e.g. C, N) into downstream intermediate metabolites. This measurement can be subsequently used to calculate metabolic fluxes (rates) directly without relying on the simplified metabolic network like the traditional MFA approach (Yuan et al. 2006, 2008,2010). 

The calculation of metabolic fluxes based on isotopomer distribution has gained importance as a quantitative and reliable tool for the study of cellular physiology. Metabolic fluxes are mainly calculated during steady-state conditions, which limits their applicability to scale-down dynamic studies. Nevertheless, research efforts with labeling experiments and mathematical models have allowed important advances in the dynamic C-based metabolic flux analysis [37-39], from which scale-down studies would be much benefited. An interesting example... 

The first example of a dynamic flux analysis was a study performed in the 1960s [269]. In the yeast Candida utilis, the authors determined metabolic fluxes via the amino acid synthesis network by applying a pulse with 15N-labeled ammonia and chasing the label with unlabeled ammonia. Differential equations were then used to calculate the isotope abundance of intermediates in these pathways, with unknown rate values fitted to experimental data. In this way, the authors could show that only glutamic acid and glutamine-amide receive their nitrogen atoms directly from ammonia, to then pass it on to the other amino acids. 

Cellular Metabolites. - A review of methods for the measurement of ml has been produced with 95 references. It examines the quantitative measurement of ml by mass spectrometry and in vivo NMR. The NMR chemical shifts and /-coupling values of 35 metabolites which can be detected by in vivo or in vitro investigations of the mammalian brain have been published. The principles and recent applications of dynamic nuclear polarisation, which combines the sensitivity to oxygen of EPR and the tractability of NMR imaging, have been reviewed with 244 references. A review of studies of intermediary metabolism, including the use of NMR in the analysis of substrate selection under in vivo conditions, has been produced. A review has been produced, with 74 references, on the study of metabolic flux and subcellular transport of metabolites using NMR. " ... 

In close analogy to flux-balance analysis, we thus extend the constraint-based description of metabolic networks to incorporate (local) dynamic properties. Recall the expansion of the mass-balance equation into a Taylor series, already given in Eq. (68)... 

We note that metabolic systems are studied often in systems biology using dynamics analysis such as flux balance analysis and differential equations. However, discussion regarding systems dynamics is beyond the scope of this current manuscript, and we refer the interested reader to the relevant literature (37, 38). This limitation, however, does not preclude these analyses from the standpoint of integrated systems analysis for understanding the metabolic pathway. 

Whereas detailed dynamic models can precisely answer questions on cellular behavior, the widespread application of such approaches has been hampered by the lack of kinetic information. In the absence of kinetic information, a method known as flux balance analysis (FBA) has been developed to analyze the metabolic capabilities of a cellular system based on mass balance constraints [Varma and Palsson 1994 Edwards et al. 1999 Edwards and Palsson 2000],... 

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