Introduction

Metabolic modeling of ¹³C turnover curves obtained during infusion of a ¹³C labeled substrate (e.g. [1,6-¹³C₂]glucose) in vivo allows measurement of compartmentalized metabolic fluxes and parameters providing basis for biomarker discovery. The goal of the present work was to extend the isotopomer metabolic model we recently developed¹ in order to simultaneously fit ¹³C MRS isotopomer curves of lactate and glutamate. Utilizing ¹³C MRS and LC-MS methods, coupled with metabolic modeling we have determined activities of key central metabolic pathways in human DB1 melanoma subcutaneous mouse model.

Methods

¹³C isotopomer MRS in vivo data acquired during [1,6-¹³C2]glucose infusion were analyzed with compartmentalized extended metabolic network models that included glucose, lactate and glutamine transport, glycolysis, TCA cycle, malate-aspartate shuttle, pyruvate carboxylase activity, other anaplerosis, pyruvate recycling through malic enzyme, lactate dehydrogenase activity, glutaminolysis, reductive carboxylation and de novo fatty acids synthesis. Isotope balance equations were derived for every network metabolite. The resulted sets of differential equations were solved to determine flux through various metabolic pathways by isotopomer/bonded cumomer analysis¹. Statistics for the obtained parameters/fluxes was calculated by Monte Carlo simulations including experimental noise levels².

Results

Metabolic Flux Analysis revealed metabolic enzymes activities in melanoma in vivo for multiple pathways and, potentially, these metabolic flux signature may serve as key biomarkers of therapeutic response and drug resistance. Some resulting fitted metabolic fluxes (see Figure 1, fluxes in mM/h) were: TCA cycle activity F_TCA= 16, mitochondrial pyruvate carrier FMPC= 34, anaplerotic flux at the SucCoA level FAna= 27, glutaminolysis activity FGls=35% of the TCA flux and mitochondrial malic enzyme activity was 50% of the TCA flux. In addition, the Warburg effect parameter (the ratio of lactate production flux to the pyruvate influx to mitochondria was estimated to be 0.25; ATP production rate in mitochondrial compartment accounted impressive 90% and only 50% of this energy from glucose-derived pyruvate; concentration of the mitochondrial glutamate was estimated as 1.1 mM and total glutamate concentration was 6mM. Additionaly Isotopomer Control Analysis³ was performed to estimate how robustly fluxes were estimated (Figure 2)

Discussion

The additional information included in the extended metabolic network has allowed simultaneously fitting of at least 6 experimental isotopomers time courses, which appear as multiplets in ¹³C spectra. In addition, the new model has allowed the measurement of some concentrations of compartmentalized metabolites which can hardly to measure with others experimental techniques. It worth to mention that using the additional information from ¹³C multiplets leads to an increase in precision for all metabolic fluxes in the model². Dynamic high-resolution MR spectra and LC-MS mass-isotopomers are very sensitive to changing/adding biochemical pathways and flux values, and metabolic modeling allows one to check precisely the feasibility of assumed general bionetworks and alterations of particular metabolic pathways. In conclusion, we were able to fit ¹³C isotopomer turnover curves simultaneously using the extended metabolic network where additional feasible fluxes and metabolite compartmentalized pools were included in the model.

Acknowledgements

No acknowledgement found.