Bonded Cumomer Analysis of Human Melanoma Metabolism Monitored by 13C NMR Spectroscopy of Perfused Tumor Cells

Abstract

A network model for the determination of tumor metabolic fluxes from 13 C NMR kinetic isotopomer data has been developed and validated with perfused human DB-1 melanoma cells carrying the BRAF V600E mutation, which promotes oxidative metabolism. The model generated in the bonded cumomer formalism describes key pathways of tumor intermediary metabolism and yields dynamic curves for positional isotopic enrichment and spin-spin multiplets. Cells attached to microcarrier beads were perfused with 26 mM [1,6-13 C 2 ]glucose under normoxic conditions at 37°C and monitored by 13 C NMR spectroscopy. Excellent agreement between model-predicted and experimentally measured values of the rates of oxygen and glucose consumption, lactate production, and glutamate pool size validated the model. ATP production by glycolytic and oxidative metabolism were compared under hyperglycemic normoxic conditions; 51% of the energy came from oxidative phosphorylation and 49% came from glycolysis. Even though the rate of glutamine uptake was ϳ50% of the tricarboxylic acid cycle flux, the rate of ATP production from glutamine was essentially zero (no glutaminolysis). De novo fatty acid production was ϳ6% of the tricarboxylic acid cycle flux. The oxidative pentose phosphate pathway flux was 3.6% of glycolysis, and three non-oxidative pentose phosphate pathway exchange fluxes were calculated. Mass spectrometry was then used to compare fluxes through various pathways under hyperglycemic (26 mM) and euglycemic (5 mM) conditions. Under euglycemic conditions glutamine uptake doubled, but ATP production from glutamine did not significantly change. A new parameter measuring the Warburg effect (the ratio of lactate production flux to pyruvate influx through the mitochondrial pyruvate carrier) was calculated to be 21, close to upper limit of oxidative metabolism.Cancer cells exhibit metabolic patterns that differ significantly from those of terminally differentiated adult tissues. They require rapid production of both energy and biosynthetic precursors to sustain a high rate of proliferation. The network of biochemical pathways underlying these processes is complex. Most of the individual enzymatic conversions involved in this network have probably already been identified, but a comprehensive and quantitative description of the fluxes involved is lacking. Methods to produce such descriptions will likely be very useful in the design of new therapeutics that specifically target the metabolic abnormalities of cancer as well as in clinical implementation of non-invasive methods for detection of cancer and its therapeutic response.The kinetics of 13 C isotope labeling as monitored by NMR spectroscopy has been used to measure flux through various metabolic pathways of perfused isolated cells and organs and in vivo (1-4). Kinetic data provide estimates of absolute flux, whereas steady-state data measure relative flux. Both types of analysis require validated mathematical models, which have been formulated for the heart (5-10), liver (11-13), brain (14 -16), a...

Mechanism of antineoplastic activity of lonidamine

“…To quantitatively model the metabolic pathways related to energy and other intermediary metabolism, Shestov et al (32, 58) have used 13 C metabolic modeling techniques called Bonded Cumomer and Fragmented Cumomer Analysis for analysis of 13 C NMR and LC-MS data, respectively. These techniques provide a detailed picture of metabolism at the level of in vivo enzyme activities, whole pathways and the integrated systems level.…”

“…Transport of the main nutrients utilized by cancer cells were included in the model: the perfused labeled glucose, lactate and glutamine were transported from the extracellular medium to the cancer cells and vice versa assuming reversible non-steady-state Michaelis-Menten transport kinetics through corresponding transporters. Isotopomer balance equations were derived for all bonded cumomers of orders 1, 2, and 3 of participating metabolites (32, 57, 58). Fine structure multiplets were completely described by each metabolite’s bonded cumomers (32, 57, 58) of order 1, 2, and 3 using connection matrices between bonded cumomers and 13 C fine multiplets (32, 57, 58).…”

“…Isotopomer balance equations were derived for all bonded cumomers of orders 1, 2, and 3 of participating metabolites (32, 57, 58). Fine structure multiplets were completely described by each metabolite’s bonded cumomers (32, 57, 58) of order 1, 2, and 3 using connection matrices between bonded cumomers and 13 C fine multiplets (32, 57, 58). The use of bonded and fragmented cumomer techniques leads to a reduced number of equations compared to a model including all possible isotopomers while retaining all the NMR- and MS-measurable isotopomer information.…”

“…Ben-Yoseph et al (28) also demonstrated selective acidification and de-energization of gliomas in rats with no significant effects on the muscle or brain. Selectivity for tumors probably results from their higher glycolytic capacity although perfused DB-1 melanoma cells derive 54% of their energy from oxidative metabolism and 46% by glycolysis as shown by Shestov et al (32). …”

Perfused Cells, Tissues and Organs by Magnetic Resonance Spectroscopy

“…There are a number of methods to do this, but most involve solving a series of coupled differential equations. 24,37 Despite the relatively small number of experimental measurements available, detailed information on metabolic flux can still be obtained. Hyperpolarization Methods for MRS) has led to new interest in 13 C-MRS for perfused cell and organs.…”