Comparison of kinetic models for analysis of pyruvate‐to‐lactate exchange by hyperpolarized ¹³C NMR

Abstract

The activity of specific enzyme-catalyzed reactions may be detected in vivo by 13C NMR of hyperpolarized (HP) substrates. The signal from HP substrates and products, acquired over time, have been fit to a number of different mathematical models to determine fluxes, but these models have not been critically compared. In this study, two-pool and three-pool first-order models were constructed to measure flux through lactate dehydrogenase in isolated glioblastoma cells by NMR detection of lactate and pyruvate following addition of hyperpolarized [1-13C]pyruvate. Mass spectrometry (MS) was used to independently monitor 13C enrichment in intra- and extracellular lactate. Six models were evaluated using time dependent pyruvate C2 and lactate C1 HP NMR data acquired by use of selective excitation pulses plus 13C enrichment data from intracellular and extracellular lactate measured by MS. A three-pool bi-directional model provided the most accurate description of pyruvate metabolism in these cells. With computed values for the T1 of pyruvate and lactate as well as the effect of pulsing, the initial flux through lactate dehydrogenase (LDH) was well-determined by both the two-pool bidirectional and unidirectional models when only HP data was available. The three-pool model was necessary to fit the combined data from both MS and HP, but the simpler two-pool exchange model was sufficient to determine the 13C lactate concentration when the lactate appearance was measured only by HP.

Simultaneous Steady-state and Dynamic 13C NMR Can Differentiate Alternative Routes of Pyruvate Metabolism in Living Cancer Cells

“…When a non-selective excitation pulse was applied, a summation of spectra revealed labeling of lactate C1 (Fig. 4A), as previously reported for these cells (31). Labeling in alanine C1 and in C1 of pyruvate hydrate was also detected, but H[…”

“…Acquisition of spectra was initiated immediately and proceeded for ϳ3.5 min (see "Hyperpolarization, Shaped Pulse, and NMR Spectroscopy in Intact Cells" below for details on cell transfer and conditions for the hyperpolarization experiment). We previously reported that cells subjected to trypsinization and hyperpolarized [1][2][3][4][5][6][7][8][9][10][11][12][13] C]pyruvate maintain rates of LDH flux similar to rates of adherent cells (31). To protect against depletion of oxygen from the medium during trypsinization and preparation of the cell suspension, the medium was mixed frequently, including immediately before transfer of the cells to the NMR tube, which occurred within a few seconds of introduction of [1][2][3][4][5][6][7][8][9][10][11][12][13] C]pyruvate.…”

“…Because the cells were injected into a waiting sample of HP pyruvate, they experience an instantaneous increase in pyruvate concentration, and there was no need for a time-dependent delivery term. Export of lactate from the cells is not included because previous experiments showed this to be unnecessary to obtain accurate flux values (31). Inclusion of a mitochondrial pool of pyruvate was required to adequately model the initial delay detected in the H[…”

“…The model fits were done using 1000 sets of initial values as described previously (31). Radiofrequency depolarization rates were measured in cell-free phantom experiments, and T 1 values were fit by the program.…”

“…These same activities were detected in mouse and human tumors by infusing 13 C-enriched glucose before surgery, extracting metabolites from surgically resected tumor tissue, and analyzing 13 C enrichment patterns by NMR (26,28). We also used hyperpolarized [1][2][3][4][5][6][7][8][9][10][11][12][13] C]pyruvate to quantify flux into lactate (31). Here, we combined these methods to study two metabolically distinct cancer cell lines.…”