Kinetic Modeling of Hyperpolarized 13C Label Exchange between Pyruvate and Lactate in Tumor Cells

Abstract

Measurements of the kinetics of hyperpolarized 13 C label exchange between [1-13 C]pyruvate and lactate in suspensions of intact and lysed murine lymphoma cells, and in cells in which lactate dehydrogenase expression had been modulated by inhibition of the PI3K pathway, were used to determine quantitatively the role of enzyme activity and membrane transport in controlling isotope flux. Both steps were shown to share in the control of isotope flux in these cells. The kinetics of label exchange were well described by a kinetic model that employed rate constants for the lactate dehydrogenase reaction that had been determined previously from steady state kinetic studies. The enzyme showed pyruvate inhibition in steady state kinetic measurements, which the kinetic model predicted should also be observed in the isotope exchange measurements. However, no such pyruvate inhibition was observed in either intact cells or cell lysates and this could be explained by the much higher enzyme concentrations present in the isotope exchange experiments. The kinetic analysis presented here shows how lactate dehydrogenase activity can be determined from the isotope exchange measurements. The kinetic model should be useful for modeling the exchange reaction in vivo, particularly as this technique progresses to the clinic.Magnetic resonance spectroscopy is a powerful tool for the non-invasive investigation of cellular metabolism, in systems ranging from isolated cells, through perfused organs (1), and small animals to humans (2, 3). A fundamental limitation of magnetic resonance spectroscopy has been a lack sensitivity, which limits temporal resolution to the minute time scale and spatial resolution to 1-10 cm 3 , depending on the magnetic field strength and nucleus detected (4). The recent introduction of dissolution dynamic nuclear polarization (5), which can increase the sensitivity of the magnetic resonance experiment by more than 10,000-fold, has had a significant impact on the field (reviewed in Refs. 6 and 7), dramatically increasing the temporal resolution to the second time scale and the spatial resolution to the millimeter scale. With this technique the 13 C nucleus, in a 13 C-labeled cell metabolite, is hyperpolarized and then injected into the biological system, where this may be an intravenous injection into a small animal or human. The gain in sensitivity, due to hyperpolarization of the 13 C nucleus, means that there is now sufficient signal to image the distribution of the labeled metabolite in the body and, more importantly, its metabolism and conversion into other cellular metabolites. The main limitation is the relatively short lifetime of the polarization, which in the 13 C-labeled metabolites, which have been used to date, is between 10 and 30 s. This means that spectroscopic imaging must be accomplished within 2-3 min and the labeled molecule must be taken up by cells and metabolized very rapidly for a significant labeling of other cell metabolites within the lifetime of the polarization. Although other nuclei ca...

Magnetic resonance imaging with hyperpolarized [1,4- 13 C 2 ]fumarate allows detection of early renal acute tubular necrosis

“…LDH is one of the genes up-regulated by hypoxia-inducible factor-1α (34), a transcription factor known to be increased in ATN (35,36). The increase in LDH activity, coupled with the expected increase in lactate concentration in the hypoxic tissue, will increase the degree of lactate labeling (37). The hyperpolarized [1-13 C]lactate signal obtained from the kidney will reflect the summed value of the increased labeled lactate labeling generated in intact, hypoxic tubular cells and the decrease attributable to loss of LDH, lactate, and NADH from frankly necrotic tubular cells.…”

Spin echo measurements of the extravasation and tumor cell uptake of hyperpolarized [1‐13C]lactate and [1‐13C]pyruvate

“…MCT activity is known to play a significant role in the observed pyruvate-lactate exchange in vivo (35,36) and we have shown previously in EL4 cells in vitro that the rate of label exchange between injected pyruvate and endogenous lactate is affected by both MCT and lactate dehydrogenase activity (26). In a spin echo experiment with injected hyperpolarized [1-13 C]pyruvate, the increase in the pyruvate echo/FID ratio was also decreased by 4-CIN, again suggesting that at least part of the increase in the ratio is due to cell uptake.…”

“…MRS was performed when the tumors had grown to a size of $2 cm 3 (which was reached typically at 10 days following implantation) (8) (18,19), were prepared by adding 1.2 mM gadolinium chelate to a solution containing 44 mg of HP001 and 15 mM OX063. Samples were hyperpolarized in an alpha-prototype hyperpolarizer (GE Healthcare, Amersham, UK), as described previously (17,26). The polarized samples were dissolved in pressurized buffer at $180 C and $1000 kPa.…”

Evaluation of Deuterated 18F- and 11C-Labeled Choline Analogs for Cancer Detection by Positron Emission Tomography

“…Western blotting was done using standard techniques (22,23). For detailed methodology, see Supplementary Materials.…”