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Measuring cerebral glucose metabolism in vivo using hyperpolarized 13C labelled glucose
Mor Mishkovsky1,2, Brian Anderson3, Magnus Karlsson4, Mathilde H Lerche4, A Dean Sherry3, Rolf Gruetter1,5,6, Zoltan Kovacs3, and Arnaud Comment2,7

1Laboratory for Functional and Metabolic Imaging, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne, Switzerland, 2Institute of Physics of Biological Systems, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne, Switzerland, 3Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, TX, United States, 4Albeda Research, ApS, Copenhagen, Denmark, 5Department of Radiology, Universite de Lausanne, Lausanne, Switzerland, 6Department of Radiology, Geneva University Hospital and Faculty of Medicine, Geneva, Switzerland, 7General Electric Healthcare, Buckinghamshire HP8 4SP, United Kingdom

Synopsis

Real-time glucose metabolism was observed in healthy mice brain following infusion of hyperpolarized [U-2H, U-13C]glucose and [U-2H, 3,4-13C]glucose. The evolution of lactate formation was readily observed. In addition, two glycolysis metabolites, namely 3-phosphoglycerate and pyruvate, were identified. Abnormalities in cerebral glucose metabolism is associated with large number of diseases so implementation of this method may prove useful in imaging brain metabolism in various animal models.

Introduction


Glucose is the primary fuel for the mammalian brain and variations in its metabolism are often indicative of pathologies1. Collection of MRS data during continuous infusion of thermally-polarized 13C-labeled glucose is a method of choice to study the kinetics of glucose metabolism and to highlight specific metabolic pathways2-4. Hyperpolarization by dissolution DNP offers dramatic enhancement of the 13C signal in small molecules5 and offers the possibility of detecting instantaneous metabolic transformations6. Despite the relatively short T1s of the 13C carbons in the glucose molecule, recent studies demonstrated the applicability of hyperpolarized [U-2H, U-13C]glucose to monitor metabolism in various in vitro systems7-11. Hyperpolarized 13C-labeled glucose has also been imaged in rats in vivo12. Real-time hyperpolarized [U-2H, U-13C]glucose metabolism in vivo has been reported in lymphoma tumors, but glucose metabolism could not be directly monitored in the brain13. The aim of the present study is to demonstrate that it is indeed possible to detect real-time cerebral metabolism of hyperpolarized glucose with a time resolution of sub-seconds.

Methods

[U-2H, U-13C]glucose and [U-2H, 3,4-13C]glucose samples were dynamically polarized at 7 T polarizer (196 GHz / 1.00±0.05 K) using trityl radical as polarizing agent and were dissolved with superheated D2O. All MR measurements were performed on a 9.4T/31cm actively shielded animal scanner (Varian/Magnex). The liquid polarization of hyperpolarized [U-2H, U-13C]glucose was measured inside an injection pump placed at the bore of the MR scanner, by comparing the hyperpolarized signal to its thermal counterpart (Figure 1). In vivo measurements were performed on C57BL/6J female mice. Animals were anesthetized using 1.5% isoflurane, a catheter was placed at their femoral vein and connected to a separator/infusion pump for automatic infusion of hyperpolarized solutions14. Field inhomogeneity was corrected using the FASTMAP protocol. In vivo data was collected using a home-built quadrature-1H single loop 13C surface coil. Acquisition was triggered 5.5s after bolus injection of hyperpolarized [U-2H, U-13C]glucose solution (n = 4) and hyperpolarized [U-2H, 3,4-13C]glucose (n = 2) using frequency selective RF pulses of 20° flip angle in the carboxyl area and 1.4° flip angle in the glucose resonances every 500 ms. Glucose levels were quantified from blood collected from the tail vein before and just after i.v. infusion. Animal physiology was measured during the entire length of the experiments. Lactate-to-pyruvate ratio was calculated from the area under the curve in the summed spectra.

Results

An average 13C glucose polarization of about 20% was measured in the liquid state at the time of injection (Figure 1). A typical time course of 13C spectra detected in the mice brain following hyperpolarized [U-2H, U-13C]glucose infusion is presented in Figure 2 (lower panel). The formation of [1-13C]lactate was readily detected at 183.5 ppm with good SNR for quantification (Figure 2 upper panel). Upon infusing of [U-2H, 3,4-13C]glucose, the lactate doublet is reduced to a single peak (Figure 3). Two additional metabolites could be identified in the summed spectra (Figure 3). The resonance at 171.1 ppm corresponds to the C1 of pyruvate with lactate-to-pyruvate ratio of 17.6 ± 2 (n = 5). The peak at 179.8 ppm was assigned to C1 of 3-phosphoglycerate (3PG). The blood glucose concentration did not exceed the typical values as in glucose tolerance test (GTT) with a peak at 16.8±2.8 mM post injection.

Discussion

We demonstrate the feasibility of using hyperpolarized 13C-glucose to measure in vivo cerebral metabolism in healthy mice using a dose of glucose similar to that used in a GTT. Replacing [U-2H, U-13C]glucose with [U-2H, 3,4-13C]glucose improves the sensitivity of the measurement due to lack of 13C-13C coupling. The glycolytic intermediate 3PG and glycolytic end product pyruvate were identified in vivo even though those metabolites cannot be detected using thermally polarized 13C MRS due to their low concentration. The observed lactate-to-pyruvate ratio is in agreement with its expected value15,16. Lactate signal was identified 10.5 s after glucose infusion, and the time course of lactate formation could be quantified. This dynamic data could provide a tool study the kinetics of cerebral glycolysis with sub-second time resolution, and is complementary to information obtained by thermally polarized 13C MRS where lactate can be identified only after 20 min post infusion even when using the an optimized setup4. Abnormalities in cerebral glucose metabolism are associated with large number of diseases so this method may prove useful for imaging brain metabolism in various animal models.

Conclusion

13C glucose can be polarized to high levels that enable its application to study real-time cerebral metabolism in healthy mice.

Acknowledgements

This work was supported by the Swiss National Science Foundation (grant PP00P2_133562 to A.C), the Centre d’Imagerie BioMédicale (CIBM) of the UNIL, UNIGE, HUG, CHUV, EPFL, the Leenards and Jeantet Foundations, and the National Institutes of Health (P41-EB015908 and R37-HL034557 to ADS).

References

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Figures

(A) 13C signal of [U-2H,U-13C]glucose enhanced by DNP at 7T measured inside a separator/infusion pump 3 seconds after dissolution and transfer (black) and the corresponding thermally polarized signal of the same solution measured overnight in the same setup (blue). (B) Usable 13C polarization of [U-2H,U-13C]glucose at the time of infusion quantified from the spectra for the different carbon positions.

Expanded region of typical dynamic spectra acquired from a mouse brain following infusion of hyperpolarized [U-2H, U-13C]glucose. Glucose C1 peaks appear at 96.8 ppm (C1-β) and 93 ppm (C1-α), lactate is observed at 183.5 ppm and can be identified 10.5 s after the infusion (bottom). The corresponding time course of the spectral data that was quantified using AMARES while fitting the lactate doublet to two separated peaks (top).

Expanded carboxyl region from summed spectra acquired after infusion of hyperpolarized [U-2H, U-13C]glucose (black) or hyperpolarized [U-2H, 3,4-13C]glucose (red). The glycolytic metabolites 3PG (179.8 ppm) and pyruvate (171.1 ppm) can be identified in addition to the lactate peak at 183.5 ppm. The resonance marked by (*) are unknown impurities. The 13C-13C coupling pattern is collapsed to a single peak when replacing [U-2H, U-13C]glucose by [U-2H, 3,4-13C]glucose.

Proc. Intl. Soc. Mag. Reson. Med. 25 (2017)
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