Elsevier

Magnetic Resonance Imaging

Volume 29, Issue 8, October 2011, Pages 1035-1040
Magnetic Resonance Imaging

Original contribution
In vivo measurement of normal rat intracellular pyruvate and lactate levels after injection of hyperpolarized [1-13C]alanine

https://doi.org/10.1016/j.mri.2011.07.001Get rights and content

Abstract

Hyperpolarized technology utilizing dynamic nuclear polarization has enabled rapid and high-sensitivity measurements of 13C metabolism in vivo. The most commonly used in vivo agent for hyperpolarized 13C metabolic imaging thus far has been [1-13C]pyruvate. In preclinical studies, not only is its uptake detected, but also its intracellular enzymatic conversion to metabolic products including [1-13C]lactate and [1-13C]alanine. However, the ratio of 13C-lactate/13C-pyruvate measured in this data does not accurately reflect cellular values since much of the [1-13C]pyruvate is extracellular depending on timing, vascular properties, and extracellular space and monocarboxylate transporter activity. In order to measure the relative levels of intracellular pyruvate and lactate, in this project we hyperpolarized [1-13C]alanine and monitored the in vivo conversion to [1-13C]pyruvate and then the subsequent conversion to [1-13C]lactate. The intracellular lactate-to-pyruvate ratio of normal rat tissue measured with hyperpolarized [1-13C]alanine was 4.89±0.61 (mean±S.E.) as opposed to a ratio of 0.41±0.03 when hyperpolarized [1-13C]pyruvate was injected.

Introduction

Hyperpolarized MR imaging technology utilizing dissolution dynamic nuclear polarization (DNP) has made feasible the rapid and high-sensitivity detection of in vivo metabolism of prepolarized 13C compounds [1]. The most widely used DNP agent for both in vitro and in vivo applications thus far has been [1-13C]pyruvate. After injection, [1-13C]pyruvate is actively transported into cells by monocarboxylate transporters (MCTs) and acts as a metabolic precursor whose flux to [1-13C]lactate and [1-13C]alanine is catalyzed by the enzymes lactate dehydrogenase (LDH) and alanine transaminase (ALT), respectively. The levels of hyperpolarized metabolites have been shown to inform on the disease state of tissue in many preclinical studies. In particular, in cancer animal models, dramatic increases in [1-13C]lactate have been observed [2], [3], [4]. Typically, [1-13C]lactate is measured relative to other hyperpolarized metabolites, for example, [1-13C]pyruvate or [1-13C]total carbon (the sum of hyperpolarized pyruvate, alanine and lactate). However, the ratio of 13C-lactate/13C-pyruvate measured typically does not accurately reflect cellular values since much of the [1-13C]pyruvate is extracellular depending on timing, vascular properties, and extracellular space and MCT activity. In order to measure the relative levels of intracellular pyruvate and lactate, we hyperpolarized [1-13C]alanine and monitored the in vivo conversion to [1-13C]pyruvate and [1-13C]lactate. With the assumption that normal cells exhibit little or no leakage of ALT, then hyperpolarized alanine must be transported into cells in order for conversion to hyperpolarized pyruvate and then lactate to take place, and, thus, the detected levels of pyruvate and lactate would reflect intracellular ratios.

Section snippets

Polarizer and preparations

All hyperpolarizations were performed using a HyperSense DNP polarizer (Oxford Instruments, Abingdon, UK). The alanine preparation was created by mixing together 350 mg [1-13C] l-alanine (Cambridge Isotope Laboratories, Andover, MA, USA), 250 μl 18.94 M NaOH and 100 μl DMSO. The resulting alanine and NaOH concentrations were ∼6.2 and ∼7.5 M, respectively. Also added were 15 mM OX063 trityl radical and 0.3 mM Dotarem gadolinium (Guerbet). A 100-μl sample of the final mixture was polarized at a

Results

Fig. 1 shows a comparison of hyperpolarized spectra obtained from injecting [1-13C]pyruvate and [1-13C]alanine. As illustrated by Fig. 1A, in the hyperpolarized [1-13C]pyruvate injection experiment, hyperpolarized [1-13C]lactate and hyperpolarized [1-13C]alanine are produced by LDH and ALT catalyzed reactions, respectively. Note that for clarity, the MCT step and the transport of pyruvate into the mitochondria/decarboxylation to acetyl-CoA and 13CO2 are not shown. In contrast, when

Discussion

Hyperpolarized 13C metabolic imaging, with its many advantages — including high sensitivity, virtually no background signal, and no ionizing radiation — has the potential to become an important research and ultimately clinical tool [7]. Use of hyperpolarized 13C technology to monitor metabolism could have a significant impact on the diagnosis/staging of disease and measurement of its response to treatment [7]. Substantial effort has already been devoted to the evaluation of this technology in

Conclusions

Uptake of hyperpolarized [1-13C]alanine and its flux to [1-13C]pyruvate and [1-13C]lactate were detected in vivo, with the enhancement from hyperpolarized technology enabling the reliable detection of low steady-state levels of pyruvate. The lactate-to-pyruvate ratios, which reflected intracellular values, were substantially higher than those measured when [1-13C]pyruvate was injected, suggesting that the majority of pyruvate detected in [1-13C]pyruvate studies is of extracellular origin.

Acknowledgments

The authors thank Dr. James Tropp for designing and building the coil used in this study and Kristen Scott for animal handling assistance.

References (13)

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This study was supported by NIH grant R01 EB007588, P41EB013598, and UC Discovery Grant ITL-BIO04-10148 in conjunction with GE Healthcare.

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