Multi-compound polarization by DNP allows simultaneous assessment of multiple enzymatic activities in vivo

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Abstract

Methods for the simultaneous polarization of multiple 13C-enriched metabolites were developed to probe several enzymatic pathways and other physiologic properties in vivo, using a single intravenous bolus. A new method for polarization of 13C sodium bicarbonate suitable for use in patients was developed, and the co-polarization of 13C sodium bicarbonate and [1-13C] pyruvate in the same sample was achieved, resulting in high solution-state polarizations (15.7% and 17.6%, respectively) and long spin–lattice relaxation times (T1) (46.7 s and 47.7 s respectively at 3 T). Consistent with chemical shift anisotropy dominating the T1 relaxation of carbonyls, T1 values for 13C bicarbonate and [1-13C] pyruvate were even longer at 3 T (49.7 s and 67.3 s, respectively). Co-polarized 13C bicarbonate and [1-13C] pyruvate were injected into normal mice and a murine prostate tumor model at 3 T. Rapid equilibration of injected hyperpolarized 13C sodium bicarbonate with 13C CO2 allowed calculation of pH on a voxel by voxel basis, and simultaneous assessment of pyruvate metabolism with cellular uptake and conversion of [1-13C] pyruvate to its metabolic products. Initial studies in a Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model demonstrated higher levels of hyperpolarized lactate and lower pH within tumor, relative to surrounding benign tissues and to the abdominal viscera of normal controls. There was no significant difference observed in the tumor lactate/pyruvate ratio obtained after the injection of co-polarized 13C bicarbonate and [1-13C] pyruvate or polarized [1-13C] pyruvate alone. The technique was extended to polarize four 13C labelled substrates potentially providing information on pH, metabolism, necrosis and perfusion, namely [1-13C]pyruvic acid, 13C sodium bicarbonate, [1,4-13C]fumaric acid, and 13C urea with high levels of solution polarization (17.5%, 10.3%, 15.6% and 11.6%, respectively) and spin–lattice relaxation values similar to those recorded for the individual metabolites. These studies demonstrated the feasibility of simultaneously measuring in vivo pH and tumor metabolism using nontoxic, endogenous species, and the potential to extend the multi-polarization approach to include up to four hyperpolarized probes providing multiple metabolic and physiologic measures in a single MR acquisition.

Introduction

A significant advantage of hyperpolarized MR metabolic imaging using dynamic nuclear polarization (DNP) is the ability to probe metabolic fluxes in real time, at high signal-to-noise [1]. In vivo hyperpolarized MR is unprecedented in its ability to characterize specific enzymatic pathways [2], [3]. Most studies to date have focused on the last step of glycolysis in which [1-13C] pyruvate is enzymatically converted to a number of products, including [1-13C] lactate mediated by the activity of lactate dehydrogenase (LDH) [3], [4], [5], [6], [7]. This pathway is associated with the Warburg effect, which postulates increased metabolism to lactate in tumor cells relative to normal tissue [8]. More recently, a number of additional pathways have been probed, for example the conversion of bicarbonate to CO2 as mediated by carbonic anhydrase, and the conversion of glutamine to glutamate catalyzed by glutaminase [9], [10]. Investigation of these processes by DNP–NMR has allowed mapping of pH in vivo and assessment of glutaminase activity in hepatic tumor cells. Additional agents showing promise in animals or perfused heart models include [2-13C] pyruvate, [2-13C] fructose, and [1-13C] lactate itself [11], [12], [13]. As the number of useful DNP agents continues to expand, the ability to probe multiple pathways and mechanisms simultaneously may provide valuable metabolic “signatures” associated with specific types of tumor and other diseased tissue. 1H MRS is well established as a means to establish metabolic profiles in diseased tissue in vivo [14], [15], but hyperpolarized MR has the additional capacity to provide kinetic information. A particular conversion pattern observed in diseased tissue may aid in targeting regions of pathology for biopsy or focal therapy, and/or better characterize the extent or aggressiveness of disease present prior to or after treatment. 13C sodium bicarbonate has special promise due to its lack of toxicity and ability to probe physiological pH [9]. A broad number of pathologic processes demonstrate alterations in pH, including neoplastic, ischemic, and inflammatory conditions [16], [17], [18], [19].

The dynamic nuclear polarization process requires the 13C labelled probe compound to be in an amorphous (glassy) solid state with the appropriate free radical concentration at low temperatures (∼1.2 K) [1]. To accomplish an optimal preparation of a new probe or combinations of probes, the concentration of the desired agent(s), solvent/glassing agent(s), presence, concentration and type of a gadolinium agent, and the concentration and type of free radical must all be optimized. In addition, appropriate dissolution media must be prepared for each agent in order to ensure physiological pH, osmolarity, and to preserve the longest possible T1. In this manuscript, a method for polarization of 13C sodium bicarbonate is reported, without requiring the removal of cesium as in the prior published method. This method has been combined with a co-polarization technique that allows simultaneous polarization of 13C bicarbonate and [1-13C] pyruvate, to perform both pH and metabolic mapping in vivo using a single intravenous bolus. The technique was subsequently extended to polarize four 13C labelled substrates, namely [1-13C] pyruvic acid, 13C sodium bicarbonate, [1,4-13C] fumaric acid, and 13C urea in vitro demonstrating the potential of obtaining information on pH, metabolism, necrosis and perfusion, in a single in vivo imaging experiment.

Section snippets

Sample preparation

In all cases the 13C compounds were purchased from Isotec (Miamisburg, OH) and used without further purification. All natural abundance chemicals and solvents were obtained from Aldrich (Miamisburg, OH). 13C sodium bicarbonate: 135 mg of 13C-sodium bicarbonate were dissolved in 1099 mg of glycerol, in a sealed flask while heating with a heat-gun. The hot solution was then passed through a 0.45 μM Millipore MCE Membrane filter (Fisher Scientific), and OX63 radical (Oxford Instruments, Abingdon, UK)

Co-polarization of 13C-sodium bicarbonate with [1-13C] pyruvic acid and T1 measurements

A preparation method for 13C sodium bicarbonate in glycerol was developed, allowing for reasonable 13C sodium bicarbonate concentrations in the preparation [1.8 M  half that achieved previously for cesium bicarbonate [9]] and a high solution-state polarization (12.7 ± 1.9%). The final concentration of sodium bicarbonate is currently limited by the lower solubility of sodium bicarbonate relative to cesium bicarbonate in water and the maximum volume of the preparation that can be polarized in the

Discussion

Multi-metabolite polarization is intended to circumvent one of the main drawbacks of DNP, namely its long polarization times, by polarizing several precursors at the same time, and then developing a biological scenario whereby the inevitably very complex metabolic data in vivo can be analyzed. In this study, a method for direct polarization of 13C sodium bicarbonate suitable for use in patients was developed, and the co-polarization of 13C sodium bicarbonate and [1-13C] pyruvate in a single

Acknowledgments

We would like to acknowledge funding from the National Institutes of Health (R21 EB005363, R01 EB007588); NIBIB T32 Training Grant 1 T32 ED001631 as well as support from GE Healthcare. Grant sponsors: National Institutes of Health (R21 EB005363, R01 EB007588); NIBIB T32 Training Grant 1 T32 ED001631.

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      Several promising HP agents are also being investigated for diverse applications, including [1,4-13C]fumarate for imaging necrosis [3], 13C-urea for perfusion [4], [2-13C]pyruvate for citric acid cycle metabolism [5], [1-13C]acetate for fatty acid metabolism [6], [1-13C]dehydroascorbate [7] and [1,3-13C]acetoacetate [8] for imaging redox potentials, and [2-13C]dihydroxyacetone [9] for measurement of gluconeogenic and glycolytic flux. Additionally, using multiple HP agents in a single experiment permits the simultaneous interrogation of different physiological processes [10]. Each HP metabolic agent produces a distinct set of downstream metabolites in tissue, requiring specialized MR methods to spectrally encode the corresponding chemical shifts that distinguish the biochemical fates of the HP 13C labels.

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