Enhanced Solubility and Polarization of ¹³C-Fumarate with Meglumine Allows for In Vivo Detection of Gluconeogenic Metabolism in Kidneys

Abstract

Hyperpolarized ¹³C-labeled fumarate probes tissue necrosis via the production of ¹³C-malate. Despite its promises in detecting tumor necrosis and kidney injuries, its clinical translation has been limited, primarily due to the low solubility in conventional glassing solvents. In this study, we introduce a new formulation of fumarate for dissolution dynamic nuclear polarization (DNP) by using meglumine as a counterion, a nonmetabolizable derivative of sorbitol. We have found that meglumine fumarate vitrifies by itself with enhanced water solubility (4.8 M), which is expected to overcome the solubility-restricted maximum concentration of hyperpolarized fumarate after dissolution. The achievable liquid-state polarization level of meglumine-fumarate is more than doubled (29.4 ± 1.3%) as compared to conventional dimethyl sulfoxide (DMSO)-mixed fumarate (13.5 ± 2.4%). In vivo comparison of DMSO- and meglumine-prepared 50-mM hyperpolarized [1,4-¹³C₂]fumarate shows that the signal sensitivity in rat kidneys increases by 10-fold. As a result, [1,4-¹³C₂]aspartate and [¹³C]bicarbonate in addition to [1,4-¹³C₂]malate can be detected in healthy rat kidneys in vivo using hyperpolarized meglumine [1,4-¹³C₂]fumarate. In particular, the appearance of [¹³C]bicarbonate indicates that hyperpolarized meglumine [1,4-¹³C₂]fumarate can be used to investigate phosphoenolpyruvate carboxykinase, a key regulatory enzyme in gluconeogenesis.

Graphical Abstract

INTRODUCTION

Hyperpolarized (HP) nuclear magnetic resonance (NMR)/magnetic resonance imaging (MRI) is a collective term for various technologies developed to improve the sensitivity of conventional NMR/MRI. These include techniques that operate on physics principles such as dynamic nuclear polarization (DNP) and spin exchange optical pumping as well as the chemistry-based approaches such as parahydrogen-induced polarization. Hyperpolarization technology dramatically widens the scope of metabolic MRI by enabling the real-time in vivo detection of nonproton nuclei that are difficult to measure otherwise (e.g., ¹³C, ¹⁵N, and ²⁹Si).^1–3 Currently, dissolution DNP is the most widely used method to enhance the sensitivity of liquid-state MR signals largely because it is generally applicable to all MR active nuclei and can increase the MR signal by several orders of magnitude. Dissolution DNP works by transferring the high electron spin polarization to coupled nuclear spins at cryogenic temperatures followed by rapid dissolution of the frozen DNP sample. The most prominent applications of dissolution DNP are to polarize ¹³C-labeled metabolic substrates for in vivo ¹³C NMR spectroscopy and imaging. In vivo HP ¹³C MR spectroscopy (MRS) can provide read-outs of metabolite levels, pH, or redox state or can be used to monitor enzyme-catalyzed biochemical processes in real time, as they happen. The most commonly used HP agent is ¹³C-labeled pyruvate because it polarizes extremely well by DNP, is quickly transported into cells, and lies at a key intersection in intermediary metabolism where it can be oxidized to acetyl-CoA + CO₂ via pyruvate dehydrogenase (PDH) in mitochondria, forms alanine via transamination, or exchanges with tissue lactate via lactate dehydrogenase.⁴ As a result, translational studies with HP [1-¹³C]pyruvate are actively being performed at multiple sites around the world.^5–10

The DNP process requires an isotropic glass matrix, in which the molecules are randomly oriented. Therefore, the composition and vitrification properties of the frozen sample are an important consideration. The DNP sample must be a homogeneous mixture and is generally composed of the substrate to be polarized, the polarizing agent, and, if necessary, glassing agents. DNP polarizers are designed to accommodate a few hundred μL of a sample volume. The SPINlab polarizer, the only available clinical DNP polarizer at this time, can polarize up to ~2 mL of sample. For human studies, the concentration of the HP substrate in the administered solution is around 250 mM. This means that the concentration of the substrate in the DNP sample must be on the order of 1 to 10 M to produce an injectable solution of the HP substrate. Some compounds such as pyruvic acid form a glass at cryogenic temperatures without adding any vitrifying agents. However, the majority of substrates need to be dissolved in a glassing matrix for DNP. A commonly used biocompatible matrix for in vivo work is a mixture of glycerol and water, but other glassing matrices such as dimethyl sulfoxide (DMSO), glycerol, choline,¹¹ and trehalose¹² are also used.

HP ¹³C-labeled fumarate is another promising probe for clinical translation. The appearance of HP [1,4-¹³C₂]malate from HP [1,4-¹³C₂]fumarate via fumarase is a sensitive indicator of cellular necrosis associated with acute kidney injury, tumor necrosis, and myocardial infarction.^13–16 It was postulated that the formation of HP [1,4-¹³C₂]malate in necrotic tissues was the result of easier access of fumarate to fumarase in damaged tissues than in normal ones. The ruptured plasma membranes of necrotic cells may allow small molecules such as fumarate to rapidly enter cells or may facilitate the release of the enzyme into the interstitial space. Despite the potential for human applications, the poor solubility of both fumaric acid and sodium fumarate in conventional glassing solvents such as water, glycerol, and DMSO is a primary limiting factor for achieving HP fumarate concentration necessary for successful in vivo studies.¹⁷

It should be noted that ¹³C-labeled fumarate has also been polarized very efficiently by parahydrogen-induced polarization (PHIP) using acetylenedicarboxylate and a water-soluble ruthenium catalyst (pentamethylcyclopentadienyltris-(acetonitrile)ruthenium(II) hexafluorophosphate). The addition of parahydrogen is performed in an aqueous solution of sodium acetylenedicarboxylate to produce 180-mM crude HP solution of [1-¹³C]fumarate with 37% ¹³C polarization. Although the limited solubility of fumarate is obviously not an issue for the PHIP method, the removal of the catalyst is challenging. To produce catalyst-free solutions, the HP fumarate is precipitated out as HP fumaric acid after acidification with HCl and redissolved in bicarbonate D₂O solution. The precipitation and purification steps reduce the polarization to about 13%.^18,19

Miller et al. reported the conversion of HP fumarate to HP malate at a low level in healthy hearts, but not any other HP products.¹⁶ No other studies have reported any HP products from HP fumarate in healthy tissues.^13–16 Blocking fumarate transport across cell membranes via the sodium-dependent dicarboxylate acid transporter (DCT) had no effect on the HP malate signal in necrotic tumor tissues,¹³ strongly suggesting that fumarate uptake and metabolism in intact cells is slow and does not contribute significantly to the HP malate signal. As a result, metabolic pathways such as malate-aspartate shuttle or gluconeogenesis that are directly associated with fumarate have not been investigated using HP fumarate in normal tissues. However, the absence of products in normal tissues may be attributed to insufficient signal sensitivity.

In this study, we introduce a new formulation of fumarate for dissolution DNP by using meglumine as a counterion to enhance the polarization performance and investigate the feasibility of assessing intracellular fumarate metabolism and its related pathways using the meglumine-prepared HP [1,4-¹³C₂]fumarate.

RESULTS

Preparation of Meglumine Fumarate.

Meglumine fumarate was conveniently prepared by mixing one equivalent of fumaric acid with two equivalents of meglumine-free base in the presence of a trace amount of water (approximately 6% by weight) followed by sonication for 90 min pH of resulting preparation was 7.0–7.4.

Solubility and Glassing.

Glassing properties of meglumine-mixed fumarate were compared with conventional, DMSO-prepared fumaric acid at two concentrations: 3.6 and 4.8 M. Both meglumine fumarate and DMSO fumaric acid were prepared from natural abundance fumaric acid. At 3.6 M, meglumine fumarate and DMSO fumaric acid solutions were transparent after 90 min of sonication in a water bath at 40 °C (Figure 1B). For testing the glassing matrix, the meglumine fumarate and DMSO fumaric acid were rapidly frozen using liquid nitrogen. Both solutions formed a clear glass (Figure 1C). However, the fumaric acid solution in DMSO became cloudy when warmed to room temperature. In contrast, the meglumine fumarate remained clear in solution after thawing (Figure 1D). Maximum fumarate concentration was also higher with meglumine (4.8 M) than DMSO (3.6 M). The solubility comparison between meglumine fumarate and DMSO fumaric acid was performed at 4.8 M using natural abundance fumaric acid. The solution of DMSO fumaric acid was cloudy after 90 min of sonication at room temperature, whereas the meglumine fumarate was clear and transparent (Figure 1F). The stability of the meglumine-mixed fumarate during the preparation, storage, and transfer processes was checked using high resolution NMR as shown in Figures S1–S4. No byproducts were detected before and after 90 min of sonication in a water bath at 40 °C and after storing the stock for 3 months. As a result, during the in vitro and in vivo comparisons between HP meglumine fumarate and DMSO fumaric acid, both samples were prepared in 3.6 M.

Figure 1.

Glassing property of meglumine fumarate and DMSO fumaric acid. (A) Chemical reaction of meglumine fumarate. (B-E) 3.6 M of meglumine-fumarate (left) and fumaric acid in DMSO (right) (B) at room temperature after 90 min sonication, (C) after freezing in liquid nitrogen, (D) after thawing to the room temperature, and (E) after thawing at room temperature and sonicating 40 min at 40 °C. (F) 4.8 M of meglumine-fumarate (left) and fumaric acid in DMSO (right) at room temperature after 90 min sonication.

T₁ and Polarization.

Immediately after dissolution, the HP signal decays exponentially in accordance with a longitudinal relaxation time (T₁), a key parameter that limits the observation time window. Thus, having a long liquid-state T₁ is of utmost importance for HP probes. The liquid-state T₁ of HP [1,4-¹³C₂]fumarate at 1 T was longer when the sample was prepared with meglumine (59.47 ± 0.61 s, n = 7; P = 1.2 × 10⁻¹⁰) as compared to the DMSO-prepared fumarate sample (50.92 ± 0.43 s, n = 5) at pH 7.0. The T₁ of meglumine [1,4-¹³C₂]fumarate at 3 T at pH 7.0 (59.77 ± 5.72 s, n = 4) was comparable to the measurements at 1 T. The polarization level of meglumine-prepared [1,4-¹³C₂]fumarate was measured as 19.39 ± 3.10% (n = 7) after 28.2 ± 4.6 s of transport time from dissolution to scan. DMSO-prepared [1,4-¹³C₂]fumarate had 9.69 ± 3.1% (n = 5) after 28.9 ± 3.9 s of transport time. The polarization level at the time of dissolution was estimated as 29.42 ± 1.31% for meglumine [1,4-¹³C₂]fumarate, which is 2.18-fold of DMSO [1,4-¹³C₂]fumarate (13.48 ± 2.40%; P = 3.6 × 10⁻⁸). The in vitro performance of 50-mM meglumine [1,4-¹³C₂]fumarate (sample concentration: 3.6 M) in comparison to 50-mM DMSO [1,4-¹³C₂]fumarate (3.6 M) is summarized in Figure 2. Hyperpolarization of 4.8-M meglumine-fumarate samples was also feasible and tested to produce 80-mM [1,4-¹³C₂]fumarate HP solution after dissolution, as shown in Figure S5.

Figure 2.

Dissolution DNP of [1,4-¹³C₂]fumarate. Time-resolved HP ¹³C normalized NMR spectra of (A) 50-mM meglumine-prepared [1,4-¹³C₂]fumarate and (B) 50-mM DMSO-prepared [1,4-¹³C₂]fumarate. Representative first ¹³C spectra of (C) HP meglumine [1,4-¹³C₂]fumarate and (D) HP DMSO [1,4-¹³C₂]fumarate. (E) Relaxation of the HP meglumine [1,4-¹³C₂]fumarate and DMSO [1,4-¹³C₂]fumarate over time. (F) Calculated T₁ values and liquid-state ¹³C polarization levels of meglumine [1,4-¹³C₂]fumarate and DMSO [1,4-¹³C₂]fumarate at the time of dissolution.

In Vivo Performance.

The signal sensitivity of HP [1,4-¹³C₂]fumarate (175.4 ppm) was evaluated in healthy rodents in vivo at 3 T, as shown in Figure 3 from a representative rat. HP fumarate images from the other rats are available in Figure S7. The HP fumarate signal intensity in the kidneys, averaged over the regions of interest (ROIs), was 4.49 × 10¹⁰ ± 1.27 × 10¹⁰ arbitrary unit (a.u.) with HP 50-mM meglumine [1,4-¹³C₂]fumarate, which is about 10-times higher than HP 50-mM DMSO [1,4-¹³C₂]fumarate (4.42 × 10⁹ ± 2.20 × 10⁹ a.u.; P = 0.006). Due to the dramatic improvement of in vivo signal sensitivity, additional peaks could be detected using meglumine [1,4-¹³C₂]fumarate (Figure 3B).

Figure 3.

Performance comparison of 50-mM meglumine [1,4-¹³C₂]fumarate and 50-mM DMSO [1,4-¹³C₂]fumarate in healthy rat kidneys in vivo. (A) Chemical shift imaging (CSI) was acquired 13 s after a bolus injection of HP [1,4-¹³C₂]fumarate. [1,4-¹³C₂]Fumarate images and averaged ¹³C spectra in the kidneys using HP [1,4-¹³C₂]fumarate when prepared using meglumine (B) and DMSO (C). The raw spectra are available in Figure S6. The kidney ROIs used for averaging are marked in the matching ¹H MRI (shaded red regions). (D) As compared to DMSO-prepared fumarate, the averaged signal sensitivity of in vivo [1,4-¹³C₂]fumarate increased by 10.2-fold for signal intensity in kidneys (P = 0.006) when meglumine was used. Due to the improved sensitivity, additional peaks could be detected using meglumine [1,4-¹³C₂]fumarate (B).

In Vivo Detection of Gluconeogenic Products in Kidneys.

The chemical shifts of the additional peaks were aligned with the resonances of [1-¹³C]malate (181.7 ppm), [4-¹³C]malate (180.3 ppm), [4-¹³C]aspartate (178.3 ppm), and [¹³C]bicarbonate (160.7 ppm). Metabolite maps of HP [1,4-¹³C₂]malate and HP [¹³C]bicarbonate could be generated by integrating the peaks in pure absorption mode after correcting the zeroth order phase (Figure 4). [1,4-¹³C₂]-Aspartate peaks were not resolvable from the large [1,4-¹³C₂]-fumarate peak due to their adjacency. A cohort of rats was imaged twice under normal fed and 24-h fasted conditions to test whether they were sensitive to the nutritional condition. Both malate ([¹³C]malate-to-[¹³C]fumarate ratio) and bicarbonate ([¹³C]bicarbonate-to-[¹³C]fumarate ratio) did not show significant differences (P = 0.23 and 0.37) between the fed (malate/fumarate = 0.0088 ± 0.0005, bicarbonate/fumarate = 0.0013 ± 0.0004) and fasted states (malate/fumarate = 0.0077 ± 0.0006, bicarbonate/fumarate = 0.0020 ± 0.0006).

Figure 4.

In vivo detection of mitochondrial products in healthy rat kidneys using HP meglumine [1,4-¹³C₂]fumarate. In addition to [1,4-¹³C₂]fumarate (A), ¹³C metabolite maps could be generated for [1,4-¹³C₂]malate (B) and [¹³C]bicarbonate (C) from the phase-corrected ¹³C spectra. [1,4-¹³C₂]Aspartate peaks were not resolvable from the large [1,4-¹³C₂]fumarate peak (A). (D) ¹H MRI of the corresponding image plane. (E) Metabolic pathway of [1,4-¹³C₂]fumarate for producing [1,4-¹³C₂]malate, [1,4-¹³C₂]aspartate, and [¹³C]bicarbonate. (F) Malate and bicarbonate production were quantified in rat kidneys under fed and fasted conditions.

DISCUSSION

Solubility and Glassing.

Fumarate requires a glassing matrix such as DMSO and glycerol for DNP. With DMSO, fumaric acid concentration can reach 3.6 M. However, due to its extremely low solubility in water (6 g/L at 25 °C),^20,21 HP fumaric acid can quickly precipitate after dissolution, limiting the final concentration of fumarate in injectate. Moreover, although fumaric acid DMSO solutions will form a glass upon rapid cooling to cryogenic temperatures, the acid can crystallize when the sample is warmed after an initial rapid freezing process, as demonstrated in this study. This creates a practical problem of using DMSO-fumaric acid mixtures for DNP. Although sodium fumarate has a solubility of 220 g/L at 30 °C,²² the solubility gets much lower when mixed with the glassing matrix. In this study, a new fumarate formulation was introduced for dissolution DNP. We demonstrated that the use of meglumine as a counterion significantly improved the solubility and glassing properties of fumarate, which resulted in increased ¹³C polarization. Meglumine is a nontoxic and nonmetabolizable derivative of sorbitol with a strong structural similarity to glycerol. Due to the extremely high solubility in water (up to 1 g/mL), meglumine is widely used to increase the aqueous solubility of drugs and contrast agents.^23,24 The high solubility of meglumine in polar protic solvents is the result of intermolecular hydrogen bonding between the meglumine and the solvent molecules. Meglumine [1,4-¹³C₂]-fumarate was conveniently prepared by neutralizing fumaric acid with meglumine. We have found that meglumine fumarate vitrifies well by itself in the presence of trace amounts of water. The DNP samples were polarized in the clinical SPINlab polarizer. Notably, fumaric acid DMSO samples had to be loaded in the SPINlab using the fast lowering procedure to avoid crystallization of fumaric acid.¹⁵ This single step lowering process is not practical for large samples (e.g., human dose) for creating excessive heat load to the polarizer. However, the superior glassing properties of meglumine fumarate overcome this limitation and these samples could be loaded in the polarizer using the standard multistep lowering procedure. Fumarate concentrations in the meglumine sample could reach 4.8 M, which is expected to achieve 120-mM HP fumarate solutions after dissolution using a clinical DNP polarizer (SPINlab, GE Healthcare, Waukesha, WI, USA).²⁵ For human studies, estimating the maximum achievable concentration after dissolution requires extensive experimental investigations due to the complex mechanism that relies on multiple equipment-dependent factors, including the sample size and the dissolution media volume.^25,26 With the maximum fumarate sample in the sample vial (4.8 M × 1.5 mL = 7.2 mmol), the typical receiver volume of 55–60 mL, and 85–90% of sample recovery,²⁵ the maximum concentration of meglumine-prepared fumarate is expected to be 102–111 mM after dissolution. The ongoing translational study of HP DMSO-prepared fumarate targets to achieve 35 mM after dissolution using the system.²⁷ In addition, since the meglumine fumarate samples are prepared by neutralizing fumaric acid with two equivalents of meglumine, further neutralization during the dissolution is not necessary. On the other hand, DMSO-fumaric acid samples must be pH-neutralized.

Longer T₁.

T₁ values of meglumine fumarate were investigated at 1 and 3 T. At 1 T, the T₁ of HP [1,4-¹³C₂]fumarate was nearly 20% longer when the sample was prepared with meglumine as compared to the DMSO-fumaric acid samples that had been neutralized with NaOH. This may be due to the quadrupolar relaxation effect of the sodium ions present in the DMSO samples. The quadrupole moment of ²³Na is approximately 5 times stronger than that of ¹⁴N,²⁸ and the negatively charged carboxylate of fumarate may interact with the Na+ cations via ion pair formation. On the other hand, ion pair formation of the much larger protonated meglumine cation is much less likely.^29,30 The T₁ was maintained at a comparable level at 3 T. Previously, Wilson et al. reported that the T₁ of [1,4-¹³C₂]fumarate at 11.7 T is 29 s using water as solvent.³¹ The longer T₁ at 1 T and 3 T than at 11.7 T is because the relaxation mechanism for the carboxylate, chemical shift anisotropy, dominates.³² The T₁ does not increase significantly at lower fields (<1 T) because an additional relaxation mechanism becomes dominant: dipole–dipole relaxation from coupling to the nearby protons. Therefore, a T₁ of ~60 s is expected in applications of meglumine [1,4-¹³C₂]fumarate in clinical imaging scanners, which typically employ field strengths of 1—3 T. Apparent in vivo T₁, estimated by fitting the fumarate decay curve of the dynamic ¹³C MRS to a mono exponential function, was 31.0 s.

Improvement of Sensitivity.

The signal intensity of HP meglumine [1,4-¹³C₂]fumarate was compared to that of [1,4-¹³C₂]fumaric acid-DMSO samples in vivo at 3 T. The signal intensity of meglumine fumarate in kidney ROIs is more than 10 times higher than that of HP DMSO [1,4-¹³C₂]-fumarate. The 10-fold signal sensitivity is likely attributed to the longer T₁ relaxation time of meglumine-prepared fumarate (by ~20%), which saves the HP signal loss during the transport time and even in vivo, in addition to the 2-fold liquid-state polarization level. Indeed, Deh et al. reported a 39% increase of in vitro T₁ enhanced the image SNR by a factor of 2.6 in vivo.³³ Due to the significant improvement of in vivo signal sensitivity of meglumine fumarate, metabolic intermediates other than [1,4-¹³C₂]malate could be observed. The additional peaks at 160.7 and 178.3 ppm were assigned to bicarbonate and C4 of aspartate, respectively. The detection of aspartate and bicarbonate peaks using HP [1,4-¹³C₂]fumarate in either normal or necrotic kidneys has not been reported. Although not investigated in this study, more pronounced product signals could be detected at a later time point, considering that metabolic products from HP agents tend to peak several seconds after the injectate’s peak.^4,34

Cellular Transport.

Fumarate transport through the cell membrane via DCT is a relatively slow process, and although the formation of malate is also observed in normal tissues, the malate/fumarate ratio is much higher (up to 300%) in regions of necrotic cells where fumarate has access to the enzyme.³⁵ A previous study using ¹³C NMR on tissue extracts and superfusates reported that [U–¹³C₄]fumarate (5 mM) metabolism to [U–¹³C₄]malate under oxygenated and hypoxic conditions suggesting uptake and metabolism of fumarate.³⁶

Probing Gluconeogenic Pathway.

Considering the slow cellular transport, the detection of malate, aspartate, and bicarbonate was not expected and thus surprising. Malate is converted to oxaloacetate in the TCA cycle and exported to the cytosol via the malate aspartate shuttle where it is converted to oxaloacetate. Alternatively, oxaloacetate can be produced from malate in the cytosol via malate dehydrogenase. In either case, the detection of HP [¹³C]bicarbonate indicates the capability of HP meglumine [1,4-¹³C₂]fumarate for probing gluconeogenic pathways. One of the labeled carbon from [1,4-¹³C₂]oxaloacetate is released as carbon dioxide via phosphoenolpyruvate carboxykinase (PEPCK) and detected as bicarbonate. The other labeled carbon follows phosphoenolpyruvate (PEP), which can be further metabolized to [1-¹³C]pyruvate and then acetyl-CoA, releasing carbon dioxide via PDH (Figure 4). Moreover, [1,4-¹³C₂]oxaloacetate can be further metabolized in the TCA cycle, producing labeled carbon dioxide via isocitrate dehydrogenase (IDH) and alpha-ketoglutarate dehydrogenase (aKDH) (Figure 4). However, the source of HP [¹³C]bicarbonate is more likely via PEPCK rather than PDH, considering that HP [1-¹³C]PEP, [1-¹³C]-pyruvate, and [1-¹³C]lactate were not detected. The contribution of IDH and aKDH to HP [¹³C]bicarbonate is also expected to be negligible as these are much farther metabolic steps. Indeed, Hikari et al. demonstrated that HP bicarbonate produced from [1,4-¹³C₂]aspartate completely disappeared after inhibiting PEPCK in rat kidneys, demonstrating that HP [¹³C]bicarbonate is primarily via PEPCK.³⁷ In vivo experiments to compare the signal of [4-¹³C]aspartate and [¹³C]bicarbonate in the kidney in fasted and fed states showed higher bicarbonate/fumarate ratio in fasted animals compared to the fed group, although this did not reach statistical significance. This rather persistent bicarbonate level is consistent with the fact that renal gluconeogenesis is maintained stable until glycogen stored in the liver is depleted, which may require more than 24 h.^38,39 HP meglumine fumarate has unique potentials for investigating metabolic syndromes such as type 2 diabetes and chronic kidney diseases, which develop dysregulated gluconeogenesis.^40–42

Feasibility of Clinical Translation.

Due to the extremely high solubility in polar protic solvents such as water (up to 1 g/mL), meglumine is widely used to increase the aqueous solubility of drugs such as quercetin in water⁴³ and both gadolinium-based MR contrast agents^44,45 and iodinated contrast agents^46–49 for intravenous injection.^23,24 Frozen, DMSO-prepared fumaric acid samples became cloudy after thawing at room temperature, demonstrating limitation in its use. Its solubility decreases exponentially as the temperature decreases,²¹ which is a primary limiting factor to get highly concentrated solutions of the substrate necessary for human usage.¹⁷

Meglumine is considered a safe, nonmetabolizable expedient by the FDA.²³ Meglumine is used as a counterion for X-ray contrast agents [e.g., Conray (iothalamate meglumine), dosage: 1.6 mmol/kg] and for gadolinium-based MRI contrast agents [e.g., DOTAREM (gadoterate meglumine), dosage: 0.1 mmol/kg]. The meglumine supplement was reported to have a beneficial effect on metabolism. Analogous to the structurally related sorbitol, meglumine supplementation (18 mM in drinking water for at least 30 days) was shown to have a favorable effect on muscle function likely via elevating the expression of the AMPK-related kinase SNARK.⁵⁰ However, at a single dose of 1.25 mmol/kg employed in HP ¹³C MRS studies, meglumine is safe and unlikely to produce any metabolic effects.

CONCLUSIONS AND FUTURE PERSPECTIVE

In conclusion, we introduced a new formulation of HP [1,4-¹³C₂]fumarate with meglumine, demonstrated that meglumine improves the solubility of fumarate and the polarization performance, and reported previously invisible products along gluconeogenesis in rat kidneys in vivo. Obviously, the solubilizing and vitrifying effect of the meglumine counterion is not limited to fumarate. It should be applicable to other ¹³C- and ¹⁵N-labeled compounds that have poor solubility (i.e., fatty acids) and can form meglumine salts or adducts.^51,52 These include other carboxylic acids and other amino acid probes with low solubility.

METHODS AND MATERIALS

Preparation of Meglumine [1,4-¹³C₂]Fumarate.

177 mg of [1,4-¹³C₂]fumaric acid (1.5 mmol; Sigma-Aldrich, St. Louis, MO, USA) and 585 mg of meglumine (3 mmol; Sigma-Aldrich) were added to 1.5 mL Eppendorf tube; 50 μL of water was added to the mixture. The mixture solution was vortexed and then sonicated in a water bath (40 °C) for 90 min to generate the final concentrations of 3.6 M with pH 7.0–7.4. The stock solution was stored in a −20 °C freezer until use, and 4.8-M meglumine fumarate samples were prepared stoichiometrically as described for 3.6-M samples.

Hyperpolarization of Meglumine [1,4-¹³C₂]Fumarate.

3.6-M meglumine [1,4-¹³C₂]fumarate, containing 15-mM trityl OXO63 (GE Healthcare, Waukesha, WI, USA), was polarized using a SPINlab DNP polarizer (GE Healthcare) that operates at ~0.8 K in a 5 T magnet. 90 μL of the meglumine [1,4-¹³C₂]fumarate was placed in the sample vial and 17 mL of dissolution media (0.1 g/L of ethylenediaminetetraacetic acid [EDTA] in water, pH 7.4) was added into the dissolution syringe of each research fluid path (GE Healthcare). The assembled research fluid path was loaded into the SPINlab. After approximately 4 h of polarization, the sample was dissolved with the heated dissolution media (130 °C), producing 6.5–7 mL of 50-mM HP meglumine [1,4-¹³C₂]fumarate (pH 7.0). The temperature of the solution after dissolution was 37–40 °C.

Hyperpolarization of Sodium [1,4-¹³C₂]Fumarate using DMSO as the Glassing Matrix.

3.6-M DMSO [1,4-¹³C₂]fumaric acid samples were prepared by dissolving [1,4-¹³C₂]fumaric acid (Sigma-Aldrich) in DMSO (Sigma-Aldrich). 90 μL of 3.6-M DMSO [1,4-¹³C₂]fumaric acid containing 15-mM trityl OXO63 was placed in the sample vial for each fluid path with 17 mL of dissolution media (0.1 g/L of EDTA in water, pH 7.4) in the dissolution syringe. The assembled fluid path was loaded into the SPINlab and lowered using a one-step lowering option to avoid the crystallization of fumaric acid. After approximately 4 h of polarization, the sample was dissolved with the dissolution media and mixed with 0.5 mL of neutralizing media (0.72 M of NaOH, 0.4 M TRIS, and 0.1 g/L EDTA), producing 50-mM HP sodium [1,4-¹³C₂]fumarate (pH 7.0).

In Vitro Measurements of Liquid-State Polarization and T₁.

For each dissolution, ~ 0.5 mL of 50 mM HP [1,4-¹³C₂]fumarate at pH 7.0 was used to measure the polarization level and the T₁ relaxation time using a 1-T ¹³C NMR spectrometer (SpinSolve, Magritek, Malvern, PA, USA). Time-resolved ¹³C spectra were acquired every 10 s using a 10° flip angle for ~8 min until the HP signals disappeared. The liquid-state polarization level (P_HP) at the time of dissolution was estimated in vitro by comparing the first time-point of peak-integrated HP signal a_HP (50 mM) with the thermal equilibrium signal a_TH of neat [¹³C₆]benzene (12 M) using eq 1.

$$P_{\text{HP}}=P_{\text{TH}}\cdot \left(\frac{a_{\text{HP}}\cdot \sin {\theta }_{\text{TH}}\cdot c_{\text{TH}}}{a_{\text{TH}}\cdot \sin {\theta }_{\text{HP}}\cdot c_{\text{HP}}}\right)\cdot {\text{e}}^{t_{\text{ds}}/T_1}$$

Here, ${\theta }_{\mathrm{TH}}$ and ${\theta }_{\mathrm{HP}}$ are the radiofrequency (RF) flip angles (10° for both), used for sampling thermal and HP signals, respectively. The ¹³C concentration of the benzene sample, c_TH and the fumarate sample, c_HP, are 72 M (12 M × 6 carbons) and 0.1 M (0.05 M × 2 carbons), respectively. The thermal polarization level (P_TH) was calculated for room temperature and 1 T. The signal decay during the transport time (t_ds) of the HP solution from the polarizer to the spectrometer was corrected by extrapolating the T₁ decay. The T₁ relaxation times of [1,4-¹³C₂]fumarate samples were measured by fitting the decay of the fumarate peak to a monoexponential function after correcting the signal loss due to the RF sampling. The T₁’s of [1,4-¹³C₂]fumarate at 3 T was calculated from in vitro ¹³C spectra acquired using a dynamic ¹³C pulse-and-acquire pulse sequence (flip angle = 5.625°, pulse width = 32 μs, repetition time [TR] =3 s) with a clinical MRI scanner (Discovery 750w, GE Healthcare) and a ¹³C/¹H dual-tuned birdcage RF rat coil (Ø = 80 mm, GE Healthcare).

Experimental Setup for In Vivo Imaging with HP [1,4-¹³C₂]-Fumarate.

Six male Wistar rats were used for the study (body weight = 338.8 ± 19.2 g). Each rat was cannulated with a tail vein catheter under anesthesia (isoflurane level = 2–3%) and then placed in the ¹³C/¹H RF coil at the clinical 3 T system. After localization using a three-plane fast gradient echo sequence and B₀ shimming using point-resolved spectroscopy in ¹H mode, 50-mM HP DMSO [1,4-¹³C₂]-fumarate or HP meglumine [1,4-¹³C₂]fumarate was injected as a bolus through the catheter intravenously (0.625 mmol/kg body weight, up to 4.0 mL, injection rate = 0.25 mL/s). ¹³C free-induction decay (FID) chemical shift imaging (CSI) was acquired 13 s after the start of the HP fumarate injection [field-of-view (FOV) = 96 mm × 96 mm, matrix size = 16 × 16, slice thickness = 15 mm, flip angle = 10°, TR = 75 ms, spectral width = 5000 Hz, #spectral point = 256, total scan time = 19 s]. For anatomical reference, T₂-weighted dual-echo ¹H fast spin echo (FSE; FOV = 96 mm × 96 mm, slice thickness = 3 mm, flip angle = 160°, TR = 5,000 ms, echo times = 12.33 ms/61.64 ms, echo train length = 8, #slice = 7) was acquired from the slab plane that was prescribed for ¹³C CSI. Three of the rats (344.7 ± 26.0 g) were imaged with HP DMSO [1,4-¹³C₂]fumarate once. The other three rats (333 ± 11.8 g) were imaged with HP meglumine [1,4-¹³C₂]fumarate twice with an interval of 2–4 days to compare fed and fasted (24 h) conditions. Two of the rats received additional bolus of HP meglumine [1,4-¹³C₂]fumarate for a dynamic ¹³C pulse-and-acquire scan (flip angle = 10°, pulse width = 1024 μs, spectral width = 5000 Hz, #spectral point = 1024, slice thickness = 15 mm, TR = 3 s) approximately 30 min after the first HP injection.

Data Reconstruction and Analysis.

The ¹³C spectra acquired from the 1-T spectrometer were processed using Mnova (Mestrelab Research, A Coruna, Spain) for integrating HP [1,4-¹³C₂]fumarate signal. The k-space data of ¹³C CSI and dynamic ¹³C FID acquired from the 3-T scanner were reconstructed using MATLAB (Mathworks Inc., Natick, MA, USA), as described previously,^53,54 with 4-fold zero-padding in both spatial and frequency domains. Metabolite maps of [1,4-¹³C₂]fumarate, [1,4-¹³C₂]malate, and [¹³C]bicarbonate were generated by integrating the corresponding peaks in the absorption mode after zeroth-order phase correction in each voxel. For comparing the signal sensitivities of HP DMSO [1,4-¹³C₂]fumarate and meglumine [1,4-¹³C₂]fumarate in the kidney, ROIs were created around the kidneys on the T₂-weighted ¹H MRI. All the presented in vivo ¹³C spectra (Figures 3 and 4) were averaged over the ROIs and phase-corrected for both zeroth and first orders. [1,4-¹³C₂]Malate and [¹³C]bicarbonate were quantified by integrating the peaks in the ROI-averaged spectra from both kidneys and then normalized by the averaged [1,4-¹³C₂]fumarate signal in the ROI. [1,4-¹³C₂]Aspartate was excluded from the analysis due to the difficulty of resolving the peaks from the large, spectrally adjacent [1,4-¹³C₂]fumarate.

Statistical Evaluation.

All results are reported as mean ± standard deviation. For evaluating statistical significance of differences in the T₁, polarization level, signal sensitivity, and SNR between DMSO-prepared [1,4-¹³C₂]fumarate and meglumine-prepared [1,4-¹³C₂]fumarate, a nonpaired t test was used (α = 0.05, two-tailed). For evaluating the changes in [1,4-¹³C₂]malate and [¹³C]-bicarbonate production due to fasting, each product in the kidney ROIs in fed and fasted conditions was compared using a paired t test (α = 0.05, two-tailed) after normalized by [1,4-¹³C₂]fumarate peak.

ABBREVIATIONS

aKDH

alpha-ketoglutarate dehydrogenase

CSI

chemical shift imaging

DCT

dicarboxylate acid transporter

DMSO

dimethyl sulfoxide

DNP

dynamic nuclear polarization

EDTA

ethylenediaminetetraacetic acid

FID

free induction decay

HP

hyperpolarized

IDH

isocitrate dehydrogenase

MRS

magnetic resonance spectroscopy

PC

pyruvate carboxylase

PDH

pyruvate dehydrogenase

PEP

phosphoenolpyruvate

PEPCK

phosphoenolpyruvate carboxykinase

PHIP

parahydrogen-induced polarization

ROI

region of interest