Observation of Glycolytic Metabolites in Tumor Cell Lysate by Using Hyperpolarization of Deuterated Glucose

Abstract

Hyperpolarization of stable isotope-labeled substrates and subsequent NMR measurement of the metabolic reactions allow for direct tracking of cellular reactions in vitro and in vivo. Here, we report the hyperpolarization of ¹³C₆-glucose-d₇ and evaluate its use as probes to observe glucose flux in cells. We measured the lifetime of the polarized signal governed by the spin–lattice relaxation time T₁. ¹³C₆-Glucose-d₇ exhibited a T₁ that was over ten times as long as that of ¹³C₆-glucose, and metabolic NMR studies of hyperpolarized ¹³C₆-glucose-d₇ using tumor cell lysate led to observation of the resonances due to phosphorylated fluctofuranoses generated through aerobic glycolysis.

Dynamic nuclear polarization (DNP) has been proposed as a method to increase the polarization of nuclear spins.^1–6) Dissolution DNP has been shown to enhance liquid-state NMR signals by more than 10000-fold when detecting ¹³C-labeled substances.⁷⁾ This technique has been applied to the real-time metabolic imaging of several ¹³C-labeled substrates in tumor cells.^8–19) One limitation of DNP is the rapid decay of nucleus spin polarization, which is attributed to the spin–lattice relaxation time (T₁) of the nucleus. Since carbonyl carbons have relatively long T₁’s (40–60 s), most DNP probes have been observed through their carbonyl carbons. The metabolism of ¹³C-labeled pyruvate has been well-investigated and reported in a number of in vivo studies of cancer, diabetes, the cardiac system, and the brain.^9,10,13,19) On the other hand, T₁’s of other carbon species such as protonated carbons are much shorter (approximately 1 s), and so compounds without a carbonyl cannot be used as a hyperpolarized metabolic probe due to the short T₁’s of their carbons.

Glucose is one of the most important biomarkers in metabolic research because elevation of aerobic glycolysis (Fig. 1) occurs with the evolution and progression of cancer as well as a number of other human diseases.²⁰⁾ The glucose analogue, ¹⁸F-2-deoxy-2-fluoroglucose (¹⁸F-FDG), has proven useful as an functional positron emission tomography (PET) probe for various malignancies on the basis of glycolysis acceleration.^21,22) Until now, conventional ¹³C-NMR studies for the glucose metabolism have been examined using extracts of cells and tissues treated with ¹³C-labeled glucose,^23–28) and these studies revealed that glucose is metabolized through the pentose phosphate pathway and aerobic glycolysis, and then converted into several amino acids.²⁶⁾ The metabolic study of DNP has shown that ¹³C-labeled glucose is unsuitable due to the short T₁’s of their carbons.²⁹⁾ However, the 2-hemiketal carbons for 2-¹³C-fructose exhibited a moderate T₁ (ca. 16 s at 11.4T), and, therefore, in vivo spectroscopic imaging study have been performed.³⁰⁾ Recently, Harris and coworkers described the living cellular metabolism of hyperpolarized ¹³C₆-glucose-C-d₇ (1, ¹³C₆-Glc-d₇),³¹⁾ and detected the conversion from glucose to lactate and dihydroxyacetone phosphate.

During our investigation of useful DNP substrates,^32,33) we have measured ¹³C-NMR spectra of several deuterated glucoses hyperpolarized by DNP, and have found that the hyperpolarized C-perdeuterated glucose exhibited a larger enhancement and slower decay of signal than those with a C-protonated glucose.³⁴⁾ This manuscript details the ¹³C-NMR profiles of hyperpolarized ¹³C₆-Glc-d₇ (1) and compares with those of ¹³C₆-glucose (2, ¹³C₆-Glc). The metabolic reactions of hyperpolarized 1 by using the lysate of the human acute monocyte leukemia THP-1 cells led to observation of the resonances due to phosphorylated fluctofuranoses generated through aerobic glycolysis.

MATERIALS AND METHODS

DNP Experiments

1 (¹³C, 99%; ²H, 97–98%) and 2 (99%) were purchased from Sigma-Aldrich/Isotec (St. Louis, MO, U.S.A. and CIL, Boston, MA, U.S.A.). Each substrate was dissolved in 50/50 v/v glycerol/D₂O or 35/65 v/v glycerol/H₂O containing 15 mM OX063 [tritylradical(tris(8-carboxy-2,2,6,6-tetra(methoxyethyl)benzo[1,2-d:4,5-d′]bis(1,3)dithiole-4-yl)methyl sodium salt, GE Healthcare, Waukesha, WI, U.S.A.). The mixture was placed in 3.35 T superconducting magnet of a DNP polarizer (Oxford Instruments, Abingdon, U.K.), frozen at 1.4 K, and irradiated with microwave at 94 GHz for 3 h. After polarization, dissolution was carried out with 2.5 or 4 mL water with 0.15% ethylenediaminetetraacetate (EDTA), and then rapidly transferred to a NMR spectrometer (Varian 400 WBMRI) for measurement. Final concentrations of ¹³C₆-Glc-d₇ (1) and ¹³C₆-Glc (2) were 2.1 and 2.2 mM for enhancement factor analysis, respectively, and 1.3 mM for T₁ measurements. The ¹³C-NMR spectrum of the hyperpolarized substrate was recorded with 60 repetitions of 1-s acquisition employing 30° radio frequency flip angle.

The enhancement factors of ¹³C₆-Glc-d₇ (1) and ¹³C₆-Glc (2) were quantified by comparing the signal intensity of the polarized spectrum with that of thermal signal obtained with 256 and 128 scans, and a repetition time of 10 and 2 s, respectively, with 60° flip angle. These values were calculated for each carbon after adjusting for the differences in concentration and the number of scan between thermal equilibrium and DNP experiments. The polarization levels were estimated against the thermal-equilibrium polarization.

Longitudinal relaxation times T₁ were analyzed with the Bruker package Inversion Recovery (IR) pulse sequence, which fits the peak integrals, I, to the expression:

  

where P* is a constant. T₁ measurements at hyperpolarized state consisted of recording the decay curve of the hyperpolarized signal. Besides the IR method, T₁ values were calculated using the method by Day et al. in DNP.³⁵⁾

Cell Culture and Lysate Preparation

Human acute monocyte leukemia THP-1 cells were cultured in RPMI medium 1640 supplemented with 10% fetal bovine serum (FBS) (Biological Industries). Cells (1×10⁷) were collected in a 15 mL polystyrene tube by centrifugation at 800×g and 4°C for 10 min, suspended in 10 mL of phosphate buffered saline (PBS) buffer, and supersonicated for 1 min.³⁶⁾ After centrifugation at 800×g for 5 min and 4°C, the insoluble materials were removed, and protease inhibitors were added to the supernatant, which was then frozen and stored at −20°C.

Metabolic Reaction

An aliquot of the cell lysate in 10 mm NMR sample tube was set to NMR magnet at 37°C, and 10 mg of thermal equilibrium or hyperpolarized ¹³C₆-Glc-d₇ (1) was added. The final concentration of 1 was 18 mM. In the thermal equilibrium NMR metabolic studies, 1 (110 mM) was incubated for 24 h in PBS buffer of THP-1 cells (1×10⁷), and ¹³C-NMR spectrum was measured with 1024 scans of 1-s acquisition employing 30° radio frequency flip angle. In the DNP-NMR metabolic studies, after addition of hyperpolarized 1, the ¹³C-NMR spectrum was immediately recorded with 60 repetitions of 1 s acquisition employing 30° radio frequency flip angle.

Fig. 1. Overview of Partial Anaerobic Glycolysis

RESULTS AND DISCUSSION

The DNP experiments were carried out using a HyperSence DNP polarizer employing OX63 as a radical source under microwave irradiation at 94 GHz in the glycerol/H₂O glassing solvent system at 1.4 K for 1 h. After dissolution with 0.15% aqueous EDTA solution, the ¹³C-NMR spectra of hyperpolarized 1 and 2 (2.1, 2.2 mM, respectively) were immediately recorded with a single acquisition using a 30° flip angle at 298 K on a 9.4 T NMR spectrometer (100 MHz for ¹³C) (Figs. 2a, b, respectively). The conventional ¹³C-NMR spectra of 1 and 2 (8.7, 8.8 mM, respectively) at the thermal equilibrium Boltzmann polarization were measured with a 60° radio frequency flip angle (Figs. 2c, d, respectively). All of the carbon signals of 1 were split multiplied by ¹³C−¹³C and ¹³C−²H couplings, and although those of C-2–C-5 heavily overlapped each other, those at C-1 (α: δ_C 92 β: δ_C 96) and C-6 (δ_C 60) could be distinguished from the C-2–C-5 signals. The magnetizations of C-1 and C-6 for 1 in the thermal equilibrium (M₀) were 2.91 and 3.65, respectively, while initial magnetizations [M(DNP)] for hyperpolarized 1 were found to be 11810 and 8749, respectively. From the comparison of magnetizations in both states, the enhancement factors of C-1 and C-6 for hyperpolarized 1 were found to be 4058 and 2397, respectively, as shown in Table 1. As the thermal-equilibrium polarization P(¹³C) was estimated to be 8.17×10⁻⁴% at 9.4 T and 298 K, the DNP-enhanced polarizations P(DNP) for C-1 and C-6 were found to be 3.31 and 1.96%, respectively. In contrast, the C-1 signal was mostly enhanced by DNP, however, the enhancement factor and polarization level P(DNP) was 598 and 0.49%, respectively. The enhancement factor and P(DNP) for the least enhanced C-6 signal was calculated as 179 and 0.14%, respectively. Thus, the hyperpolarized signals for ¹³C₆-Glc-d₇ (1) show nearly a 10-fold enhancement than ¹³C₆-Glc (2).

Fig. 2. ¹³C-NMR Spectra of Hyperpolarized (a) ¹³C₆-Glucose-d₇ (1, 2.1 mM) and (b)

¹³C₆-Glucose (2, 2.2 mM), respectively, measured with a single acquisition and 30° radio frequency flip angle. ¹³C-NMR spectrum of thermal-equilibrium (c) ¹³C₆-glucose-d₇ (1, 8.7 mM, 60° radio frequency flip angle, 10 s repetition time, and 128 scans) and (d) ¹³C₆-glucose (2, 8.8 mM, 60° radio frequency flip angle, 2 s repetition time, and 256 scans), respectively. The signals marked by asterisks are due to the hyperpolarized carbons of glycerol as a glassing solvent.

Table 1. Enhancement Factors and Polarizations for ¹³C₆-Glucose-d₇ (1) and ¹³C₆-Glucose (2) by DNP

		M0a)	M(DNP)b)	Enhancement factor	P(DNP) (%)
13C6-Glc-d7 (1)	C-1	2.91	11810	4058	3.31
	C-6	3.65	8749	2397	1.96
13C6-Glc (2)	C-1	12.3	7371	598	0.49
	C-6	12.3	2208	179	0.14

a) Tharmal-equilibrium magnetizations (M₀) were calculated from conventional ¹³C-NMR spectra measured with a 60° radio frequency flip angle. The conventional ¹³C-NMR spectra were obtained with 256 scans and 75 s repetition time for ¹³C₆-Glc-d₇ (1, 8.7 mM) and 128 scans and 2 s repetition time for ¹³C₆-Glc (2, 8.8 mM). b) Initial magnetizations for DNP [M(DNP)] were calculated from signal intensities of initial ¹³C-NMR spectra of hyperpolarized ¹³C₆-Glc-d₇ (1, 2.1 mM) and ¹³C₆-Glc (2, 2.2 mM) measured with a 30° radio frequency flip angle.

The DNP-enhanced sequential ¹³C-NMR spectra of 1 and 2 shown in Fig. 3 were measured every 1 s for 60 s. The signal intensity of all carbons for 1 at 10 s retained one-third of that at the first acquisition as shown in Fig. 3a, while all carbon signals for 2 completely disappeared at 5 s (Fig. 3b). The carbon signals for 1 were observed even at 30 s. The current studies have revealed that there was a significant advantage in using deuterated glucose for DNP because of slow decays and large enhancements of their resonances.

Fig. 3. Time-Dependent ¹³C-NMR Spectra of Hyperpolarized (a) ¹³C₆-Glucose-d₇ (1, 2.1 mM) and (b) ¹³C₆-Glucose (2, 2.2 mM) Measured by a 7.4T NMR Spectroscopy Equipped with 5 mm CH Probe

Individual spectra were processed with 5.0 Hz Gaussian line broadening. The signals marked by asterisks are due to the hyperpolarized carbons of glycerol as a glassing solvent.

The T₁’s for C-1 and C-6 for 1 and 2 were calculated according to comparison of decays of hyperpolarized signals with the simulation curves reported by Day et al.,³⁵⁾ as shown in Fig. 4. The signal decays of C-1 and C-6 for 2 corresponded to the T₁ simulation curve for 1.1 s, which is in agreement with previously reported data, while those of C-1 and C-6 for 1 were estimated to be 11 s. These values for 1 and 2 were equivalent to those measured using the inversion recovery method in Table 2.

Fig. 4. Time-Dependent Decays of C-1 and C-6 Signals for Hyperpolarized ¹³C₆-Glucose-d₇ (1) and ¹³C₆-Glucose (2)

The observed signal intensities of C-1 and C-6 for hyperpolarized 1 are represented by opened and filled triangles, respectively. Open and filled circles show the signal intensities of C-1 and C-6 for hyperpolarized 2, respectively. Solid and dotted lines represent the fitting curve calculated for T₁ at 11 s and 1.1 s, respectively.

Table 2. Spin–Lattice Relaxation Times (T₁) and the T₁(1)/T₁(2) Ratios for Carbon Signals in ¹³C₆-Glucose-d₇ (1) and 13C6-Glucose (2)

		C-1	C-2	C-3	C-4	C-5	C-6
T1a) (s)	13C6-Glc-d7 (1)	12.5	11.3	10.8	10.5	10.6	7.23
	13C6-Glc (2)	1.26	1.16	1.13	1.06	1.12	0.68
T1(1)/T1(2) ratio		9.9	9.7	9.6	9.9	9.5	10.6

a) Obtained from inversion recovery method using a 11.4T NMR spectroscopy.

Assuming the dipole–dipole (DD) interaction is the most important mechanism for inducing T₁ relaxation, the theoretical ¹³C T₁ values for protonated and perdeuterated glucoses [(¹H) and (²H)] may be calculated from the equations^37,38):

  

(1)

  

(2)

Where µ₀/4π is the magnetic permeability of vacuum, γ_H, γ_D, and γ_C are the gyromagnetic ratios for ¹H, ²H, and ¹³C, and h is Planck’s constant, Σ r_CH⁻⁶ and Σ r_CD⁻⁶ are the sums of all of the inverse sixth distances from the observed carbon to ¹H and ²H nuclei, respectively. In the molecule, τ_C is the correlation time; i.e., average time for the C–H vector to rotate 1 radian. Bond lengths for C–H and C–D were slightly deferent by almost 4 pm.^39,40) In Eq. 2, measurements of T₁’s in D₂O allowed us to ignore contribution from the hydroxyl protons. The calculated ratio for T₁(²H)/T₁(¹H) was found to be approximately 11,³⁸⁾ which corresponds to the observed ratios for T₁(1)/T₁(2) calculated using Day’s method (ca. 10) and inversion recovery (9.5–10.6).

We performed the cellular metabolism experiments of hyperpolarized glucose using human acute monocyte leukemia THP-1 cells, to compare thermal equilibrium glucose. In the ¹³C-NMR spectrum (Fig. 5) of the cellular reaction of thermal equilibrium 1 using a PBS buffer of THP-1 cells, some carbon signals of glucose metabolites were newly observed in addition to those of 1. The signal at δ_C 118 corresponded to that of carbon dioxide generated from decarboxylation of pyruvate. Splitting ¹³C resonances at δ_C 183 (C-1, d), 68 (C-2, dd), and 19 ppm (C-3, m) are probably due to ¹³C₃-lactate-3,3,3-d₃^27,31) (3) generated through glycolysis of 1 following the reduction of ¹³C₃-pyruvate-3,3,3-d₃.

Fig. 5. Structure of ¹³C₃-Lactate-3,3,3-d₃ (3) and ¹³C-NMR Spectrum of Thermal Equilibrium ¹³C₆-Glucose-d₇ (1) Treated with THP Cell Lysate for 24 h

Splat-marked signals [δ_C 183 (d, C-1), δ_C 68 (dd, C-2), δ_C 19 (m, C-3)] are derived from 3 generated from 1 through glycolysis and reduction with lactate dehydrogenase.

The metabolic reaction of hyperpolarized ¹³C₆-Glc-d₇ (1) was carried out using THP-1 cell lysate. ¹³C₆-Glc-d₇ (1) in glycerol/H₂O was hyperpolarized with OX63 for 3 h at 1.4 K, dissolved with PBS buffer, added immediately into THP-1 cell lysate, at which point 60 repetitions of single-scan ¹³C-NMR spectrum was performed and quantified with a 1-s acquisition using a 30° radio frequency flip angle. The initial spectrum of 60 spectra for hyperpolarized 1 (finally 18 mM) treated by the cell lysate, as shown in Fig. 6, revealed new minor resonances at δ_C 104 and 82, although their intensities were only 0.02% compared to the C-1 intensity (100%) of glucose. Considering the chemical shifts as well as the glucose metabolism, these resonances might appreciate to C-2 (δ_C 104) and C-5 (δ_C 82) of deuterio ¹³C₆-β-fructofuranose derivative(s)³⁰⁾ as seen with the 6-phosphate (4) or 1,6-bisphosphate form (5). The former of which is generated by phosphorylation of glucose followed by isomerization. Though signals due to fructofuranose disappeared only at 6 s and C₃ intermediates of glycolysis were not detected in this DNP study, ¹³C signals of ¹³C₃-lactate-3,3,3-d₃ (3) were observed in the thermal spectrum of the same solution at 5 min post DNP. On the other hand, the resonances of the phosphorylated fructofuranoses were not observed directly in the metabolic studies of hyperpolarized 1 using living mammalian tumor cells reported by Harris and coworkers,³¹⁾ although the resonances of dihydroacetone phosphate, 3-phosphoglycerate, and lactate were detected. This metabolic difference may be caused by the relatively low or slow glucose-uptake into cells and the smooth conversion of glucose to C₃ intermediates. Our result is a significant observation as it highlights the preparatory phase of mammalian cellular glycolysis. For the microbial metabolic studies of hyperpolarized 1 using Escherichia coli and yeast reported by Meier and coworkers,^41–44) signals of several metabolites containing phosphorylated β-fructofuranoses generated through glycolysis, pentose phosphate pathway, and alcohol fermentation are detected. To analyze these metabolic differences in living microorganisms observed directly by feeding the hyperpolarized glucose can lead to understand the phenotypic and genotypic differences.⁴³⁾ Therefore, accumulation and analysis of the real-time metabolic profiles for various living tumor cells and their lysates by using hyperpolarized glucose is essential for not only understanding of the phenotype and the gene expression in the cells, but also development of non-invasive metabolic imaging using DNP glucose.

Fig. 6. Structures of Deterio ¹³C₆-β-Fructofranose-6-phospahate (4) and/or ¹³C₆-β-Fructose-1,6-bisphospahate (5) and ¹³C-NMR Spectrum of Hyperpolarized ¹³C₆-Glucose-d₇ (1, 18 mM) Treated with the Lysate of THP Cells for 2 s

Two splat-marked signals (δ_C 104 and 82) may be derived from deuterated ¹³C₆-β-fructofranose derivative such as 4 and/or 5 generated from 1 through hexokinase, glucose-6-phosphate isomerase, and/or phosphofructokinase. The singlet signal at δ_C 65 is attributed to C-1 and C-3 of glycerol as a glassing solvent.

The metabolic imaging of glucose in tumor cells is appealing due to its application as a therapeutic treatment and cancer diagnostic tool. However, the application of DNP using glucose such as glycolytic imaging has not been possible because ¹³C nuclei of glucose hyperpolarized by DNP rapidly decays yielding short signals due to a short T₁ relaxation time. Our study demonstrates the effects of C-perdeuterated glucose for the enhancement of hyperpolarized signals and the prolongation of T₁ relaxation time. Although the T₁ (11 s) for 1 is shorter than those (40–60 s) for carbonyl carbons of pyruvate,¹⁹⁾ the polarization level (3.3%) of 1 is equal to one third of that of ¹³C-urea,⁸⁾ and sufficient for detection of resonances by using ¹³C-MRI. So, we expect that these hyperpolarized glucose metabolites could also be detected in vivo.

Acknowledgment

We thank Prof. S. Sando and Dr. H. Nonaka of Kyushu University for methodological advise of cell lysate experiments, Prof. A Tominaga and Dr. Y. Konishi of Kochi University for kindly gift of THP cells and technical support of cell cultivation, Dr. J. Kurita and T. Hino of Agilent Technologies Japan Ltd. for assistance of array NMR measurements, and S. Goto of Oxford Instruments KK for assistance for DNP operation. This study was partially supported by a Grant-in-Aid for Young Scientists (B) (Grant No. 25860079) from Japan Society for the Promotion of Science. K.I. was in part supported by the funding program ‘Creation of Innovation Centers for Advanced Interdisciplinary Research Areas’ from JST, commissioned by MEXT.

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