Background

[PubMed]

Pyruvate (Pyr) is an endogenous substrate enriched at a crossroad of the major energy-generating metabolic pathways in mammalian cells (1). Under most physiological conditions, the bulk of intracellular Pyr is generated endogenously by glycolysis of glucose or oxidation of lactate (Lac) (2). Pyr is then converted into several products through different metabolic pathways in mitochondria or cytoplasm (2, 3). In mitochondria, one of the major pathways is the irreversible transformation of Pyr into acetyl-coenzyme A (acetyl-CoA) and CO₂ by pyruvate dehydrogenase (PDH) in the presence of coenzyme nicotinamide adenine dinucleotide (NAD⁺/NADH). Another major pathway is the conversion of Pyr into oxaloacetate by PDH. Both acetyl-CoA and oxaloacetate are substrates of the tricarboxylic acid cycle (TCA), also known as the Krebs cycle, which generates adenosine triphosphate (ATP) and provides energy to the cell. In the cytoplasm, Pyr is converted into alanine (Ala) by glutamine-Pyr transaminase (GPT) and reversibly oxidized back to Lac by lactate dehydrogenase (LDH). With its ability to increase cardiac mechanical performance and energy reserves, Pyr has been an important therapeutic agent in the treatment of diseases such as dysfunctional myocardium (2). Pyr is also a diagnostic marker in cancers because it is abundantly converted to Lac through anaerobic glycolysis (1). The abnormality of Pyr metabolism in diseased tissue can be detected through quantification of its metabolites. ¹³C Magnetic resonance spectroscopic imaging (MRSI) is suitable for in vivo quantification because of the inherent chemical shift dispersion of the major metabolites of Pyr (4). The chemical shift value is 178 ppm for Ala, 185 ppm for Lac, 124.5 ppm for CO₂, and 173 ppm for Pyr itself (4). Since ¹³C has low natural abundance (1.1%) and a low gyromagnetic ratio (one quarter compared to proton spin), direct measurement of these metabolites is extremely difficult even with high magnetic fields. The recently developed hyperpolarization technique can increase the signal/noise ratio (SNR) by 10,000-fold (5), allowing direct detection of these metabolites via ¹³C MRSI.

The signal of nuclear magnetic resonance (NMR) imaging is proportional to the thermal equilibrium polarization of nuclear spins. As a function of magnetic field strength and temperature, the thermal equilibrium polarization normally is very low (i.e., 1×10^-6 for ¹³C at 1.5 T and at body temperature). The hyperpolarization technique increases the polarization of spins by creating an artificial, non-equilibrium distribution of nuclei. Dynamic nuclear polarization (DNP) is an effective technique to hyperpolarize the ¹³C nuclei of Pyr. Electron spins have a large magnetic moment that is ~660 times stronger than the magnetic moment of proton spins. This strong magnetic moment can be transferred to proton spins through the DNP technique. Theoretically, the DNP enhancement factor for NMR signals depends on the ratio of the electronic gyromagnetic ratio to the nuclear gyromagnetic ratio (γ_e/γ_n) in high magnetic fields, which corresponds to a DNP enhancement factor of ~660 for ¹H nuclei and ~2600 for ¹³C nuclei (6). There are four mechanisms accounted for in the DNP process: the Overhauser effect, the solid effect, thermal mixing, and the cross effect or electronuclear cross-polarization (7). The DNP experiments are generally conducted at low temperatures to attenuate competing spin-lattice relaxation processes and avoid loss of efficiency during the polarization transfer (9). Radicals containing unpaired electrons, which have a thermal equilibrium polarization of almost unity when exposed to a magnetic field of ~3 T at a temperature of ~1 K, are added (8). Microwave irradiation near the electron resonance frequency of the radical is used to transfer the polarization from the unpaired electrons on the radicals to the ¹³C nuclei on substrates. As a result of the short T₁ of the electrons (~1 μs), the electrons rapidly regain their polarization. This continuous pumping process can increase the nuclear polarization in the solid material by 20–40% (8). By rapid dissolution, the solid is transformed into an injectable liquid with small polarization losses. The use of DNP produces an enhancement factor of 10⁵ in ¹³C polarization compared to the thermal equilibrium state at typical magnetic field strengths (8), allowing for direct imaging of metabolites at mM in vivo.

A major difference between the signal of hyperpolarized ¹³C and the signal of conventional NMR is that the magnetization of hyperpolarized ¹³C is created outside the magnetic field in a polarizer system (9). Once the hyperpolarization has been created, the polarization will strive to return to the thermal equilibrium level at a rate governed by T₁ relaxation time, which typically ranges from a few seconds to several minutes for ¹³C. The corresponding time window for imaging is approximately a few minutes. Because the 1.1% natural abundance of ¹³C produces a negligible ¹³C signal, there is virtually no background signal other than noise from the patient and the coil/receiver system. The injected hyperpolarized ¹³C-labeled Pyr (HP[¹³C]Pyr) generates a ¹³C signal that is linearly proportional to its concentration. In this respect, hyperpolarized ¹³C MRI behaves in a manner similar to modalities such as positron emission tomography (PET) and single-photon emission tomography (SPECT). Neither PET nor SPECT can distinguish between metabolites and substrates. However, the large chemical shift dispersion in ¹³C MRSI can be used for quantification of each metabolite (10). The measurement of spatial distribution of metabolites via ¹³C MRSI allows the establishment of enzyme activity maps (11).

Synthesis

[PubMed]

The preparation of HP[¹³C]Pyr was described by several authors (10, 12). Two starting materials, [1-¹³C]Pyr and Tris{8-carboxyl-2,2,6,6-tetra[2-(1-hydroxyethyl)]-benzo(1,2-d:4,5-d’)bis(1,3)dithiole-4-yl}methyl sodium (trityl radical), were commercially available. Both chemicals were dissolved in an aqueous solvent and loaded into a chamber where the sample was cooled to 1.2 K at a pressure of 0.8 mbar. The sample was then irradiated with microwaves (~93.9 GHz) in a helium bath at a magnetic field of 3.35 T for >1 h. After the polarization, the sample was dissolved with hot solvents to produce an injectable solution at approximately 37°C. The level of polarization was estimated by measuring the free induction decays of an aliquot in a NMR spectrometer. The typical polarization was 20–30%.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The evaluation of HP[¹³C]Pyr influx in EL-4 mouse lymphoma cells was conducted with a 9.4-T imager (11). After adding HP[¹³C]Pyr into the cells, the conversion of Pyr to Lac produced an increased Lac carboxyl signal that decayed at an apparent T₁ of 40 s. The ¹³C peak intensities for Pyr and Lac were fitted to a modified two-site exchange Bloch equation to extract the apparent exchange rate constants. These rate constants were used to evaluate the LDH activity and Pyr influx during drug treatment. Etoposide was an inhibitor of the enzyme topoisomerase II used in chemotherapy for various malignant carcinomas. A 16-h treatment of the tumor cells with etoposide caused ~30–40% apoptosis and 10–15% necrosis in the cells. This induced ~80% reduction in Pyr flux, which was decreased from 54 ± 15 nmol/s per 10⁸ cells to 9.0 ± 1.5 nmol/s per 10⁸ cells. The LDH activity was nearly the same: 5.43 ± 0.34 units/10⁷ cells in control cells and 5.37 ± 0.27 units/10⁷ cells in drug-treated cells. A 20-h treatment with etoposide produced a 45% necrosis in the cells. The LDH activity also decreased significantly to 2.53 ± 0.32 units/10⁷ cells. The levels of necrosis and apoptosis were further confirmed by flow cytometry.

The metabolism of HP[¹³C]Pyr in isolated rat hearts was examined at 14.1 T (10). Hearts from adult rats were rapidly excised and perfused at 37°C using standard Langendorff methods. After each aorta was cannulated, each heart was placed in a NMR tube that was then filled with recirculated medium containing fatty acids and 2 mM HP[¹³C]Pyr with 20–30% polarization. Fatty acids with even or odd carbons were used to modulate the influx of PDH. Propionate (a three-carbon fatty acid) activated 90% PDH and stimulated PDH flux, whereas even-carbon fatty acid strongly inhibited PDH. Experiments were performed under ideal conditions for detected downstream metabolites such as H¹³CO₃^- (160.9 ppm) and ¹³CO₂ (124.5 ppm) in the presence of HP[¹³C]Pyr alone as well as in the presence of HP[¹³C]Pyr with even or odd fatty acids. The intensity of the H¹³CO₃^- and ¹³CO₂ resonances in the hearts reached a maximum of ~20 s after infusion of HP[¹³C]Pyr. The ratio of the H¹³CO₃^- resonance area to the ¹³CO₂ area was 7.03 ± 0.5 and remained constant until T₁ relaxation began to destroy the ¹³CO₂ signal. In the presence of octanoate, a medium chain fatty acid, H¹³CO₃^- and ¹³CO₂ were nearly invisible and did not recover even in the presence of propionate. This result suggested an effective competition of octanoate with Pyr for supplying acetyl-CoA to the heart.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

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