Background

[PubMed]

Magnetic resonance spectroscopy (MRS) has been noninvasively used to detect small molecules in tissues in vivo (1-4). Proton (¹H) and carbon-13 (¹³C) are the most frequently investigated MRS applications (5-7). One technique has been often used to enhance ¹³C nuclear spins with dynamic nuclear polarization (DNP), which dramatically increases sensitivities in MRS spectra of ¹³C-labeled substrates in solutions and tissues (2, 3, 8). DNP transfers high electron-spin polarization to nuclear spins via microwave irradiation. The ¹³C MRS signals from tissues are near background levels, whereas regions with hyperpolarized ¹³C-labeled substrates provide strong signals. Thus, hyperpolarized ¹³C-labeled substrates can provide >10,000-fold enhancement of the ¹³C MRS signals from the substrate and its subsequent metabolites. ¹³C-Labeled substrates must exhibit long spin-lattice relaxation time (T₁) values, be rapidly metabolized, and produce an observable change in the chemical shift (δ) value between the substrate and its metabolite(s). ¹³C-Labeled substrates, such as [1-¹³C]pyruvate, [2-1³C]fructose, [¹³C]bicarbonate, α-keto[1-¹³C]isocaproate, and [1,4-¹³C₂]fumarate have been studied using the DNP technique in tumors in preclinical studies to measure changes in metabolism in vivo through glycolysis, citric acid cycle, amino acid metabolism and fatty acid synthesis (1-3).

Vitamin C (ascorbic acid, AA) is reversibly oxidized to DHA, which is a substrate of glucose transporters GLUT1 and GLUT3 (9). Epithelial cells of the intestine, liver, and kidney transport AA directly through sodium-dependent transporters, whereas most normal tissues acquire AA through transport of DHA (10). Intracellular DHA is then converted to AA through reaction with glutathione (GSH) or GSH-dependent enzymes and through NADPH-dependent thioredoxin reductases (11). On the other hand, tumor cells have been found to lack the capacity to transport AA and to exhibit upregulation of the GSH and thioredoxin antioxidant systems, reflecting high oxidative stress in tumor cells (10). High oxidative stress has also been implicated in neurodegenerative and cardiovascular conditions (12). Bohndiek et al. (13) generated a hyperpolarized small molecule, [1-¹³C]dehydroascorbic acid ([1-¹³C]DHA), as an imaging agent of ¹³C MRS for evaluation of intracellular reduction/oxidation (redox) status in tumors and other pathological conditions. [1-¹³C]DHA has been evaluated as an intracellular redox probe using tumor cells in vitro and in vivo. Keshari et al. (14) have performed MRS imaging of redox status in normal organs and prostate tumor in mice using hyperpolarized [1-¹³C]DHA.

Synthesis

[PubMed]

[1-¹³C]DHA was prepared by charcoal oxidation of commercially available [1-¹³C]AA. [1-¹³C]AA 1 (2.9 mmol) was dissolved in 15 mL methanol, and then highly purified activated charcoal Norit (0.75 g) was added as a catalyst (13). The mixture was stirred vigorously for 90 minutes at room temperature, with oxygen bubbled through at 0.2 L/minute. Charcoal was removed from the product with filtration. The final product, [1-¹³C]DHA, was then lyophilized to give a yield of 70% with >99% chemical purity. [1-¹³C]DHA (8.2 ± 1.1%, n = 8) exhibited a significantly higher degree of polarization (P < 0.05) than [1-¹³C]AA (5.1 ± 0.6%, n = 6) at pH 7 at 8 s after dissolution in phosphate-buffered saline (PBS) measured at 9.4 T. The δ values for [1-¹³C]AA and [1-¹³C]DHA were 179 ppm and 175 ppm, respectively. The T₁ values for [1-¹³C]AA and [1-¹³C]DHA were 15.9 ± 0.7 s and 20.5 ± 0.9 s, respectively.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Bohndiek et al. (13) analyzed the conversion of [1-¹³C]DHA to [1-¹³C]AA in PBS containing 1, 5, 10, 25, and 50 mM GSH. ¹³C MRS spectra were acquired at 9.4 T. The [1-¹³C]AA signal (179 ppm) reached maximal intensity within 7–16 s. Reduction of [1-¹³C]DHA to [1-¹³C]AA was observed in RPMI medium, which contains 3.3 nM GSH and other factors. The rate of conversion observed in RPMI medium and EL4 mouse lymphoma cell suspension (1 × 10⁸ cells in 2 mL) in RPMI medium were 99 ± 6 nmol/s (n = 2) and 223 ± 18 nmol/s (n = 3), respectively. On the other hand, no oxidation of [1-¹³C]AA to [1-¹³C]DHA was observed in either RPMI medium alone or in EL4 cell suspension in RPMI medium within 60 s.

Animal Studies

Human Studies

[PubMed]

No references are currently available.

References

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