Carbonic Anhydrase Activity Monitored In Vivo by Hyperpolarized 13C-Magnetic Resonance Spectroscopy Demonstrates Its Importance for pH Regulation in Tumors.

Carbonic anhydrase buffers tissue pH by catalyzing the rapid interconversion of carbon dioxide (CO2) and bicarbonate (HCO3 (-)). We assessed the functional activity of CAIX in two colorectal tumor models, expressing different levels of the enzyme, by measuring the rate of exchange of hyperpolarized (13)C label between bicarbonate (H(13)CO3(-)) and carbon dioxide ((13)CO2), following injection of hyperpolarized H(13)CO3(-), using (13)C-magnetic resonance spectroscopy ((13)C-MRS) magnetization transfer measurements. (31)P-MRS measurements of the chemical shift of the pH probe, 3-aminopropylphosphonate, and (13)C-MRS measurements of the H(13)CO3(-)/(13)CO2 peak intensity ratio showed that CAIX overexpression lowered extracellular pH in these tumors. However, the (13)C measurements overestimated pH due to incomplete equilibration of the hyperpolarized (13)C label between the H(13)CO3(-) and (13)CO2 pools. Paradoxically, tumors overexpressing CAIX showed lower enzyme activity using magnetization transfer measurements, which can be explained by the more acidic extracellular pH in these tumors and the decreased activity of the enzyme at low pH. This explanation was confirmed by administration of bicarbonate in the drinking water, which elevated tumor extracellular pH and restored enzyme activity to control levels. These results suggest that CAIX expression is increased in hypoxia to compensate for the decrease in its activity produced by a low extracellular pH and supports the hypothesis that a major function of CAIX is to lower the extracellular pH.

Detection of tumor glutamate metabolism in vivo using (13)C magnetic resonance spectroscopy and hyperpolarized [1-(13)C]glutamate.

Dynamic nuclear polarization can be used to increase the sensitivity of solution state (13)C magnetic resonance spectroscopy by four orders of magnitude. We show here that [1-(13)C]glutamate can be polarized to 28%, representing a 35,000-fold increase in its sensitivity to detection at 9.4 T and 37°C. The metabolism of hyperpolarized glutamate to α-ketoglutarate, catalyzed by the enzyme alanine transaminase, was detected in vitro in human hepatoma cells (HepG2). Incubation of the cells with sodium pyruvate increased the level of the hyperpolarized label in the α-ketoglutarate pool, with an associated increase in the apparent rate constant describing flux of hyperpolarized (13)C label between glutamate and α-ketoglutarate. The metabolism of hyperpolarized glutamate was observed in vivo following coadministration of pyruvate in a murine lymphoma model. This represents a new method to probe glutamate metabolism and citric acid cycle activity in vivo; as glutamate is an endogenous molecule, it has the potential to be used in the clinic.

Detecting response of rat C6 glioma tumors to radiotherapy using hyperpolarized [1- 13C]pyruvate and 13C magnetic resonance spectroscopic imaging.

We show here that hyperpolarized [1-(13) C]pyruvate can be used to detect treatment response in a glioma tumor model; a tumor type where detection of response with (18) fluoro-2-deoxyglucose, using positron emission tomography, is limited by the high background signals from normal brain tissue. (13) C chemical shift images acquired following intravenous injection of hyperpolarized [1-(13) C]pyruvate into rats with implanted C6 gliomas showed significant labeling of lactate within the tumors but comparatively low levels in surrounding brain.Labeled pyruvate was observed at high levels in blood vessels above the brain and from other major vessels elsewhere but was detected at only low levels in tumor and brain.The ratio of hyperpolarized (13) C label in tumor lactate compared to the maximum pyruvate signal in the blood vessels was decreased from 0.38 ± 0.16 to 0.23 ± 0.13, (a reduction of 34%) by 72 h following whole brain irradiation with 15 Gy.

Magnetization transfer measurements of exchange between hyperpolarized [1-13C]pyruvate and [1-13C]lactate in a murine lymphoma.

Measurements of the conversion of hyperpolarized [1-(13)C]pyruvate into lactate, in the reaction catalyzed by lactate dehydrogenase, have shown promise as a metabolic marker for the presence of disease and response to treatment. However, it is unclear whether this represents net flux of label from pyruvate to lactate or exchange of isotope between metabolites that are close to chemical equilibrium. Using saturation and inversion transfer experiments, we show that there is significant exchange of label between lactate and pyruvate in a murine lymphoma in vivo. The rate constants estimated from the magnetization transfer experiments, at specific points during the time course of label exchange, were similar to those obtained by fitting the changes in peak intensities during the entire exchange time course to a kinetic model for two-site exchange. These magnetization transfer experiments may therefore provide an alternative and more rapid way of estimating flux between pyruvate and lactate to serial measurements of pyruvate and lactate (13)C peak intensities following injection of hyperpolarized [1-(13)C]pyruvate.

13C MR spectroscopy measurements of glutaminase activity in human hepatocellular carcinoma cells using hyperpolarized 13C-labeled glutamine.

Dynamic nuclear polarization (DNP) is an emerging technique for increasing the sensitivity of (13)C MR spectroscopy (MRS). [5-(13)C(1)]Glutamine was hyperpolarized using this technique by up to 5%, representing a 6000-fold increase in sensitivity. The conversion of hyperpolarized glutamine to glutamate by mitochondrial glutaminase was demonstrated using (13)C-MRS measurements in cultured human hepatoma cells (HepG2). These results represent the first step in developing an imaging technique for detecting glutamine metabolism in vivo. Furthermore, since glutamine utilization has been correlated with cell proliferation, the study suggests a new technique for detecting changes in tumor cell proliferation.

Magnetic resonance imaging of pH in vivo using hyperpolarized 13C-labelled bicarbonate.

As alterations in tissue pH underlie many pathological processes, the capability to image tissue pH in the clinic could offer new ways of detecting disease and response to treatment. Dynamic nuclear polarization is an emerging technique for substantially increasing the sensitivity of magnetic resonance imaging experiments. Here we show that tissue pH can be imaged in vivo from the ratio of the signal intensities of hyperpolarized bicarbonate (H(13)CO(3)(-)) and (13)CO(2) following intravenous injection of hyperpolarized H(13)CO(3)(-). The technique was demonstrated in a mouse tumour model, which showed that the average tumour interstitial pH was significantly lower than the surrounding tissue. Given that bicarbonate is an endogenous molecule that can be infused in relatively high concentrations into patients, we propose that this technique could be used clinically to image pathological processes that are associated with alterations in tissue pH, such as cancer, ischaemia and inflammation.

Detecting tumor response to treatment using hyperpolarized 13C magnetic resonance imaging and spectroscopy.

Measurements of early tumor responses to therapy have been shown, in some cases, to predict treatment outcome. We show in lymphoma-bearing mice injected intravenously with hyperpolarized [1-(13)C]pyruvate that the lactate dehydrogenase-catalyzed flux of (13)C label between the carboxyl groups of pyruvate and lactate in the tumor can be measured using (13)C magnetic resonance spectroscopy and spectroscopic imaging, and that this flux is inhibited within 24 h of chemotherapy. The reduction in the measured flux after drug treatment and the induction of tumor cell death can be explained by loss of the coenzyme NAD(H) and decreases in concentrations of lactate and enzyme in the tumors. The technique could provide a new way to assess tumor responses to treatment in the clinic.