A method for obtaining strongly polarized nuclear spins in solution has been developed. The method uses low temperature, high magnetic field, and dynamic nuclear polarization (DNP) to strongly polarize nuclear spins in the solid state. The solid sample is subsequently dissolved rapidly in a suitable solvent to create a solution of molecules with hyperpolarized nuclear spins. The polarization is performed in a DNP polarizer, consisting of a superconducting magnet (3.35 T) and a liquid-helium cooled sample space. The sample is irradiated with microwaves at Ϸ94 GHz. Subsequent to polarization, the sample is dissolved by an injection system inside the DNP magnet. The dissolution process effectively preserves the nuclear polarization. The resulting hyperpolarized liquid sample can be transferred to a high-resolution NMR spectrometer, where an enhanced NMR signal can be acquired, or it may be used as an agent for in vivo imaging or spectroscopy. In this article we describe the use of the method on aqueous solutions of [ 13 C]urea. Polarizations of 37% for 13 C and 7.8% for 15 N, respectively, were obtained after the dissolution. These polarizations correspond to an enhancement of 44,400 for 13 C and 23,500 for 15 N, respectively, compared with thermal equilibrium at 9.4 T and room temperature. The method can be used generally for signal enhancement and reduction of measurement time in liquid-state NMR and opens up for a variety of in vitro and in vivo applications of DNP-enhanced NMR. T wo major applications exist for NMR: spectroscopy and imaging. NMR spectroscopy has gained acceptance as one of the major analytical techniques due to the detailed information that can be obtained about molecular structure, dynamics, and intra-and intermolecular interactions. MRI is a noninvasive technique with superior soft-tissue contrast and broad diagnostic value. The technique has gained wide clinical acceptance and is of great importance in diagnostic medicine.However, despite significant technological advancements (increasing field strength and cooling of electronics), the application of NMR is limited by an intrinsically low sensitivity as compared with other analytical methods. Fundamentally, the low sensitivity originates from the low magnetic energy of nuclear spins compared with the thermal energy at room temperature. At a magnetic field strength of 1.5 T and room temperature, the 1 H spins are polarized to only 5 ppm, and an improvement of 200,000 is thus theoretically possible. The two most sensitive nuclei are 1 H and 19 F, which have large magnetic moments and 100% abundance. But, even at the largest field strength available today (21 T), these nuclei are polarized to only 70 and 67 ppm, respectively. For other nuclei bearing lower magnetic moments (1͞4 for 13 C and 1͞10 for 15 N compared with 1 H), the theoretical enhancement factor is proportionally greater. The weak nuclear polarization is generally compensated by a high concentration (i.e., a large number of nuclear spins). However, the sensitivity of several other ...

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.

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.

The ''Warburg effect,'' an elevation in aerobic glycolysis, may be a fundamental property of cancer cells. For cancer diagnosis and treatment, it would be valuable if elevated glycolytic metabolism could be quantified in an image in animals and humans. The pyruvate molecule is at the metabolic crossroad for energy delivery inside the cell, and with a noninvasive measurement of the relative transformation of pyruvate into lactate and alanine within a biologically relevant time frame (seconds), it may be possible to quantify the glycolytic status of the cells. We have examined the metabolism after i.v. injection of hyperpolarized 13 C-pyruvate in rats with implanted P22 tumors. The strongly enhanced nuclear magnetic resonance signal generated by the hyperpolarization techniques allows mapping of pyruvate, lactate, and alanine in a 5 Â 5 Â 10 mm 3 imaging voxel using a 1.5 T magnetic resonance scanner. The magnetic resonance scanning (chemical shift imaging) was initiated 24 seconds after the pyruvate injection and had a duration of 14 seconds. All implanted tumors showed significantly higher lactate content than the normal tissue. The results indicate that noninvasive quantification of localized Warburg effect may be possible.

Dynamic nuclear polarization of 13 C-labeled cell substrates has been shown to massively increase their sensitivity to detection in NMR experiments. The sensitivity gain is sufficiently large that if these polarized molecules are injected intravenously, their spatial distribution and subsequent conversion into other cell metabolites can be imaged. We have used this method to image the conversion of fumarate to malate in a murine lymphoma tumor in vivo after i.v. injection of hyperpolarized [1,4-13 C2]fumarate. In isolated lymphoma cells, the rate of labeled malate production was unaffected by coadministration of succinate, which competes with fumarate for transport into the cell. There was, however, a correlation with the percentage of cells that had lost plasma membrane integrity, suggesting that the production of labeled malate from fumarate is a sensitive marker of cellular necrosis. Twenty-four hours after treating implanted lymphoma tumors with etoposide, at which point there were significant levels of tumor cell necrosis, there was a 2.4-fold increase in hyperpolarized [1,4-13 C2]malate production compared with the untreated tumors. Therefore, the formation of hyperpolarized 13 C-labeled malate from [1,4-13 C2]fumarate appears to be a sensitive marker of tumor cell death in vivo and could be used to detect the early response of tumors to treatment. Given that fumarate is an endogenous molecule, this technique has the potential to be used clinically. magnetic resonance imaging ͉ spectroscopy ͉ metabolism ͉ cell death ͉ lymphoma