In Situ Generation and High Bioresistance of Trityl-based Semiquinone Methide Radicals Under Anaerobic Conditions in Cellular Systems.

Bioreduction of spin labels and polarizing agents (generally stable radicals) has been an obstacle limiting the in-cell applications of pulsed electron paramagnetic resonance (EPR) spectroscopy and dynamic nuclear polarization (DNP). In this work, we have demonstrated that two semiquinone methide radicals (OXQM⋅ and CTQM⋅) can be easily produced from the trityl-based quinone methides (OXQM and CTQM) via reduction by various reducing agents including biothiols and ascorbate under anaerobic conditions. Both radicals have relatively low pKa's and exhibit EPR single line signals at physiological pH. Moreover, the bioreduction of OXQM in three cell lysates enables quantitative generation of OXQM⋅ which was most likely mediated by flavoenzymes. Importantly, the resulting OXQM⋅ exhibited extremely high stability in the E.coli lysate under anaerobic conditions with 76- and 14.3-fold slower decay kinetics as compared to the trityl OX063 and a gem-diethyl pyrrolidine nitroxide, respectively. Intracellular delivery of OXQM into HeLa cells was also achieved by covalent conjugation with a cell-permeable peptide as evidenced by the stable intracellular EPR signal from the OXQM⋅ moiety. Owing to extremely high resistance of OXQM⋅ towards bioreduction, OXQM and its derivatives show great application potential in in-cell EPR and in-cell DNP studies for various cells which can endure short-term anoxic treatments.

Frontiers in the Application of RF Vacuum Electronics.

The application of radio frequency (RF) vacuum electronics for the betterment of the human condition began soon after the invention of the first vacuum tubes in the 1920s and has not stopped since. Today, microwave vacuum devices are powering important applications in health treatment, material and biological science, wireless communication-terrestrial and space, Earth environment remote sensing, and the promise of safe, reliable, and inexhaustible energy. This article highlights some of the exciting application frontiers of vacuum electronics.

Complete resonance assignment of a pharmaceutical drug at natural isotopic abundance from DNP-Enhanced solid-state NMR.

Solid-state dynamic nuclear polarization enhanced magic angle spinning (DNP-MAS) NMR measurements coupled with density functional theory (DFT) calculations enable the full resonance assignment of a complex pharmaceutical drug molecule without the need for isotopic enrichment. DNP dramatically enhances the NMR signals, thereby making possible previously intractable two-dimensional correlation NMR spectra at natural abundance. Using inputs from DFT calculations, herein we describe a significant improvement to the structure elucidation process for complex organic molecules. Further, we demonstrate that a series of two-dimensional correlation experiments, including 15N-13C TEDOR, 13C-13C INADEQUATE/SARCOSY, 19F-13C HETCOR, and 1H-13C HETCOR, can be obtained at natural isotopic abundance within reasonable experiment times, thus enabling a complete resonance assignment of sitagliptin, a pharmaceutical used for the treatment of type 2 diabetes.

JOM-4S Overhauser Magnetometer and Sensitivity Estimation.

The Overhauser magnetometer is a scalar quantum magnetometer based on the dynamic nuclear polarization (DNP) effect in the Earth's magnetic field. Sensitivity is a key technical specification reflecting the ability of instruments to sense small variations of the Earth's magnetic field and is closely related to the signal-to-noise ratio (SNR) of the free induction decay (FID) signal. In this study, deuterated 15N TEMPONE radical is used in our sensor to obtain high DNP enhancement. The measured SNR of the FID signal is approximately 63/1, and the transverse relaxation time T2 is 2.68 s. The direct measurement method with a single instrument and the synchronous measurement method with two instruments are discussed for sensitivity estimation in time and frequency domains under different electromagnetic interference (EMI) environments and different time periods. For the first time, the correlation coefficient of the magnetic field measured by the two instruments is used to judge the degree of the influence of the environmental noise on the sensitivity estimation. The sensitivity evaluation in the field environment is successfully realized without electrical and magnetic shields. The direct measurement method is susceptible to EMI and cannot work in general electromagnetic environments, except it is sufficiently quiet. The synchronous measurement method has an excellent ability to remove most natural and artificial EMIs and can be used under noisy environments. Direct and synchronous experimental results show that the estimated sensitivity of the JOM-4S magnetometer is approximately 0.01 nT in time domain and approximately 0.01 nT/Hz in frequency domain at a 3 s cycling time. This study provides a low-cost, simple, and effective sensitivity estimation method, which is especially suitable for developers and users to estimate the performance of the instrument.

Absolute 1H polarization measurement with a spin-correlated component of magnetization by hyperpolarized MAS-DNP solid-state NMR.

Sensitivity of magic-angle spinning (MAS) NMR spectroscopy has been dramatically improved by the advent of high-field dynamic nuclear polarization (DNP) technique and its rapid advances over the past decades. In this course, discussions on ways to improve the DNP enhancement factor or the overall sensitivity gain have been numerous, and led to a number of methodological and instrumental breakthroughs. Beyond the sensitivity gain, however, discussions on accurate quantification of the 1H polarization amplitude achievable in a sample with DNP have been relatively rare. Here, we propose a new method for quantifying the local 1H hyperpolarization amplitude, which is applicable to un-oriented/powdered solid samples under MAS NMR conditions. The method is based on the ability to observe the high-order spin-correlated term (2IzSz) intrinsic to a hyperpolarized IS two-spin state, separately from the lowest-order Zeeman term (Sz) in quasi-equilibrium magnetization. The quantification procedure does not require evaluation of signal amplitudes for a "microwave-off" condition and for an un-doped reference sample, and thus enables quick and accurate quantification unaffected by the effects of the paramagnetic quenching and the MAS-induced depolarization. The method is also shown to elucidate spatial polarization distribution through the 2IzSz term prepared domain-selectively. As a potential application, we also demonstrate 2D DQ-SQ spectroscopy utilizing the 2IzSz term that is generated in a spatially selective manner without using IS dipolar or J coupling. These salient features may be evolved into a way for characterizing mesoscopic molecular assemblies of medical/biological importance.

Metabolic biomarkers for non-alcoholic fatty liver disease induced by high-fat diet: In vivo magnetic resonance spectroscopy of hyperpolarized [1-13C] pyruvate.

Hyperpolarized 13C magnetic resonance spectroscopy (MRS) to assess hepatic metabolism in non-alcoholic fatty liver disease (NAFLD) has not been reported. This study searched for cellular metabolism-based biomarkers for NAFLD induced by a high-fat diet (HFD) in rats. Also, correlations of the biomarkers with enzyme levels and histopathology were identified during a 6-week follow-up. Six rats were fed a control diet (CD) and seven rats were fed the HFD for 6 weeks. Hyperpolarized 13C dynamic MRS was performed on rat liver following an injection of hyperpolarized [1-13C] pyruvate. Compared with CD-fed rats, HFD-fed rats showed significant increases in the levels of serum alanine aminotransferase and low-density lipoprotein cholesterol at weeks 4 and 6 of follow-up. After the 6-week HFD, the ratios of [1-13C] alanine/pyruvate and [1-13C] lactate/pyruvate were significantly increased, as were the levels of alanine aminotransferase and lactate dehydrogenase, which are potentially associated with hepatosteatosis. The results implicate [1-13C] alanine and [1-13C] lactate as potentially useful noninvasive biomarkers of hepatosteatosis occurring in NAFLD.

Modeling non-linear kinetics of hyperpolarized [1-(13)C] pyruvate in the crystalloid-perfused rat heart.

Hyperpolarized (13)C MR measurements have the potential to display non-linear kinetics. We have developed an approach to describe possible non-first-order kinetics of hyperpolarized [1-(13)C] pyruvate employing a system of differential equations that agrees with the principle of conservation of mass of the hyperpolarized signal. Simultaneous fitting to a second-order model for conversion of [1-(13)C] pyruvate to bicarbonate, lactate and alanine was well described in the isolated rat heart perfused with Krebs buffer containing glucose as sole energy substrate, or glucose supplemented with pyruvate. Second-order modeling yielded significantly improved fits of pyruvate-bicarbonate kinetics compared with the more traditionally used first-order model and suggested time-dependent decreases in pyruvate-bicarbonate flux. Second-order modeling gave time-dependent changes in forward and reverse reaction kinetics of pyruvate-lactate exchange and pyruvate-alanine exchange in both groups of hearts during the infusion of pyruvate; however, the fits were not significantly improved with respect to a traditional first-order model. The mechanism giving rise to second-order pyruvate dehydrogenase (PDH) kinetics was explored experimentally using surface fluorescence measurements of nicotinamide adenine dinucleotide reduced form (NADH) performed under the same conditions, demonstrating a significant increase of NADH during pyruvate infusion. This suggests a simultaneous depletion of available mitochondrial NAD(+) (the cofactor for PDH), consistent with the non-linear nature of the kinetics. NADH levels returned to baseline following cessation of the pyruvate infusion, suggesting this to be a transient effect.

Dynamic nuclear polarization of nucleic acid with endogenously bound manganese.

We report the direct dynamic nuclear polarization (DNP) of (13)C nuclei of a uniformly [(13)C,(15)N]-labeled, paramagnetic full-length hammerhead ribozyme (HHRz) complex with Mn(2+) where the enhanced polarization is fully provided by the endogenously bound metal ion and no exogenous polarizing agent is added. A (13)C enhancement factor of ε = 8 was observed by intra-complex DNP at 9.4 T. In contrast, "conventional" indirect and direct DNP experiments were performed using AMUPol as polarizing agent where we obtained a (1)H enhancement factor of ε ≈ 250. Comparison with the diamagnetic (Mg(2+)) HHRz complex shows that the presence of Mn(2+) only marginally influences the (DNP-enhanced) NMR properties of the RNA. Furthermore two-dimensional correlation spectra ((15)N-(13)C and (13)C-(13)C) reveal structural inhomogeneity in the frozen, amorphous state indicating the coexistence of several conformational states. These demonstrations of intra-complex DNP using an endogenous metal ion as well as DNP-enhanced MAS NMR of RNA in general yield important information for the development of new methods in structural biology.

Real-time measurement of hyperpolarized lactate production and efflux as a biomarker of tumor aggressiveness in an MR compatible 3D cell culture bioreactor.

We have developed a 3D cell/tissue culture bioreactor compatible with hyperpolarized (HP) (13)C MR and interrogated HP [1-(13)C]lactate production and efflux in human renal cell carcinoma (RCC) cells. This platform is capable of resolving intracellular and extracellular HP lactate pools, allowing the kinetic measurement of lactate production and efflux in the context of cancer aggressiveness and response to therapy. HP (13)C MR studies were performed on three immortalized human renal cell lines: HK2, a normal renal proximal tubule cell line from which a majority of RCCs arise, UMRC6, a cell line derived from a localized RCC, and UOK262, an aggressive and metastatic RCC. The intra- (Lacin ) and extracellular (Lacex ) HP lactate signals were robustly resolved in dynamic (13)C spectra of the cell lines due to a very small but reproducible chemical shift difference (0.031 ± 0.0005 ppm). Following HP [1-(13)C]pyruvate delivery, the ratio of HP Lacin /Lacex was significantly lower for UOK262 cells compared with both UMRC6 and HK2 cells due to a significant (p < 0.05) increase in the Lacex pool size. Lacin /Lacex correlated with the MCT4 mRNA expression of the cell lines, and inhibition of MCT4 transport using DIDS resulted in a significant reduction in the HP Lacex pool size. The extension of these studies to living patient-derived RCC tissue slices using HP [1,2-(13)C2]pyruvate demonstrated a similarly split lactate doublet with a high Lacex pool fraction; in contrast, only a single NMR resonance is noted for HP [5-(13)C]glutamate, consistent with intracellular localization. These studies support the importance of lactate efflux as a biomarker of cancer aggressiveness and metastatic potential, and the utility of the MR compatible 3D cell/tissue culture bioreactor to study not only cellular metabolism but also transport. Additionally, this platform offers a sophisticated way to follow therapeutic interventions and screen novel therapies that target lactate export.

Hyperpolarized [1,(13)C]pyruvate in lysed human erythrocytes: effects of co-substrate supply on reaction time courses.

Hyperpolarized [1,(13)C]pyruvate was injected rapidly into haemolysates in which hydrolysis of nicotinamide adenine dinucleotide (phosphate) (NAD(P))/NAD(P)H had been inhibited with nicotinamide. Haemolysates provide a stable glycolytic system in which membrane permeability is not a flux-controlling step, and they enable the concentration of NADH to be adjusted experimentally while keeping the rest of the sample with the same composition as that of the cytoplasm of the cell (albeit diluted twofold at the time of injection of the [1,(13)C]pyruvate). We showed that the maximum amplitude of the (13)C NMR signal from the [1,(13)C]L-lactate, produced from [1,(13)C]pyruvate, and the time at which it occurred was dependent on NADH concentration, as predicted by enzyme-kinetic analysis. The main feature of such curves was dictated by the immediacy of the supply of the co-substrate of lactate dehydrogenase (LDH, EC 1.1.1.27), and we posit that this also pertains in vivo in various tissues including neoplasms. By constructing an appropriate mathematical model and by using a Markov-chain Monte Carlo approach, we fitted experimental data to estimate LDH and NADH concentrations. Experiments carried out with only endogenous NADH present enabled the estimation of its effective concentration in human RBCs; the ability to make this estimate is a special feature of the rapid-dissolution dynamic nuclear polarization method. We found an endogenous NADH concentration in human RBCs two to four times higher than previously reported.

Recent developments in materials and applications of triplet dynamic nuclear polarization.

Dynamic nuclear polarization (DNP) is a method for achieving high levels of nuclear spin polarization by transferring spin polarization from electrons to nuclei by microwave irradiation, resulting in higher sensitivity in NMR/MRI. In particular, DNP using photoexcited triplet electron spins (triplet-DNP) can provide a hyperpolarized nuclear spin state at room temperature and in low magnetic field. In this review article, we highlight recent developments in materials and instrumentation for the application of triplet-DNP. First, a brief history and principles of triplet-DNP will be presented. Next, important advances in recent years will be outlined: new materials to hyperpolarize water and biomolecules; high-sensitivity solution NMR by dissolution triplet-DNP; and strategies for further improvement of the polarization. In view of these developments, future directions to widen the range of applications of triplet-DNP will be discussed.

Triplet-DNP in magnetically oriented microcrystal arrays.

We explore dynamic nuclear polarization using electron spins in the photo-excited triplet state (Triplet-DNP) in magnetically oriented microcrystal arrays (MOMAs) of pentacene-doped p-terphenyl, in which the individual crystallites are magnetically aligned and UV-cured. In contrast to the conventional approach to Triplet-DNP in powder, which suffers from reduced nuclear polarization due to the averaged electron polarization and the broadening of electron-spin resonance, Triplet-DNP of the MOMAs offers as high dynamic polarization as that attainable in single-crystals. In the case of pentacene-doped p-terphenyl, the enhanced 1H polarization in the one-dimensional MOMA, prepared simply by leaving the suspension in a stationary magnetic field before UV curation, can be higher than that attainable in the powder sample by an order of magnitude and comparable to that in single crystals and in the three-dimensional MOMA made using a modulational rotating field. Triplet-DNP of the MOMAs may find potential applications, such as the polarization of the co-doped target molecules and dissolution experiments.

Watch-sized 12 Tesla all-high-temperature-superconducting magnet.

We demonstrate the construction of 7 Tesla and 12 Tesla all high-temperature-superconducting (HTS) magnets, small enough to fit on your wrist. The size of the magnet reduces the cost of fabrication, decreases the fringe field to permit facile siting of magnets, and decreases the stored energy of high field magnets. These small HTS-based magnets are being developed for gyrotron microwave sources for use in high-field nuclear magnetic resonance applications. The 7 Tesla and 12 Tesla magnets employ a no-insulation winding technique and are cooled to 4.2 Kelvin in a liquid helium cryostat. The 7 Tesla magnet is a single pancake coil, made of only 9.4 m of HTS tape, with an inner diameter of 8 mm and an outer diameter of 24 mm. This magnet was charged up to 1168 Amperes, generating a field of 7.3 Tesla. The 12 Tesla magnet is comprised of two pancake coils (inner diameter of 10 mm and outer diameter of 27 mm) connected in series. This magnet reached its maximum field at a current of 850 Amperes.

Evolution of α-synuclein conformation ensemble toward amyloid fibril via liquid-liquid phase separation (LLPS) as investigated by dynamic nuclear polarization-enhanced solid-state MAS NMR.

Protein fibrillation and human neurodegenerative diseases, with a profound underlying connection suggested between them, have been the subject of intense investigations in the medical, biophysical and bio-engineering sciences. For gaining the molecular mechanistic insights into such connection, i.e., the cause and effect, atomic-resolution molecular structure information especially on the initial oligomeric states is of paramount importance, not only that on the mature amyloid fibrils. α-Synuclein (αSyn) and its amyloid fibril has a direct relevance to the Parkinson's disease and other synucleinopathies, but what triggers the fibrillation is still not entirely clear. We here describe the liquid-liquid phase separation (LLPS) of αSyn and investigate its conformational evolution from its monomeric state into oligomer state within the early-stage of the phase-separated droplets, mainly using solution and magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectroscopies, aided with optical and fluorescent microscopies and CD spectroscopy. Based on the analysis of the intricately broadened shapes of the MAS NMR peaks observed for isotopically 13C-labeled His-50 of αSyn, we show that the distribution of the αSyn conformation is skewed from the initial completely random state to a loose ß-rich ensembles at/around His-50 as early as day-3 (d3) within the droplet. This intra-droplet loose ß-rich assembly showed a very slow progression until d8, and eventually maturated into ThT-positive, long and unbranched amyloid fibrils after 8 weeks. The obtained information on the evolution of the distribution of the conformation ensemble is unique, and difficult to obtain with X-ray crystallography and cryo-electron microscopy (cryoEM). In particular, the sensitivity-enhanced MAS NMR based on the low-temperature dynamic nuclear polarization (DNP) technique was proven to be a key tool in characterizing the conformational ensemble with dilute protein samples such as the liquid-phase droplets.

Sample volume effects in optical overhauser dynamic nuclear polarization.

The optical dynamic nuclear polarization (DNP) method has been proposed as an alternative to microwave pumping as a hyperpolarization method for solution-state NMR studies. Using continuous laser illumination to photogenerate triplet states in the presence of a persistent radical produces chemically-induced dynamic electron polarization (CIDEP) via the radical-triplet pair mechanism (RTPM), with cross-relaxation transferring this to nuclear hyperpolarization via an Overhauser mechanism. Numerical simulations have previously indicated that reducing the sample volume while maintaining a constant optical density can significantly increase the NMR signal enhancement, due to the larger steady-state concentration of triplets obtained. Here we provide the first experimental confirmation of these effects, producing a nearly five-fold increase in the optical DNP enhancement factor just by reducing the sample volume with optimal dye and radical concentrations adjusted for each optical path length. The results are supported with an in depth analysis of volume effects in the numerical model, with which they are in good qualitative agreement. These important observations will impact on the future development of the technique, with particular significance for attempts to apply DNP methods to increase sensitivity for volume-limited biological samples.

THz-enhanced dynamic nuclear polarized liquid spectrometer.

Dynamic nuclear polarization (DNP) technology can be utilized to dramatically enhance NMR signal. In this paper, we report on the development of a self-constructed 5 T DNP spectrometer for liquid samples and the 13C DNP enhancement achieved with this spectrometer. The DNP spectrometer is comprised of a wide-bore superconducting magnet, a home-made console, a dual resonance probe and a self-built 140 GHz microwave source for the spectrometer. Specifically, a microwave source of traveling wave tube (TWT) amplifier has been developed, which can provide a maximum power output of 4.4 W and a wide frequency tuning range of 1 GHz. The excellent performance of our built liquid-state DNP spectrometer is verified by the observation of more than 100-fold DNP enhancement of the 13C NMR signal for liquid 13CCl4 sample. Our result shows the superiority of DNP technology in the liquid-state high-field NMR spectrometer.

In-Cell NMR of Intact Mammalian Cells Preserved with the Cryoprotectants DMSO and Glycerol Have Similar DNP Performance.

NMR has the resolution and specificity to determine atomic-level protein structures of isotopically-labeled proteins in complex environments and, with the sensitivity gains conferred by dynamic nuclear polarization (DNP), NMR has the sensitivity to detect proteins at their endogenous concentrations. Prior work established that DNP MAS NMR is compatible with cellular viability. However, in that work, 15% glycerol, rather than the more commonly used 10% DMSO, was used as the cellular cryoprotectant. Moreover, incubation of cells cryoprotected 15% glycerol with the polarization agent, AMUPol, resulted in an inhomogeneous distribution of AMUPol through the cellular biomass, which resulted in a spatial bias of the NMR peak intensities. Because 10% DMSO is not only the most used cryoprotectant for mammalian cells, but also because DMSO is often used to improve delivery of molecules to cells, we sought to characterize the DNP performance of cells that were incubated with AMUPol and cryoprotected with 10% DMSO. We found that, like cells preserved with 15% glycerol, cells preserved with 10% DMSO retain high viability during DNP MAS NMR experiments if they are frozen at a controlled rate. However, DMSO did not improve the dispersion of AMUPol throughout the cellular biomass. Cells preserved with 15% glycerol and with 10% DMSO had similar DNP performance for both the maximal DNP enhancements as well as the inhomogeneous dispersion of AMUPol throughout the cellular biomass. Therefore, 10% DMSO and 15% glycerol are both appropriate cryoprotectant systems for DNP-assisted MAS NMR of intact viable mammalian cells.

Analysis Methods for Hyperpolarized Carbon (13C) MRI of the Kidney.

Hyperpolarized 13C MR is a novel medical imaging modality with substantially different signal dynamics as compared to conventional 1H MR, thus requiring new methods for processing the data in order to access and quantify the embedded metabolic and functional information. Here we describe step-by-step analysis protocols for functional renal hyperpolarized 13C imaging. These methods are useful for investigating renal blood flow and function as well as metabolic status of rodents in vivo under various experimental physiological conditions.This chapter is based upon work from the COST Action PARENCHIMA, a community-driven network funded by the European Cooperation in Science and Technology (COST) program of the European Union, which aims to improve the reproducibility and standardization of renal MRI biomarkers. This analysis protocol chapter is complemented by two separate chapters describing the basic concept and experimental procedure.

Solid-state NMR of unlabeled plant cell walls: high-resolution structural analysis without isotopic enrichment.

BACKGROUND: Multidimensional solid-state nuclear magnetic resonance (ssNMR) spectroscopy has emerged as an indispensable technique for resolving polymer structure and intermolecular packing in primary and secondary plant cell walls. Isotope (13C) enrichment provides feasible sensitivity for measuring 2D/3D correlation spectra, but this time-consuming procedure and its associated expenses have restricted the application of ssNMR in lignocellulose analysis. RESULTS: Here, we present a method that relies on the sensitivity-enhancing technique Dynamic Nuclear Polarization (DNP) to eliminate the need for 13C-labeling. With a 26-fold sensitivity enhancement, a series of 2D 13C-13C correlation spectra were successfully collected using the unlabeled stems of wild-type Oryza sativa (rice). The atomic resolution allows us to observe a large number of intramolecular cross peaks for fully revealing the polymorphic structure of cellulose and xylan. NMR relaxation and dipolar order parameters further suggest a sophisticated change of molecular motions in a ctl1 ctl2 double mutant: both cellulose and xylan have become more dynamic on the nanosecond and microsecond timescale, but the motional amplitudes are uniformly small for both polysaccharides. CONCLUSIONS: By skipping isotopic labeling, the DNP strategy demonstrated here is universally extendable to all lignocellulose materials. This time-efficient method has landed the technical foundation for understanding polysaccharide structure and cell wall assembly in a large variety of plant tissues and species.

Imaging 6-Phosphogluconolactonase Activity in Brain Tumors In Vivo Using Hyperpolarized δ-[1-13C]gluconolactone.

INTRODUCTION: The pentose phosphate pathway (PPP) is essential for NADPH generation and redox homeostasis in cancer, including glioblastomas. However, the precise contribution to redox and tumor proliferation of the second PPP enzyme 6-phosphogluconolactonase (PGLS), which converts 6-phospho-δ-gluconolactone to 6-phosphogluconate (6PG), remains unclear. Furthermore, non-invasive methods of assessing PGLS activity are lacking. The goal of this study was to examine the role of PGLS in glioblastomas and assess the utility of probing PGLS activity using hyperpolarized δ-[1-13C]gluconolactone for non-invasive imaging. METHODS: To interrogate the function of PGLS in redox, PGLS expression was silenced in U87, U251 and GS2 glioblastoma cells by RNA interference and levels of NADPH and reduced glutathione (GSH) measured. Clonogenicity assays were used to assess the effect of PGLS silencing on glioblastoma proliferation. Hyperpolarized δ-[1-13C]gluconolactone metabolism to 6PG was assessed in live cells treated with the chemotherapeutic agent temozolomide (TMZ) or with vehicle control. 13C 2D echo-planar spectroscopic imaging (EPSI) studies of hyperpolarized δ-[1-13C]gluconolactone metabolism were performed on rats bearing orthotopic glioblastoma tumors or tumor-free controls on a 3T spectrometer. Longitudinal 2D EPSI studies of hyperpolarized δ-[1-13C]gluconolactone metabolism and T2-weighted magnetic resonance imaging (MRI) were performed in rats bearing orthotopic U251 tumors following treatment with TMZ to examine the ability of hyperpolarized δ-[1-13C]gluconolactone to report on treatment response. RESULTS: PGLS knockdown downregulated NADPH and GSH, elevated oxidative stress and inhibited clonogenicity in all models. Conversely, PGLS expression and activity and steady-state NADPH and GSH were higher in tumor tissues from rats bearing orthotopic glioblastoma xenografts relative to contralateral brain and tumor-free brain. Importantly, [1-13C]6PG production from hyperpolarized δ-[1-13C]gluconolactone was observed in live glioblastoma cells and was significantly reduced by treatment with TMZ. Furthermore, hyperpolarized δ-[1-13C]gluconolactone metabolism to [1-13C]6PG could differentiate tumor from contralateral normal brain in vivo. Notably, TMZ significantly reduced 6PG production from hyperpolarized δ-[1-13C]gluconolactone at an early timepoint prior to volumetric alterations as assessed by anatomical imaging. CONCLUSIONS: Collectively, we have, for the first time, identified a role for PGLS activity in glioblastoma proliferation and validated the utility of probing PGLS activity using hyperpolarized δ-[1-13C]gluconolactone for non-invasive in vivo imaging of glioblastomas and their response to therapy.