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Purpose: Serine is a major source of one-carbon units needed for the synthesis of nucleotides and the production of intramitochondrial nicotinamide adenine dinucleotide phosphate (NADPH), and it plays an important role in cancer cell proliferation. The aim of this study was to develop a deuterium ( 2 H) MRS imaging method for imaging tumor serine metabolism., Methods: Sequential ( 2 H) spectra and spectroscopic images were used to monitor the metabolism of [2,3,3- 2 H 3 ]serine in patient-derived glioblastoma cells in vitro and in tumors obtained by their orthotopic implantation in mouse brain., Results: [14,14- 2 H 2 ] 5,10-methylene-tetrahydrofolate, [ 2 H]glycine, [ 2 H]formate, and labeled water were detected in cell suspensions and water labeling in spectroscopic images of tumors. Studies in cells and tumors with variable mitochondrial content and inhibitor studies in cells demonstrated that most of the labeled serine was metabolized in the mitochondria. Water labeling in the cell suspensions was correlated with formate labeling; therefore, water labeling observed in tumors could be used to provide a surrogate measure of flux in the pathway of one-carbon metabolism in vivo., Conclusion: The method has the potential to be used clinically to select patients for treatment with inhibitors of one-carbon metabolism and subsequently to detect their early responses to such treatment., (© 2024 The Author(s). Magnetic Resonance in Medicine published by Wiley Periodicals LLC on behalf of International Society for Magnetic Resonance in Medicine.)

Imaging the metabolism of [2,3- 2 H 2 ]fumarate to produce malate can be used to detect tumor cell death post-treatment. Here, we assess the sensitivity of the technique for detecting cell death by lowering the concentration of injected [2,3- 2 H 2 ]fumarate and by varying the extent of tumor cell death through changes in drug concentration. Mice were implanted subcutaneously with human triple negative breast cancer cells (MDA-MB-231) and injected with 0.1, 0.3, and 0.5 g/kg [2,3- 2 H 2 ]fumarate before and after treatment with a multivalent TRAlL-R2 agonist (MEDI3039) at 0.1, 0.4, and 0.8 mg/kg. Tumor conversion of [2,3- 2 H 2 ]fumarate to [2,3- 2 H 2 ]malate was assessed from a series of 13 spatially localized 2 H MR spectra acquired over 65 min using a pulse-acquire sequence with a 2-ms BIR4 adiabatic excitation pulse. Tumors were then excised and stained for histopathological markers of cell death: cleaved caspase 3 (CC3) and DNA damage (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]). The rate of malate production and the malate/fumarate ratio plateaued at tumor fumarate concentrations of 2 mM, which were obtained with injected [2,3- 2 H 2 ]fumarate concentrations of 0.3 g/kg and above. Tumor malate concentration and the malate/fumarate ratio increased linearly with the extent of cell death determined histologically. At an injected [2,3- 2 H 2 ]fumarate concentration of 0.3 g/kg, 20% CC3 staining corresponded to a malate concentration of 0.62 mM and a malate/fumarate ratio of 0.21. Extrapolation indicated that there would be no detectable malate at 0% CC3 staining. The use of low and nontoxic fumarate concentrations and the production of [2,3- 2 H 2 ]malate at concentrations that are within the range that can be detected clinically suggest this technique could translate to the clinic., (© 2023 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)

Purpose: There is an unmet clinical need for direct and sensitive methods to detect cell death in vivo, especially with regard to monitoring tumor treatment response. We have shown previously that tumor cell death can be detected in vivo from 2 H MRS and MRSI measurements of increased [2,3- 2 H 2 ]malate production following intravenous injection of [2,3- 2 H 2 ]fumarate. We show here that cell death can be detected with similar sensitivity following oral administration of the 2 H-labeled fumarate., Methods: Mice with subcutaneously implanted EL4 tumors were fasted for 1 h before administration (200 μl) of [2,3- 2 H 2 ]fumarate (2 g/kg bodyweight) via oral gavage without anesthesia. The animals were then anesthetized, and after 30 min, tumor conversion of [2,3- 2 H 2 ]fumarate to [2,3- 2 H 2 ]malate was assessed from a series of 13 2 H spectra acquired over a period of 65 min. The 2 H spectra and 2 H spectroscopic images were acquired using a surface coil before and at 48 h after treatment with a chemotherapeutic drug (etoposide, 67 mg/kg)., Results: The malate/fumarate signal ratio increased from 0.022 ± 0.03 before drug treatment to 0.12 ± 0.04 following treatment (p = 0.023, n = 4). Labeled malate was undetectable in spectroscopic images acquired before treatment and increased in the tumor area following treatment. The increase in the malate/fumarate signal ratio was similar to that observed previously following intravenous administration of labeled fumarate., Conclusion: Orally administered [2,3- 2 H 2 ]fumarate can be used to detect tumor cell death noninvasively following treatment with a sensitivity that is similar to that obtained with intravenous administration., (© 2022 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals LLC on behalf of International Society for Magnetic Resonance in Medicine.)

Purpose: The performance of pulse sequences in vivo can be limited by fast relaxation rates, magnetic field inhomogeneity, and nonuniform spin excitation. We describe here a method for pulse sequence optimization that uses a stochastic numerical solver that in principle is capable of finding a global optimum. The method provides a simple framework for incorporating any constraint and implementing arbitrarily complex cost functions. Efficient methods for simulating spin dynamics and incorporating frequency selectivity are also described., Methods: Optimized pulse sequences for polarization transfer between protons and X-nuclei and excitation pulses that eliminate J-coupling modulation were evaluated experimentally using a surface coil on phantoms, and also the detection of hyperpolarized [2- 13 C]lactate in vivo in the case of J-coupling modulation-free excitation., Results: The optimized polarization transfer pulses improved the SNR by ~50% with a more than twofold reduction in the B 1 field, and J-coupling modulation-free excitation was achieved with a more than threefold reduction in pulse length., Conclusion: This process could be used to optimize any pulse when there is a need to improve the uniformity and frequency selectivity of excitation as well as to design new pulses to steer the spin system to any desired achievable state., (© 2021 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals LLC on behalf of International Society for Magnetic Resonance in Medicine.)

Purpose: To compare carbon-13 ( 13 C) MRSI of hyperpolarized [1- 13 C]pyruvate metabolism in a murine tumor model with mass spectrometric (MS) imaging of the corresponding tumor sections in order to cross validate these metabolic imaging techniques and to investigate the effects of pyruvate delivery and tumor lactate concentration on lactate labeling., Methods: [1- 13 C]lactate images were obtained from tumor-bearing mice, following injection of hyperpolarized [1- 13 C]pyruvate, using a single-shot 3D 13 C spectroscopic imaging sequence in vivo and using desorption electrospray ionization MS imaging of the corresponding rapidly frozen tumor sections ex vivo. The images were coregistered, and levels of association were determined by means of Spearman rank correlation and Cohen kappa coefficients as well as linear mixed models. The correlation between [1- 13 C]pyruvate and [1- 13 C]lactate in the MRS images and between [ 12 C] and [1- 13 C]lactate in the MS images were determined by means of Pearson correlation coefficients., Results: [1- 13 C]lactate images generated by MS imaging were significantly correlated with the corresponding MRS images. The correlation coefficient between [1- 13 C]lactate and [1- 13 C]pyruvate in the MRS images was higher than between [1- 13 C]lactate and [ 12 C]lactate in the MS images., Conclusion: The inhomogeneous distribution of labeled lactate observed in the MRS images was confirmed by MS imaging of the corresponding tumor sections. The images acquired using both techniques show that the rate of 13 C label exchange between the injected pyruvate and endogenous tumor lactate pool is more correlated with the rate of pyruvate delivery to the tumor cells and is less affected by the endogenous lactate concentration., (© 2021 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals LLC on behalf of International Society for Magnetic Resonance in Medicine.)

Purpose: Imaging tumor metabolism in vivo using hyperpolarized [1- 13 C]pyruvate is a promising technique for detecting disease, monitoring disease progression, and assessing treatment response. However, the transient nature of the hyperpolarization and its depletion following excitation limits the available time for imaging. We describe here a single-shot multi spin echo sequence, which improves on previously reported sequences, with a shorter readout time, isotropic point spread function (PSF), and better signal-to-noise ratio., Methods: The sequence uses numerically optimized spectrally selective excitation pulses set to the resonant frequencies of pyruvate and lactate and a hyperbolic secant adiabatic refocusing pulse, all applied in the absence of slice selection gradients. The excitation pulses were designed to be resistant to the effects of B 0 and B 1 field inhomogeneity. The gradient readout uses a 3D cone trajectory composed of 13 cones, all fully refocused and distributed among 7 spin echoes. The maximal gradient amplitude and slew rate were set to 4 G/cm and 20 G/cm/ms, respectively, to demonstrate the feasibility of clinical translation., Results: The pulse sequence gave an isotropic PSF of 2.8 mm. The excitation profiles of the optimized pulses closely matched simulations and a 46.10 ± 0.04% gain in image SNR was observed compared to a conventional Shinnar-Le Roux excitation pulse. The sequence was demonstrated with dynamic imaging of hyperpolarized [1- 13 C]pyruvate and [1- 13 C]lactate in vivo., Conclusion: The pulse sequence was capable of dynamic imaging of hyperpolarized 13 C labeled metabolites in vivo with relatively high spatial and temporal resolution and immunity to system imperfections., (© 2020 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine.)

Purpose: Hyperpolarized 15 N-labeled molecules have been proposed as imaging agents for investigating tissue perfusion and pH. However, the sensitivity of direct 15 N detection is limited by the isotope's low gyromagnetic ratio. Sensitivity can be increased by transferring 15 N hyperpolarization to spin-coupled protons provided that there is not significant polarization loss during transfer. However, complete polarization transfer would limit the temporal window for imaging to the order of the proton T 1 (2-3 s). To exploit the long T 1 offered by storing polarization in 15 N and the higher sensitivity of 1 H detection, we have developed a pulse sequence for partial polarization transfer., Methods: A polarization transfer pulse sequence was modified to allow partial polarization transfer, as is required for dynamic measurements, and that can be implemented with inhomogeneous B 1 fields, as is often the case in vivo. The sequence was demonstrated with dynamic spectroscopy and imaging measurements with [ 15 N 2 ]urea., Results: When compared to direct 15 N detection, the sequence increased the signal-to-noise ratio (SNR) by a factor of 1.72 ± 0.25, where both experiments depleted ~20% of the hyperpolarization (>10-fold when 100% of the hyperpolarization is used). Simulations with measured cross relaxation rates showed that this sequence gave up to a 50-fold increase in urea proton polarization when compared to spontaneous polarization transfer via cross relaxation., Conclusion: The sequence gave an SNR increase that was close to the theoretical limit and can give a significant SNR benefit when compared to direct 13 C detection of hyperpolarized [ 13 C]urea., (© 2020 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine.)

Proton MRS ( 1 H MRS) provides noninvasive, quantitative metabolite profiles of tissue and has been shown to aid the clinical management of several brain diseases. Although most modern clinical MR scanners support MRS capabilities, routine use is largely restricted to specialized centers with good access to MR research support. Widespread adoption has been slow for several reasons, and technical challenges toward obtaining reliable good-quality results have been identified as a contributing factor. Considerable progress has been made by the research community to address many of these challenges, and in this paper a consensus is presented on deficiencies in widely available MRS methodology and validated improvements that are currently in routine use at several clinical research institutions. In particular, the localization error for the PRESS localization sequence was found to be unacceptably high at 3 T, and use of the semi-adiabatic localization by adiabatic selective refocusing sequence is a recommended solution. Incorporation of simulated metabolite basis sets into analysis routines is recommended for reliably capturing the full spectral detail available from short TE acquisitions. In addition, the importance of achieving a highly homogenous static magnetic field (B 0 ) in the acquisition region is emphasized, and the limitations of current methods and hardware are discussed. Most recommendations require only software improvements, greatly enhancing the capabilities of clinical MRS on existing hardware. Implementation of these recommendations should strengthen current clinical applications and advance progress toward developing and validating new MRS biomarkers for clinical use., (© 2019 International Society for Magnetic Resonance in Medicine.)

Hyperpolarized MRI with 13 C-labeled metabolites has enabled metabolic imaging of tumors in vivo. The heterogeneous nature of tumors and the limited lifetime of the hyperpolarization require high resolution, both temporally and spatially. We describe two sequences that make more efficient use of the 13 C polarization than previously described single-shot 3D sequences. With these sequences, the target metabolite resonances were excited using spectral-spatial pulses and the data acquired using spiral readouts from a series of echoes created using a fast-spin-echo sequence employing adiabatic 180° pulses. The third dimension was encoded with blipped gradients applied in an interleaved order to the echo train. Adiabatic inversion pulses applied in the absence of slice selection gradients allowed acquisition of signal from odd echoes, formed by unpaired adiabatic pulses, as well as from even echoes. The sequences were tested on tumor-bearing mice following intravenous injection of hyperpolarized [1- 13 C]pyruvate. [1- 13 C] pyruvate and [1- 13 C] lactate images were acquired in vivo with a 4 × 4 × 2 cm 3 field of view and a 32 × 32 × 16 matrix, leading to a nominal resolution of 1.25 × 1.25 × 1.25 mm 3 and an effective resolution of 1.25 × 1.25 × 4.5 mm 3 when the z-direction point spread function was taken into account. The acquisition of signal from more echoes also allowed for an improvement in the signal-to-noise ratio for resonances with longer T 2 relaxation times. The pulse sequences described here produced hyperpolarized 13 C images with improved resolution and signal-to-noise ratio when compared with similar sequences described previously., (© 2018 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)

Measurements of hyperpolarized 13 C label exchange between injected [1- 13 C]pyruvate and the endogenous tumor lactate pool can give an apparent first-order rate constant for the exchange. The determination of the isotope flux, however, requires an estimate of the labeled pyruvate concentration in the tumor. This was achieved here by measurement of the tumor uptake of [1- 14 C]pyruvate, which showed that <2% of the injected pyruvate reached the tumor site. Multiplication of this estimated labeled pyruvate concentration in the tumor with the apparent first-order rate constant for hyperpolarized 13 C label exchange gave an isotope flux that showed good agreement with a flux determined directly by the injection of non-polarized [3- 13 C]pyruvate, rapid excision of the tumor after 30 s and measurement of 13 C-labeled lactate concentrations in tumor extracts. The distribution of labeled lactate between intra- and extracellular compartments and the blood pool was investigated by imaging, by measurement of the labeled lactate concentration in blood and tumor, and by examination of the effects of a gadolinium contrast agent and a lactate transport inhibitor on the intensity of the hyperpolarized [1- 13 C]lactate signal. These measurements showed that there was significant export of labeled lactate from the tumor, but that labeled lactate in the blood pool produced by the injection of hyperpolarized [1- 13 C]pyruvate showed only relatively low levels of polarization. This study shows that measurements of hyperpolarized 13 C label exchange between pyruvate and lactate in a murine tumor model can provide an estimate of the true isotope flux if the concentration of labeled pyruvate that reaches the tumor can be determined., (© 2018 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)

Arthritic conditions are a major source of chronic pain. Furthering our understanding of disease mechanisms creates the opportunity to develop more targeted therapeutics. In rheumatoid arthritis (RA), measurements of pH in human synovial fluid suggest that acidosis occurs, but that this is highly variable between individuals. Here we sought to determine if tissue acidosis occurs in a widely used rodent arthritis model: complete Freund's adjuvant (CFA)-induced inflammation. CFA robustly evoked paw and ankle swelling, concomitant with worsening clinical scores over time. We used magnetic resonance spectroscopic imaging of hyperpolarized [1- 13 C]pyruvate metabolism to demonstrate that CFA induces an increase in the lactate-to-pyruvate ratio. This increase is indicative of enhanced glycolysis and an increased lactate concentration, as has been observed in the synovial fluid from RA patients, and which was correlated with acidosis. We also measured the 13 CO 2 /H 13 CO 3 - ratio, in animals injected with hyperpolarized H 13 CO 3 - , to estimate extracellular tissue pH and showed that despite the apparent increase in glycolytic activity in CFA-induced inflammation there was no accompanying decrease in extracellular pH. The pH was 7.23 ± 0.06 in control paws and 7.32 ± 0.09 in inflamed paws. These results could explain why mice lacking acid-sensing ion channel subunits 1, 2 and 3 do not display any changes in mechanical or thermal hyperalgesia in CFA-induced inflammation., (© 2018 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)

Fumarate is an important probe of metabolism in hyperpolarized magnetic resonance imaging and spectroscopy. It is used to detect the release of fumarase in cancer tissues, which is associated with necrosis and drug treatment. Nevertheless, there are limited reports describing the detailed kinetic studies of this enzyme in various cells and tissues. Thus, we aimed to evaluate the sub-minute kinetics of human red blood cell fumarase using nuclear magnetic resonance (NMR) spectroscopy, and to provide a quantitative description of the enzyme that is relevant to the use of fumarate as a probe of cell rupture. The fumarase reaction was studied using time courses of 1 H spin-echo and 13 C-NMR spectra. 1 H-NMR experiments showed that the fumarase reaction in hemolysates is sufficiently rapid to make its kinetics amenable to study in a period of approximately 3 min, a timescale characteristic of hyperpolarized 13 C-NMR spectroscopy. The rapid-dissolution dynamic nuclear polarization (RD-DNP) technique was used to hyperpolarize [1,4- 13 C]fumarate, which was injected into concentrated hemolysates. The kinetic data were analyzed using recently developed FmR α analysis and modeling of the enzymatic reaction using Michaelis-Menten equations. In RD-DNP experiments, the decline in the 13 C-NMR signal from fumarate, and the concurrent rise and fall of that from malate, were captured with high spectral resolution and signal-to-noise ratio, which allowed the robust quantification of fumarase kinetics. The kinetic parameters obtained indicate the potential contribution of hemolysis to the overall rate of the fumarase reaction when 13 C-NMR RD-DNP is used to detect necrosis in animal models of implanted tumors. The analytical procedures developed will be applicable to studies of other rapid enzymatic reactions using conventional and hyperpolarized substrate NMR spectroscopy., (Copyright © 2018 John Wiley & Sons, Ltd.)

Purpose: Dynamic magnetic resonance spectroscopic imaging of hyperpolarized 13 C-labeled cell substrates has enabled the investigation of tissue metabolism in vivo. Currently observation of these hyperpolarized substrates is limited mainly to 13 C detection. We describe here an imaging pulse sequence that enables proton observation by using polarization transfer from the hyperpolarized 13 C nucleus to spin-coupled protons., Methods: The pulse sequence transfers 13 C hyperpolarization to 1 H using a modified reverse insensitive nuclei enhanced by polarization transfer (INEPT) sequence that acquires a fully refocused echo. The resulting hyperpolarized 1 H signal is acquired using a 2D echo-planar trajectory. The efficiency of polarization transfer was investigated using simulations with and without T 1 and T 2 relaxation of both the 1 H and 13 C nuclei., Results: Simulations showed that 1 H detection of the hyperpolarized 13 C nucleus in lactate should increase significantly the signal-to-noise ratio when compared with direct 13 C detection at 3T. However the advantage of 1 H detection is expected to disappear at higher fields. Dynamic 1 H images of hyperpolarized [1- 13 C]lactate, with a spatial resolution of 1.25 × 1.25 mm 2 , were acquired from a phantom injected with hyperpolarized [1- 13 C]lactate and from tumors in vivo following injection of hyperpolarized [1- 13 C]pyruvate., Conclusions: The sequence allows 1 H imaging of hyperpolarized 13 C-labeled substrates in vivo. Magn Reson Med 79:741-747, 2018. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited., (© 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine.)

Single-shot echo planar imaging (EPI), which allows an image to be acquired using a single excitation pulse, is used widely for imaging the metabolism of hyperpolarized 13 C-labelled metabolites in vivo as the technique is rapid and minimizes the depletion of the hyperpolarized signal. However, EPI suffers from Nyquist ghosting, which normally is corrected for by acquiring a reference scan. In a dynamic acquisition of a series of images, this results in the sacrifice of a time point if the reference scan involves a full readout train with no phase encoding. This time penalty is negligible if an integrated navigator echo is used, but at the cost of a lower signal-to-noise ratio (SNR) as a result of prolonged T 2 * decay. We describe here a workflow for hyperpolarized 13 C EPI that requires no reference scan. This involves the selection of a ghost-containing background from a 13 C image of a single metabolite at a single time point, the identification of phase correction coefficients that minimize signal in the selected area, and the application of these coefficients to images acquired at all time points and from all metabolites. The workflow was compared in phantom experiments with phase correction using a 13 C reference scan, and yielded similar results in situations with a regular field of view (FOV), a restricted FOV and where there were multiple signal sources. When compared with alternative phase correction methods, the workflow showed an SNR benefit relative to integrated 13 C reference echoes (>15%) or better ghost removal relative to a 1 H reference scan. The residual ghosting in a slightly de-shimmed B 0 field was 1.6% using the proposed workflow and 3.8% using a 1 H reference scan. The workflow was implemented with a series of dynamically acquired hyperpolarized [1- 13 C]pyruvate and [1- 13 C]lactate images in vivo, resulting in images with no observable ghosting and which were quantitatively similar to images corrected using a 13 C reference scan., (© 2017 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)

Purpose: Metabolic imaging with hyperpolarized 13 C-labeled cell substrates is a promising technique for imaging tissue metabolism in vivo. However, the transient nature of the hyperpolarization, and its depletion following excitation, limits the imaging time and the number of excitation pulses that can be used. We describe here a single-shot three-dimensional (3D) imaging sequence and demonstrate its capability to generate 13 C MR images in tumor-bearing mice injected with hyperpolarized [1- 13 C]pyruvate., Methods: The pulse sequence acquires a stack-of-spirals at two spin echoes after a single excitation pulse and encodes the kz-dimension in an interleaved manner to enhance robustness to B 0 inhomogeneity. Spectral-spatial pulses are used to acquire dynamic 3D images from selected hyperpolarized 13 C-labeled metabolites., Results: A nominal spatial/temporal resolution of 1.25 × 1.25 × 2.5 mm 3  × 2 s was achieved in tumor images of hyperpolarized [1- 13 C]pyruvate and [1- 13 C]lactate acquired in vivo. Higher resolution in the z-direction, with a different k-space trajectory, was demonstrated in measurements on a thermally polarized [1- 13 C]lactate phantom., Conclusion: The pulse sequence is capable of imaging hyperpolarized 13 C-labeled substrates at relatively high spatial and temporal resolutions and is robust to moderate system imperfections. Magn Reson Med 77:740-752, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited., (© 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine.)

Purpose: Dissolution dynamic nuclear polarization can increase the sensitivity of the (13) C magnetic resonance spectroscopy experiment by at least four orders of magnitude and offers a novel approach to the development of MRI gene reporters based on enzymes that metabolize (13) C-labeled tracers. We describe here a gene reporter based on the enzyme pyruvate decarboxylase (EC 4.1.1.1), which catalyzes the decarboxylation of pyruvate to produce acetaldehyde and carbon dioxide., Methods: Pyruvate decarboxylase from Zymomonas mobilis (zmPDC) and a mutant that lacked enzyme activity were expressed using an inducible promoter in human embryonic kidney (HEK293T) cells. Enzyme activity was measured in the cells and in xenografts derived from the cells using (13) C MRS measurements of the conversion of hyperpolarized [1-(13) C] pyruvate to H(13) CO3-., Results: Induction of zmPDC expression in the cells and in the xenografts derived from them resulted in an approximately two-fold increase in the H(13) CO3-/[1-(13) C] pyruvate signal ratio following intravenous injection of hyperpolarized [1-(13) C] pyruvate., Conclusion: We have demonstrated the feasibility of using zmPDC as an in vivo reporter gene for use with hyperpolarized (13) C MRS. Magn Reson Med 76:391-401, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited., (© 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine.)

Imaging of the metabolism of hyperpolarized [1-(13) C]pyruvate has shown considerable promise in preclinical studies in oncology, particularly for the assessment of early treatment response. The repeatability of measurements of (13) C label exchange between pyruvate and lactate was determined in a murine lymphoma model in fasted and non-fasted animals. The fasted state showed lower intra-individual variability, although the [1-(13) C]lactate/[1-(13) C]pyruvate signal ratio was significantly greater in fasted than in non-fasted mice, which may be explained by the higher tumor lactate concentrations in fasted animals. These results indicate that the fasted state may be preferable for the measurement of (13) C label exchange between pyruvate and lactate, as it reduces the variability and therefore should make it easier to detect the effects of therapy. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd., (© 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)

The 24th annual symposium of the International Isotope Society's United Kingdom Group took place at the Møller Centre, Churchill College, Cambridge, UK on Friday 6th November 2015. The meeting was attended by 77 delegates from academia and industry, the life sciences, chemical, radiochemical and scientific instrument suppliers. Delegates were welcomed by Dr Ken Lawrie (GlaxoSmithKline, UK, chair of the IIS UK group). The subsequent scientific programme consisted of oral presentations, short 'flash' presentations in association with particular posters and poster presentations. The scientific areas covered included isotopic synthesis, regulatory issues, applications of labelled compounds in imaging, isotopic separation and novel chemistry with potential implications for isotopic synthesis. Both short-lived and long-lived isotopes were represented, as were stable isotopes. The symposium was divided into a morning session chaired by Dr Rebekka Hueting (University of Oxford, UK) and afternoon sessions chaired by Dr Sofia Pascu (University of Bath, UK) and by Dr Alan Dowling (Syngenta, UK). The UK meeting concluded with remarks from Dr Ken Lawrie (GlaxoSmithKline, UK)., (Copyright © 2016 John Wiley & Sons, Ltd.)

Purpose: To assess the potential of an MRI gene reporter based on the ferritin receptor Timd2 (T-cell immunoglobulin and mucin domain containing protein 2), using T1- and T2-weighted imaging., Methods: Pellets of cells that had been modified to express the Timd2 transgene, and incubated with either iron-loaded or manganese-loaded ferritin, were imaged using T1- and T2-weighted MRI. Mice were also implanted subcutaneously with Timd2-expressing cells and the resulting xenograft tissue imaged following intravenous injection of ferritin using T2-weighted imaging., Results: Timd2-expressing cells, but not control cells, showed a large increase in both R2 and R1 in vitro following incubation with iron-loaded and manganese-loaded ferritin, respectively. Expression of Timd2 had no effect on cell viability or proliferation; however, manganese-loaded ferritin, but not iron-loaded ferritin, was toxic to Timd2-expressing cells. Timd2-expressing xenografts in vivo showed much smaller changes in R2 following injection of iron-loaded ferritin than the same cells incubated in vitro with iron-loaded ferritin., Conclusion: Timd2 has demonstrated potential as an MRI reporter gene, producing large increases in R2 and R1 with ferritin and manganese-loaded ferritin respectively in vitro, although more modest changes in R2 in vivo. Manganese-loaded apoferritin was not used in vivo due to the toxicity observed in vitro. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance., (© 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine.)

Dissolution dynamic nuclear polarization (DNP) enables the metabolism of hyperpolarized (13)C-labelled molecules, such as the conversion of [1-(13)C]pyruvate to [1-(13)C]lactate, to be dynamically and non-invasively imaged in tissue. Imaging of this exchange reaction in animal models has been shown to detect early treatment response and correlate with tumour grade. The first human DNP study has recently been completed, and, for widespread clinical translation, simple and reliable methods are necessary to accurately probe the reaction in patients. However, there is currently no consensus on the most appropriate method to quantify this exchange reaction. In this study, an in vitro system was used to compare several kinetic models, as well as simple model-free methods. Experiments were performed using a clinical hyperpolarizer, a human 3 T MR system, and spectroscopic imaging sequences. The quantitative methods were compared in vivo by using subcutaneous breast tumours in rats to examine the effect of pyruvate inflow. The two-way kinetic model was the most accurate method for characterizing the exchange reaction in vitro, and the incorporation of a Heaviside step inflow profile was best able to describe the in vivo data. The lactate time-to-peak and the lactate-to-pyruvate area under the curve ratio were simple model-free approaches that accurately represented the full reaction, with the time-to-peak method performing indistinguishably from the best kinetic model. Finally, extracting data from a single pixel was a robust and reliable surrogate of the whole region of interest. This work has identified appropriate quantitative methods for future work in the analysis of human hyperpolarized (13)C data., (© 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)

Purpose: A resonance at ∼181 ppm in the (13) C spectra of tumors injected with hyperpolarized [U-(2) H, U-(13) C]glucose was assigned to 6-phosphogluconate (6PG), as in previous studies in yeast, whereas in breast cancer cells in vitro this resonance was assigned to 3-phosphoglycerate (3PG). These peak assignments were investigated here using measurements of 6PG and 3PG (13) C-labeling using liquid chromatography tandem mass spectrometry (LC-MS/MS) METHODS: Tumor-bearing mice were injected with (13) C6 glucose and the (13) C-labeled and total 6PG and 3PG concentrations measured. (13) C MR spectra of glucose-6-phosphate dehydrogenase deficient (zwf1Δ) and wild-type yeast were acquired following addition of hyperpolarized [U-(2) H, U-(13) C]glucose and again (13) C-labeled and total 6PG and 3PG were measured by LC-MS/MS RESULTS: Tumor (13) C-6PG was more abundant than (13) C-2PG/3PG and the resonance at ∼181 ppm matched more closely that of 6PG. (13) C MR spectra of wild-type and zwf1Δ yeast cells showed a resonance at ∼181 ppm after labeling with hyperpolarized [U-(2) H, U-(13) C]glucose, however, there was no 6PG in zwf1Δ cells. In the wild-type cells 3PG was approximately four-fold more abundant than 6PG CONCLUSION: The resonance at ∼181 ppm in (13) C MR spectra following injection of hyperpolarized [U-(2) H, U-(13) C]glucose originates predominantly from 6PG in EL4 tumors and 3PG in yeast cells., (© 2014 Wiley Periodicals, Inc.)

Purpose: Aldehyde dehydrogenase (ALDH2) is an emerging drug target for the treatment of heart disease, cocaine and alcohol dependence, and conditions caused by genetic polymorphisms in ALDH2. Noninvasive measurement of ALDH2 activity in vivo could inform the development of these drugs and accelerate their translation to the clinic., Methods: [1-(13) C, U-(2) H5 ] ethanol was hyperpolarized using dynamic nuclear polarization, injected into mice and its oxidation in the liver monitored using (13) C MR spectroscopy and spectroscopic imaging., Results: Oxidation of [1-(13) C, U-(2) H5 ] ethanol to [1-(13) C] acetate was observed. Saturation of the acetaldehyde resonance, which was below the level of detection in vivo, demonstrated that acetate was produced via acetaldehyde. Irreversible inhibition of ALDH2 activity with disulfiram resulted in a proportional decrease in the amplitude of the acetate resonance., Conclusion: (13) C magnetic resonance spectroscopy measurements of hyperpolarized [1-(13) C, U-(2) H5 ] ethanol oxidation allow real-time assessment of ALDH2 activity in liver in vivo., (© 2014 The Authors. Magnetic Resonance in Medicine Published by Wiley Periodicals, Inc. on behalf of International Society of Medicine in Resonance.)

Purpose: To assess the potential of a gene reporter system, based on a urea transporter (UTB) and hyperpolarized [(13) C]urea., Methods: Mice were implanted subcutaneously with either unmodified control cells or otherwise identical cells expressing UTB. After injection of hyperpolarized [(13) C]urea, a spin echo sequence was used to measure urea concentration, T1 , and diffusion in control and UTB-expressing tissue., Results: The apparent diffusion coefficient of hyperpolarized urea was 21% lower in tissue expressing UTB, in comparison with control tissue (P < 0.05, 1-tailed t-test, n = 6 in each group). No difference in water apparent diffusion coefficient or cellularity between these tissues was found, indicating that they were otherwise similar in composition., Conclusion: Expression of UTB, by mediating cell uptake of urea, lowers the apparent diffusion coefficient of hyperpolarized (13) C urea in tissue and thus the transporter has the potential to be used as a magnetic resonance-based gene reporter in vivo. Magn Reson Med 73:1401-1406, 2015. © 2014 Wiley Periodicals, Inc., (© 2014 Wiley Periodicals, Inc.)

Purpose: The acquisition of ever increasing volumes of high resolution magnetic resonance imaging (MRI) data has created an urgent need to develop automated and objective image analysis algorithms that can assist in determining tumor margins, diagnosing tumor stage, and detecting treatment response., Methods: We have shown previously that Minkowski functionals, which are precise morphological and structural descriptors of image heterogeneity, can be used to enhance the detection, in T1 -weighted images, of a targeted Gd(3+) -chelate-based contrast agent for detecting tumor cell death. We have used Minkowski functionals here to characterize heterogeneity in T2 -weighted images acquired before and after drug treatment, and obtained without contrast agent administration., Results: We show that Minkowski functionals can be used to characterize the changes in image heterogeneity that accompany treatment of tumors with a vascular disrupting agent, combretastatin A4-phosphate, and with a cytotoxic drug, etoposide., Conclusions: Parameterizing changes in the heterogeneity of T2 -weighted images can be used to detect early responses of tumors to drug treatment, even when there is no change in tumor size. The approach provides a quantitative and therefore objective assessment of treatment response that could be used with other types of MR image and also with other imaging modalities., (Copyright © 2013 Wiley Periodicals, Inc.)

Hyperpolarized NMR is a promising technique for non-invasive imaging of tissue metabolism in vivo. However, the pathways that can be studied are limited by the fast T1 decay of the nuclear spin order. In metabolites containing pairs of coupled nuclear spins-1/2, the spin order may be maintained by exploiting the non-magnetic singlet (spin-0) state of the pair. This may allow preservation of the hyperpolarization in vivo during transport to tissues of interest, such as tumors, or to detect slower metabolic reactions. We show here that in human blood and in a mouse in vivo at millitesla fields the (13)C singlet lifetime of [1,2-(13)C2]pyruvate was significantly longer than the (13)C T1, although it was shorter than the T1 at field strengths of several tesla. We also examine the singlet-derived NMR spectrum observed for hyperpolarized [1,2-(13)C2]lactate, originating from the metabolism of [1,2-(13)C2]pyruvate., (© 2013 The Authors. NMR in Biomedicine published by John Wiley & Sons, Ltd.)

Purpose: To assess the blood-tissue distribution of hyperpolarized (13) C-labeled molecules in vivo., Methods: Spin-echo experiments with simultaneous acquisition of the free induction decay (FID) signal following the excitation pulse and the spin-echo signal, were used to monitor hyperpolarized [1-(13) C]lactate, [1-(13) C]pyruvate, and the perfusion marker, [(13) C]HP001, following their intravenous injection into tumor-bearing mice. Apparent T2 relaxation times and diffusion coefficients were also measured., Results: An increasing tumor echo/FID ratio was observed for all three molecules, which could be explained by their extravasation into the tumor interstitial space, where T2 relaxation times were longer and diffusion coefficients smaller. Inhibition of the monocarboxylate transporter, which decreased by 40% the label exchange between pyruvate and lactate, reduced the increase in the echo/FID ratio for pyruvate and lactate, but not for HP001, demonstrating that some of the increase in the echo/FID ratio was due to cell uptake of lactate and pyruvate. The different relaxation and diffusion behavior of the intravascular and extravascular signals affected measurements of the apparent label exchange rate constants., Conclusion: Simultaneous collection of both FID and echo signals can provide information on cell uptake thus giving further insight into the kinetics of hyperpolarized (13) C label exchange. Care is needed when comparing exchange rate constants determined in spin-echo-based studies., (Copyright © 2012 Wiley Periodicals, Inc.)

pH is a fundamental physiological parameter that is tightly controlled by endogenous buffers. The acid-base balance is altered in many disease states, such as inflammation, ischemia and cancer. Despite the importance of pH, there are currently no routine methods for imaging the spatial distribution of pH in humans. The enormous gain in sensitivity afforded by dynamic nuclear polarization (DNP) has provided a novel way in which to image tissue pH using MR, which has the potential to be translated into the clinic. This review explores the advantages and disadvantages of current pH imaging techniques and how they compare with DNP-based approaches for the measurement and imaging of pH with hyperpolarized (13)C. Intravenous injection of hyperpolarized (13)C-labeled bicarbonate results in the rapid production of hyperpolarized (13)CO(2) in the reaction catalyzed by carbonic anhydrase. As this reaction is close to equilibrium in the body and is pH dependent, the ratio of the (13)C signal intensities from H(13)CO(3)(-) and (13)CO(2), measured using MRS, can be used to calculate pH in vivo. The application of this technique to a murine tumor model demonstrated that it measured predominantly extracellular pH and could be mapped in the animal using spectroscopic imaging techniques. A second approach has been to use the production of hyperpolarized (13)CO(2) from hyperpolarized [1-(13)C]pyruvate to measure predominantly intracellular pH. In tissues with a high aerobic capacity, such as the heart, the hyperpolarized [1-(13)C]pyruvate undergoes rapid oxidative decarboxylation, catalyzed by intramitochondrial pyruvate dehydrogenase. Provided that there is sufficient carbonic anhydrase present to catalyze the rapid equilibration of the hyperpolarized (13)C label between CO(2) and bicarbonate, the ratio of their resonance intensities may again be used to estimate pH, which, in this case, is predominantly intracellular. As both pyruvate and bicarbonate are endogenous molecules they have the potential to image tissue pH in the clinic., (Copyright © 2011 John Wiley & Sons, Ltd.)

Dynamic nuclear polarization is an emerging technique for increasing the sensitivity of magnetic resonance imaging and spectroscopy, particularly for low-γ nuclei. The technique has been applied recently to a number of 13C-labeled cell metabolites in biological systems: the increase in signal-to-noise allows the spatial distribution of an injected molecule to be imaged as well as its metabolic product or products. This review highlights the most significant molecules investigated to date in preclinical cancer models, either in terms of their demonstrated metabolism in vivo or the biological processes that they can probe. In particular, label exchange between hyperpolarized 13C-labeled pyruvate and lactate, catalyzed by lactate dehydrogenase, has been shown to have a number of potential applications. Finally, techniques to image these molecules are also discussed as well as methods that may extend the lifetime of the hyperpolarized signal. Hyperpolarized magnetic resonance imaging and magnetic resonance spectroscopic imaging have shown great promise for the imaging of cancer in preclinical work, both for diagnosis and for monitoring therapy response. If the challenges in translating this technique to human imaging can be overcome, then it has the potential to significantly alter the management of cancer patients., (Copyright © 2011 Wiley-Liss, Inc.)

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., (Copyright © 2011 Wiley-Liss, Inc.)

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., (Copyright © 2010 Wiley-Liss, Inc.)

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.

A targeted Gd(3+)-based contrast agent has been developed that detects tumor cell death by binding to the phosphatidylserine (PS) exposed on the plasma membrane of dying cells. Although this agent has been used to detect tumor cell death in vivo, the differences in signal intensity between treated and untreated tumors was relatively small. As cell death is often spatially heterogeneous within tumors, we investigated whether an image analysis technique that parameterizes heterogeneity could be used to increase the sensitivity of detection of this targeted contrast agent. Two-dimensional (2D) Minkowski functionals (MFs) provided an automated and reliable method for parameterization of image heterogeneity, which does not require prior assumptions about the number of regions or features in the image, and were shown to increase the sensitivity of detection of the contrast agent as compared to simple signal intensity analysis., ((c) 2009 Wiley-Liss, Inc.)

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., ((c) 2008 Wiley-Liss, Inc.)

Activated T cells, isolated from animals that had rejected a tumor (E.G7-OVA) expressing chicken ovalbumin, were labeled with citrated superparamagnetic iron oxide nanoparticles at an intracellular iron concentration of up to 0.5 pg/cell. Injection of these labeled T cells into animals bearing E.G7-OVA tumors undergoing immune rejection resulted in tumor infiltration of these cells, which was detectable as a heterogeneous decrease in intensity in T(2)-weighted MR images. T-cell infiltration was confirmed by immunohistochemical staining of tumor sections obtained postmortem and was shown to colocalize with iron that had been stained using Prussian blue. Tumor rejection was correlated with the uptake of labeled T cells, since the infiltration of labeled T cells was only observed in those tumors that went on to regress. This technique should assist in the elucidation of those factors that are important in mediating tumor immune rejection.

Proton MRS detection of cellular lipid accumulation has been suggested as a noninvasive method for detecting apoptosis or programmed cell death (PCD) in vivo. The spectral changes that have been observed in apoptotic cells include a general increase in lipid signals and a specific increase in the ratio of the lipid methylene-to-methyl peak intensities. These changes were investigated here following drug-induced apoptosis, both in vitro with a murine lymphoma cell line (EL-4) and in vivo following implantation of these cells to form subcutaneous tumors. Fluorescence microscopy and flow cytometric measurements with a lipophilic dye revealed an accumulation of cytoplasmic lipid droplets in isolated EL-4 cells undergoing etoposide-induced apoptosis. (1)H MR spectra (both diffusion-weighted (DW) and unweighted) showed an increase in lipid signals. However, the methylene/methyl peak ratio showed only minimal changes. Localized in vivo spectroscopy of EL-4 tumors also showed an increase in lipid signals, including a signal from polyunsaturated lipid at 2.8 ppm, after 16-24 h of drug treatment. Again there was no significant change in the methylene/methyl peak ratio. This study confirms that MRS-detectable lipids accumulate in tumor cells undergoing apoptosis, and therefore may be usable as a marker for the noninvasive detection of tumor cell apoptosis in the clinic.

The effects of two anti-vascular agents, combretastatin A4 phosphate (CA4P), and 5,6-dimethylxanthenone-4-acetic acid (DMXAA), on the perfusion of two human colon adenocarcinomas implanted in SCID mice, were assessed for up to 3 h using non-invasive magnetic resonance imaging (MRI) and spectroscopy techniques (MRS). MRI measurements of GdDTPA inflow showed that treatment with CA4P had little effect on the perfusion of HT29 tumours. Localized (31)P MRS measurements also showed that the drug had no significant effect on tumour cell energy status, as assessed from the ratio of the integrals of the signals from inorganic phosphate (P(i)) and nucleoside triphosphates. However, after treatment with DMXAA, perfusion was reduced and the P(i)/NTP ratio increased, indicating that the HT29 tumour is susceptible to the action of this drug. The LS174T tumour model was susceptible to both CA4P and DMXAA, using the criteria of changes in GdDTPA inflow and P(i)/NTP ratio., (Copyright 2002 John Wiley & Sons, Ltd.)

Dynamic magnetic resonance imaging was used to monitor solute diffusion through aggregates of Chinese hamster ovary cells growing on macroporous carriers in a fixed-bed bioreactor. Diffusion-weighted (1)H magnetic resonance imaging (MRI) and scanning electron microscopy demonstrated that cell growth in the bioreactor was heterogeneous, with the highest cell densities being found at the periphery of the carriers. T(1)-weighted magnetic resonance imaging measurements of the inflow of a commonly used magnetic resonance contrast agent, gadolinium-diethylenetriaminopentaacetic acid (Gd-DTPA), showed that migration of the agent through the peripheral cell masses could be explained by diffusion. However, appearance of the contrast agent in the center of the carriers was too fast to be explained by simple diffusion and indicated that these regions were perfused by convective flow. The average diffusivity of Gd-DTPA through the cell mass was found to be (2.4 +/- 0.2) x 10(-10) m(2) sec(-) (mean +/- SEM). This technique will be useful in the characterization and development of high-cell-density bioreactor systems, in which solute transport plays a critical role in cell growth and physiology., (Copyright 2001 John Wiley & Sons, Inc.)

Programmed cell death or apoptosis was induced in human promyelocytic leukemia (HL-60) and Chinese hamster ovary (CHO-K1) cells using several cytotoxic drugs that have different modes of action, including camptothecin, ceramide, chelerythrine, etoposide, farnesol, geranyl geraniol, and hexadecylphosphocholine. The consequent changes in cellular metabolism were monitored using 31P MRS measurements on intact cells and cell extracts. Cells undergoing programmed cell death exhibited characteristic changes in the levels of glycolytic and phospholipid metabolites. The most significant changes were increases in the concentration of the glycolytic intermediate, fructose-1,6-bisphosphate and in the concentration of CDP-choline, which is an intermediate in phosphatidylcholine biosynthesis. In HL-60 cells, the increase in fructose-1,6-bisphosphate levels could be explained by depletion of cellular NAD(H) levels. All of the agents used to induce apoptosis caused the accumulation of CDP-choline. Since the resonances of this compound occur in a relatively well resolved region of tissue spectra, it could provide a marker for apoptosis that would allow the noninvasive detection of the process in vivo using 31P MRS measurements.

We mapped the distribution of dissolved oxygen and mammalian cells in a hollow-fiber bioreactor (HFBR) using (19)F NMR T(1) relaxation time imaging measurements on an infused perfluorocarbon probe molecule and diffusion-weighted (1)H NMR imaging of water. This study shows how cell density influences dissolved oxygen concentration in the reactor and demonstrates that NMR can play an important role in defining the biochemical engineering parameters required for optimization of HFBR design and operation.

31P NMR measurements on extracts prepared from a variety of cultured mammalian cell lines and primary rat hepatocytes have shown changes in the levels of several phospholipid metabolites after incubation of cells with unsaturated fatty acids. These data suggest a possible link between the accumulation of neutral lipid and the changes in phospholipid metabolite concentrations that have been observed in some tumor cells and other rapidly growing tissues such as the regenerating liver and mitogen-stimulated lymphocytes.

The appearance of high resolution neutral lipid signals in the 1H NMR spectra of myeloma cells grown in the presence of oleate was shown to correlate with the appearance of cytoplasmic lipid droplets observable by electron microscopy. The spin-spin relaxation times of these lipid signals were similar to those measured previously for lipid resonances in other cell types. These data suggest that cytoplasmic lipid droplets could make a significant contribution to the neutral lipid signals observed in the 1H NMR spectra of some cells.

The magnetic field-dependent phosphodiester (PDE) signal found in 31P NMR spectra of liver and brain has been studied using saturation transfer and proton decoupling techniques. This PDE component, which accounts for as much as 45% of the signal in vivo, has been identified as primarily phospholipid bilayer with a small contribution from a motionally averaged macromolecule(s).