Temporal and regional expression changes and co-staining patterns of metabolic and stemness-related markers during glioblastoma progression.

Glioblastomas (GBMs) are characterized by high heterogeneity, involving diverse cell types, including those with stem-like features contributing to GBM's malignancy. Moreover, metabolic alterations promote growth and therapeutic resistance of GBM. Depending on the metabolic state, antimetabolic treatments could be an effective strategy. Against this background, we investigated temporal and regional expression changes and co-staining patterns of selected metabolic markers [pyruvate kinase muscle isozyme 1/2 (PKM1/2), glucose transporter 1 (GLUT1), monocarboxylate transporter 1/4 (MCT1/4)] in a rodent model and patient-derived samples of GBM. To understand the cellular sources of marker expression, we also examined the connection of metabolic markers to markers related to stemness [Nestin, Krüppel-like factor 4 (KLF4)] in a regional and temporal context. Rat tumour biopsies revealed a temporally increasing expression of GLUT1, higher expression of MCT1/4, Nestin and KLF4, and lower expression of PKM1 compared to the contralateral hemisphere. Patient-derived tumours showed a higher expression of PKM2 and Nestin in the tumour centre vs. edge. Whereas rare co-staining of GLUT1/Nestin was found in tumour biopsies, PKM1/2 and MCT1/4 showed a more distinct co-staining with Nestin in rats and humans. KLF4 was mainly co-stained with GLUT1, MCT1 and PKM1/2 in rat and human tumours. All metabolic markers yielded individual co-staining patterns among themselves. Co-staining mainly occurred later in tumour progression and was more pronounced in tumour centres. Also, positive correlations were found amongst markers that showed co-staining. Our results highlight a link between metabolic alterations and stemness in GBM progression, with complex distinctions depending on studied markers, time points and regions.

Modeling Ligand Exchange Kinetics in Iridium Complexes Catalyzing SABRE Nuclear Spin Hyperpolarization.

Large signal enhancements can be obtained for NMR analytes using the process of nuclear spin hyperpolarization. Organometallic complexes that bind parahydrogen can themselves become hyperpolarized. Moreover, if parahydrogen and a to-be-hyperpolarized analyte undergo chemical exchange with the organometallic complex it is possible to catalytically sensitize the detection of the analyte via hyperpolarization transfer through spin-spin coupling in this organometallic complex. This process is called Signal Amplification By Reversible Exchange (SABRE). Signal intensity gains of several orders of magnitude can thus be created for various compounds in seconds. The chemical exchange processes play a defining role in controlling the efficiency of SABRE because the lifetime of the complex must match the spin-spin couplings. Here, we show how analyte dissociation rates in the key model substrates pyridine (the simplest six-membered heterocycle), 4-aminopyridine (a drug), and nicotinamide (an essential vitamin biomolecule) can be examined. This is achieved for the most widely employed SABRE motif that is based on IrIMes-derived catalysts by 1H 1D and 2D exchange NMR spectroscopy techniques. Several kinetic models are evaluated for their accuracy and simplicity. By incorporating variable temperature analysis, the data yields key enthalpies and entropies of activation that are critical for understanding the underlying SABRE catalyst properties and subsequently optimizing behavior through rational chemical design. While several studies of chemical exchange in SABRE have been reported, this work also aims to establish a toolkit on how to quantify chemical exchange in SABRE and ensure that data can be compared reliably.

Current methods for hyperpolarized [1-13C]pyruvate MRI human studies.

MRI with hyperpolarized (HP) 13C agents, also known as HP 13C MRI, can measure processes such as localized metabolism that is altered in numerous cancers, liver, heart, kidney diseases, and more. It has been translated into human studies during the past 10 years, with recent rapid growth in studies largely based on increasing availability of HP agent preparation methods suitable for use in humans. This paper aims to capture the current successful practices for HP MRI human studies with [1-13C]pyruvate-by far the most commonly used agent, which sits at a key metabolic junction in glycolysis. The paper is divided into four major topic areas: (1) HP 13C-pyruvate preparation; (2) MRI system setup and calibrations; (3) data acquisition and image reconstruction; and (4) data analysis and quantification. In each area, we identified the key components for a successful study, summarized both published studies and current practices, and discuss evidence gaps, strengths, and limitations. This paper is the output of the "HP 13C MRI Consensus Group" as well as the ISMRM Hyperpolarized Media MR and Hyperpolarized Methods and Equipment study groups. It further aims to provide a comprehensive reference for future consensus, building as the field continues to advance human studies with this metabolic imaging modality.

Modern Manufacturing Enables Magnetic Field Cycling Experiments and Parahydrogen-Induced Hyperpolarization with a Benchtop NMR.

Benchtop NMR (btNMR) spectrometers are revolutionizing the way we use NMR and lowering the cost drastically. Magnetic field cycling (MFC) experiments with precise timing and control over the magnetic field, however, were hitherto not available on btNMRs, although some systems exist for high-field, high-resolution NMR spectrometers. Still, the need and potential for btNMR MFC is great─e.g., to perform and analyze parahydrogen-induced hyperpolarization, another method that has affected analytical chemistry and NMR beyond expectations. Here, we describe a setup that enables MFC on btNMRs for chemical analysis and hyperpolarization. Taking full advantage of the power of modern manufacturing, including computer-aided design, three-dimensional printing, and microcontrollers, the setup is easy to reproduce, highly reliable, and easy to adjust and operate. Within 380 ms, the NMR tube was shuttled reliably from the electromagnet to the NMR isocenter (using a stepper motor and gear rod). We demonstrated the power of this setup by hyperpolarizing nicotinamide using signal amplification by reversible exchange (SABRE), a versatile method to hyperpolarize a broad variety of molecules including metabolites and drugs. Here, the standard deviation of SABRE hyperpolarization was between 0.2 and 3.3%. The setup also allowed us to investigate the field dependency of the polarization and the effect of different sample preparation protocols. We found that redissolution of the activated and dried Ir catalyst always reduced the polarization. We anticipate that this design will greatly accelerate the ascension of MFC experiments for chemical analysis with btNMR─adding yet another application to this rapidly developing field.

Quantification of Prostate Cancer Metabolism Using 3D Multiecho bSSFP and Hyperpolarized [1-13 C] Pyruvate: Metabolism Differs Between Tumors of the Same Gleason Grade.

BACKGROUND: Three-dimensional (3D) multiecho balanced steady-state free precession (ME-bSSFP) has previously been demonstrated in preclinical hyperpolarized (HP) 13 C-MRI in vivo experiments, and it may be suitable for clinical metabolic imaging of prostate cancer (PCa). PURPOSE: To validate a signal simulation framework for the use of sequence parameter optimization. To demonstrate the feasibility of ME-bSSFP for HP 13 C-MRI in patients. To evaluate the metabolism in PCa measured by ME-bSSFP. STUDY TYPE: Retrospective single-center cohort study. PHANTOMS/POPULATION: Phantoms containing aqueous solutions of [1-13 C] lactate (2.3 M) and [13 C] urea (8 M). Eight patients (mean age 67 ± 6 years) with biopsy-confirmed Gleason 3 + 4 (n = 7) and 4 + 3 (n = 1) PCa. FIELD STRENGTH/SEQUENCES: 1 H MRI at 3 T with T2 -weighted turbo spin-echo sequence used for spatial localization and spoiled dual gradient-echo sequence used for B0 -field measurement. ME-bSSFP sequence for 13 C MR spectroscopic imaging with retrospective multipoint IDEAL metabolite separation. ASSESSMENT: The primary endpoint was the analysis of pyruvate-to-lactate conversion in PCa and healthy prostate regions of interest (ROIs) using model-free area under the curve (AUC) ratios and a one-directional kinetic model (kP ). The secondary objectives were to investigate the correlation between simulated and experimental ME-bSSFP metabolite signals for HP 13 C-MRI parameter optimization. STATISTICAL TESTS: Pearson correlation coefficients with 95% confidence intervals and paired t-tests. The level of statistical significance was set at P < 0.05. RESULTS: Strong correlations between simulated and empirical ME-bSSFP signals were found (r > 0.96). Therefore, the simulation framework was used for sequence optimization. Whole prostate metabolic HP 13 C-MRI, observing the conversion of pyruvate into lactate, with a temporal resolution of 6 seconds was demonstrated using ME-bSSFP. Both assessed metrics resulted in significant differences between PCa (mean ± SD) (AUC = 0.33 ± 012, kP  = 0.038 ± 0.014) and healthy (AUC = 0.15 ± 0.10, kP  = 0.011 ± 0.007) ROIs. DATA CONCLUSION: Metabolic HP 13 C-MRI in the prostate using ME-bSSFP allows for differentiation between aggressive PCa and healthy tissue. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 1.

Exceptionally Mild and High-Yielding Synthesis of Vinyl Esters of Alpha-Ketocarboxylic Acids, Including Vinyl Pyruvate, for Parahydrogen-Enhanced Metabolic Spectroscopy and Imaging.

Metabolic changes often occur long before pathologies manifest and treatment becomes challenging. As key elements of energy metabolism, α-ketocarboxylic acids (α-KCA) are particularly interesting, e.g., as the upregulation of pyruvate to lactate conversion is a hallmark of cancer (Warburg effect). Magnetic resonance imaging with hyperpolarized metabolites has enabled imaging of this effect non-invasively and in vivo, allowing the early detection of cancerous tissue and its treatment. Hyperpolarization by means of dynamic nuclear polarization, however, is complex, slow, and expensive, while available precursors often limit parahydrogen-based alternatives. Here, we report the synthesis for novel 13C, deuterated ketocarboxylic acids, and a much-improved synthesis of 1-13C-vinyl pruvate-d6, arguably the most promising tracer for hyperpolarizing pyruvate using parahydrogen-induced hyperpolarization by side arm hydrogenation. The new synthesis is scalable and provides a high yield of 52%. We elucidated the mechanism of our Pd-catalyzed trans-vinylation reaction. Hydrogenation with parahydrogen allowed us to monitor the addition, which was found to depend on the electron demand of the vinyl ester. Electron-poor α-keto vinyl esters react slower than "normal" alkyl vinyl esters. This synthesis of 13C, deuterated α-ketocarboxylic acids opens up an entirely new class of biomolecules for fast and cost-efficient hyperpolarization with parahydrogen and their use for metabolic imaging.

In Vivo Metabolic Imaging of [1-13 C]Pyruvate-d3 Hyperpolarized By Reversible Exchange With Parahydrogen.

Metabolic magnetic resonance imaging (MRI) using hyperpolarized (HP) pyruvate is becoming a non-invasive technique for diagnosing, staging, and monitoring response to treatment in cancer and other diseases. The clinically established method for producing HP pyruvate, dissolution dynamic nuclear polarization, however, is rather complex and slow. Signal Amplification By Reversible Exchange (SABRE) is an ultra-fast and low-cost method based on fast chemical exchange. Here, for the first time, we demonstrate not only in vivo utility, but also metabolic MRI with SABRE. We present a novel routine to produce aqueous HP [1-13 C]pyruvate-d3 for injection in 6 minutes. The injected solution was sterile, non-toxic, pH neutral and contained ≈30 mM [1-13 C]pyruvate-d3 polarized to ≈11 % (residual 250 mM methanol and 20 µM catalyst). It was obtained by rapid solvent evaporation and metal filtering, which we detail in this manuscript. This achievement makes HP pyruvate MRI available to a wide biomedical community for fast metabolic imaging of living organisms.

Through-Space Multinuclear Magnetic Resonance Signal Enhancement Induced by Parahydrogen and Radiofrequency Amplification by Stimulated Emission of Radiation.

Hyperpolarized (i.e., polarized far beyond the thermal equilibrium) nuclear spins can result in the radiofrequency amplification by stimulated emission of radiation (RASER) effect. Here, we show the utility of RASER to amplify nuclear magnetic resonance (NMR) signals of solute and solvent molecules in the liquid state. Specifically, parahydrogen-induced RASER was used to spontaneously enhance nuclear spin polarization of protons and heteronuclei (here 19F and 31P) in a wide range of molecules. The magnitude of the effect correlates with the T1 relaxation time of the target nuclear spins. A series of control experiments validate the through-space dipolar mechanism of the RASER-assisted polarization transfer between the parahydrogen-polarized compound and to-be-hyperpolarized nuclei of the target molecule. Frequency-selective saturation of the RASER-active resonances was used to control the RASER and the amplitude of spontaneous polarization transfer. Spin dynamics simulations support our experimental RASER studies. The enhanced NMR sensitivity may benefit various NMR applications such as mixture analysis, metabolomics, and structure determination.

Synthesis of 13 C and 2 H Labeled Vinyl Pyruvate and Hyperpolarization of Pyruvate.

The hyperpolarization of nuclear spins has enabled unique applications in chemistry, biophysics, and particularly metabolic imaging. Parahydrogen-induced polarization (PHIP) offers a fast and cost-efficient way of hyperpolarization. Nevertheless, PHIP lags behind dynamic nuclear polarization (DNP), which is already being evaluated in clinical studies. This shortcoming is mainly due to problems in the synthesis of the corresponding PHIP precursor molecules. The most widely used DNP tracer in clinical studies, particularly for the detection of prostate cancer, is 1-13 C-pyruvate. The ideal derivative for PHIP is the deuterated vinyl ester because the spin physics allows for 100 % polarization. Unfortunately, there is no efficient synthesis for vinyl esters of ß-ketocarboxylic acids in general and pyruvate in particular. Here, we present an efficient new method for the preparation of vinyl esters, including 13 C labeled, fully deuterated vinyl pyruvate using a palladium-catalyzed procedure. Using 50 % enriched parahydrogen and mild reaction conditions, a 13 C polarization of 12 % was readily achieved; 36 % are expected with 100 % pH2 . Higher polarization values can be potentially achieved with optimized reaction conditions.

Frequency-Selective Manipulations of Spins allow Effective and Robust Transfer of Spin Order from Parahydrogen to Heteronuclei in Weakly-Coupled Spin Systems.

We present a selectively pulsed (SP) generation of sequences to transfer the spin order of parahydrogen (pH2 ) to heteronuclei in weakly coupled spin systems. We analyze and discuss the mechanism and efficiency of SP spin order transfer (SOT) and derive sequence parameters. These new sequences are most promising for the hyperpolarization of molecules at high magnetic fields. SP-SOT is effective and robust despite the symmetry of the 1 H-13 C J-couplings even when precursor molecules are not completely labeled with deuterium. As only one broadband 1 H pulse is needed per sequence, which can be replaced for instance by a frequency-modulated pulse, lower radiofrequency (RF) power is required. This development will be useful to hyperpolarize (new) agents and to perform the hyperpolarization within the bore of an MRI system, where the limited RF power has been a persistent problem.

Parahydrogen-Induced Polarization Relayed via Proton Exchange.

The hyperpolarization of nuclear spins is a game-changing technology that enables hitherto inaccessible applications for magnetic resonance in chemistry and biomedicine. Despite significant advances and discoveries in the past, however, the quest to establish efficient and effective hyperpolarization methods continues. Here, we describe a new method that combines the advantages of direct parahydrogenation, high polarization (P), fast reaction, and low cost with the broad applicability of polarization transfer via proton exchange. We identified the system propargyl alcohol + pH2 → allyl alcohol to yield 1H polarization in excess of P ≈ 13% by using only 50% enriched pH2 at a pressure of ≈1 bar. The polarization was then successfully relayed via proton exchange from allyl alcohol to various target molecules. The polarizations of water and alcohols (as target molecules) approached P ≈ 1% even at high molar concentrations of 100 mM. Lactate, glucose, and pyruvic acid were also polarized, but to a lesser extent. Several potential improvements of the methodology are discussed. Thus, the parahydrogen-induced hyperpolarization relayed via proton exchange (PHIP-X) is a promising approach to polarize numerous molecules which participate in proton exchange and support new applications for magnetic resonance.

Pseudo-Enhancement in Intracranial Aneurysms on Black-Blood MRI: Effects of Flow Rate, Spatial Resolution, and Additional Flow Suppression.

BACKGROUND: Vessel-wall enhancement (VWE) on black-blood MRI (BB MRI) has been proposed as an imaging marker for a higher risk of rupture and associated with wall inflammation. Whether VWE is causally linked to inflammation or rather induced by flow phenomena has been a subject of debate. PURPOSE: To study the effects of slow flow, spatial resolution, and motion-sensitized driven equilibrium (MSDE) preparation on signal intensities in BB MRI of patient-specific aneurysm flow models. STUDY TYPE: Prospective. SUBJECTS/FLOW ANEURYSM MODEL/VIRTUAL VESSELS: Aneurysm flow models based on 3D rotational angiography datasets of three patients with intracranial aneurysms were 3D printed and perfused at two different flow rates, with and without Gd-containing contrast agent. FIELD STRENGTH/SEQUENCE: Variable refocusing flip angle 3D fast-spin echo sequence at 3 T with and without MSDE with three voxel sizes ((0.5 mm)3 , (0.7 mm)3 , and (0.9 mm)3 ); time-resolved with phase-contrast velocity-encoding 3D spoiled gradient echo sequence (4D flow MRI). ASSESSMENT: Three independent observers performed a qualitative visual assessment of flow patterns and signal enhancement. Quantitative analysis included voxel-wise evaluation of signal intensities and magnitude velocity distributions in the aneurysm. STATISTICAL TESTS: Kruskal-Wallis test, potential regressions. RESULTS: A hyperintense signal in the lumen and adjacent to the aneurysm walls on BB MRI was colocalized with slow flow. Signal intensities increased by a factor of 2.56 ± 0.68 (P < 0.01) after administering Gd contrast. After Gd contrast administration, the signal was suppressed most in conjunction with high flows and with MSDE (2.41 ± 2.07 for slow flow without MSDE, and 0.87 ± 0.99 for high flow with MSDE). A clear result was not achieved by modifying the spatial resolution . DATA CONCLUSIONS: Slow-flow phenomena contribute substantially to aneurysm enhancement and vary with MRI parameters. This should be considered in the clinical setting when assessing VWE in patients with an unruptured aneurysm. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 2.

Catalytic Hydrogenation of Trivinyl Orthoacetate: Mechanisms Elucidated by Parahydrogen Induced Polarization.

Parahydrogen (pH2 ) induced polarization (PHIP) is a unique method that is used in analytical chemistry to elucidate catalytic hydrogenation pathways and to increase the signal of small metabolites in MRI and NMR. PHIP is based on adding or exchanging at least one pH2 molecule with a target molecule. Thus, the spin order available for hyperpolarization is often limited to that of one pH2 molecule. To break this limit, we investigated the addition of multiple pH2 molecules to one precursor. We studied the feasibility of the simultaneous hydrogenation of three arms of trivinyl orthoacetate (TVOA) intending to obtain hyperpolarized acetate. It was found that semihydrogenated TVOA underwent a fast decomposition accompanied by several minor reactions including an exchange of geminal methylene protons of a vinyl ester with pH2 . The study shows that multiple vinyl ester groups are not suitable for a fast and clean (without any side products) hydrogenation and hyperpolarization that is desired in biochemical applications.

Coherent Evolution of Signal Amplification by Reversible Exchange in Two Alternating Fields (alt-SABRE).

Parahydrogen (pH2 ) is a convenient and cost-efficient source of spin order to enhance the magnetic resonance signal. Previous work showed that transient interaction of pH2 with a metal organic complex in a signal amplification by reversible exchange (SABRE) experiment enabled more than 10 % polarization for some 15 N molecules. Here, we analyzed a variant of SABRE, consisting of a magnetic field alternating between a low field of ∼1 µT, where polarization transfer is expected to take place, and a higher field >50 µT (alt-SABRE). These magnetic fields affected the amplitude and frequency of polarization transfer. Deviation of a lower magnetic field from a "perfect" condition of level anti-crossing increases the frequency of polarization transfer that can be exploited for polarization of short-lived transient SABRE complexes. Moreover, the coherences responsible for polarization transfer at a lower field persisted during magnetic field variation and continued their spin evolution at higher field with a frequency of 2.5 kHz at 54 µT. The latter should be taken into consideration for an efficient alt-SABRE. Theoretical and experimental findings were exemplified with Iridium N-heterocyclic carbene SABRE complex and 15 N-acetonitrole, where a 30 % higher 15 N polarization with alt-SABRE compared to common SABRE was reached.

Selective excitation doubles the transfer of parahydrogen-induced polarization to heteronuclei.

In this work, we present a new pulse sequence to transform the spin order added to a molecule after the pairwise addition of parahydrogen into 13C polarization. Using a selective 90° preparation instead of a non-selective 45° excitation, the new variant performed twice as well as previous implementations in both simulations and experiments, exemplified with hyperpolarized ethyl acetate. This concept is expected to extend to other nuclei and other spin order transfer schemes that use non-selective excitation.

High field parahydrogen induced polarization of succinate and phospholactate.

The signal enhancement provided by the hyperpolarization of nuclear spins of metabolites is a promising technique for diagnostic magnetic resonance imaging (MRI). To date, most 13C-contrast agents are hyperpolarized utilizing a complex or cost-intensive polarizer. Recently, the in situ parahydrogen-induced 13C hyperpolarization was demonstrated. Hydrogenation, spin order transfer (SOT) by a pulsed NMR sequence, in vivo administration, and detection was achieved within the magnet bore of a 7 Tesla MRI system. So far, the hyperpolarization of the xenobiotic molecule 1-13C-hydroxyethylpropionate (HEP) and the biomolecule 1-13C-succinate (SUC) through the PH-INEPT+ sequence and a SOT scheme proposed by Goldman et al., respectively, was shown. Here, we investigate further the hyperpolarization of SUC at 7 Tesla and study the performance of two additional SOT sequences. Moreover, we present first results of the hyperpolarization at high magnetic field of 1-13C-phospholactate (PLAC), a derivate to obtain the metabolite lactate, employing the PH-INEPT+ sequence. For SUC and PLAC, 13C polarizations of about 1-2% were achieved within seconds and with minimal equipment. Effects that potentially may explain loss of 13C polarization have been identified, i.e. low hydrogenation yield, fast T1/T2 relaxation and the rarely considered 13C isotope labeling effect.

Selective excitation of hydrogen doubles the yield and improves the robustness of parahydrogen-induced polarization of low-γ nuclei.

We describe a new method for pulsed spin order transfer of parahydrogen-induced polarization (PHIP) that enables high polarization in incompletely 2H-labeled molecules by exciting only the desired protons in a frequency-selective manner. This way, the effect of selected J-couplings is suspended. Experimentally 1.25% 13C polarization were obtained for 1-13C-ethyl pyruvate and 50% pH2 at 9.4 Tesla.

Parahydrogen-Induced Polarization of Amino Acids.

Nuclear magnetic resonance (NMR) has become a universal method for biochemical and biomedical studies, including metabolomics, proteomics, and magnetic resonance imaging (MRI). By increasing the signal of selected molecules, the hyperpolarization of nuclear spin has expanded the reach of NMR and MRI even further (e.g. hyperpolarized solid-state NMR and metabolic imaging in vivo). Parahydrogen (pH2 ) offers a fast and cost-efficient way to achieve hyperpolarization, and the last decade has seen extensive advances, including the synthesis of new tracers, catalysts, and transfer methods. The portfolio of hyperpolarized molecules now includes amino acids, which are of great interest for many applications. Here, we provide an overview of the current literature and developments in the hyperpolarization of amino acids and peptides.

Pulse-Programmable Magnetic Field Sweeping of Parahydrogen-Induced Polarization by Side Arm Hydrogenation.

Among the hyperpolarization techniques geared toward in vivo magnetic resonance imaging, parahydrogen-induced polarization (PHIP) shows promise due to its low cost and fast speed of contrast agent preparation. The synthesis of 13C-labeled, unsaturated precursors to perform PHIP by side arm hydrogenation has recently opened new possibilities for metabolic imaging owing to the biological compatibility of the reaction products, although the polarization transfer between the parahydrogen-derived protons and the 13C heteronucleus must yet be better understood, characterized, and eventually optimized. In this realm, a new experimental strategy incorporating pulse-programmable magnetic field sweeping and in situ detection has been developed. The approach is evaluated by measuring the 13C polarization of ethyl acetate-1-13C, i.e., the product of pairwise addition of parahydrogen to vinyl acetate-1-13C, resulting from zero-crossing magnetic field ramps of various durations, amplitudes, and step sizes. The results demonstrate (i) the profound effect these parameters have on the 1H to 13C polarization transfer efficiency and (ii) the high reproducibility of the technique.

Dynamic 2D and 3D mapping of hyperpolarized pyruvate to lactate conversion in vivo with efficient multi-echo balanced steady-state free precession at 3 T.

The aim of this study was to acquire the transient MRI signal of hyperpolarized tracers and their metabolites efficiently, for which specialized imaging sequences are required. In this work, a multi-echo balanced steady-state free precession (me-bSSFP) sequence with Iterative Decomposition with Echo Asymmetry and Least squares estimation (IDEAL) reconstruction was implemented on a clinical 3 T positron-emission tomography/MRI system for fast 2D and 3D metabolic imaging. Simulations were conducted to obtain signal-efficient sequence protocols for the metabolic imaging of hyperpolarized biomolecules. The sequence was applied in vitro and in vivo for probing the enzymatic exchange of hyperpolarized [1-13 C]pyruvate and [1-13 C]lactate. Chemical shift resolution was achieved using a least-square, iterative chemical species separation algorithm in the reconstruction. In vitro, metabolic conversion rate measurements from me-bSSFP were compared with NMR spectroscopy and free induction decay-chemical shift imaging (FID-CSI). In vivo, a rat MAT-B-III tumor model was imaged with me-bSSFP and FID-CSI. 2D metabolite maps of [1-13 C]pyruvate and [1-13 C]lactate acquired with me-bSSFP showed the same spatial distributions as FID-CSI. The pyruvate-lactate conversion kinetics measured with me-bSSFP and NMR corresponded well. Dynamic 2D metabolite mapping with me-bSSFP enabled the acquisition of up to 420 time frames (scan time: 180-350 ms/frame) before the hyperpolarized [1-13 C]pyruvate was relaxed below noise level. 3D metabolite mapping with a large field of view (180 × 180 × 48 mm3 ) and high spatial resolution (5.6 × 5.6 × 2 mm3 ) was conducted with me-bSSFP in a scan time of 8.2 seconds. It was concluded that Me-bSSFP improves the spatial and temporal resolution for metabolic imaging of hyperpolarized [1-13 C]pyruvate and [1-13 C]lactate compared with either of the FID-CSI or EPSI methods reported at 3 T, providing new possibilities for clinical and preclinical applications.