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.

Functional activation of pyruvate dehydrogenase in human brain using hyperpolarized [1-13 C]pyruvate.

PURPOSE: Pyruvate, produced from either glucose, glycogen, or lactate, is the dominant precursor of cerebral oxidative metabolism. Pyruvate dehydrogenase (PDH) flux is a direct measure of cerebral mitochondrial function and metabolism. Detection of [13 C]bicarbonate in the brain from hyperpolarized [1-13 C]pyruvate using carbon-13 (13 C) MRI provides a unique opportunity for assessing PDH flux in vivo. This study is to assess changes in cerebral PDH flux in response to visual stimuli using in vivo 13 C MRS with hyperpolarized [1-13 C]pyruvate. METHODS: From seven sedentary adults in good general health, time-resolved [13 C]bicarbonate production was measured in the brain using 90° flip angles with minimal perturbation of its precursors, [1-13 C]pyruvate and [1-13 C]lactate, to test the hypothesis that the appearance of [13 C]bicarbonate signals in the brain reflects the metabolic changes associated with neuronal activation. With a separate group of healthy participants (n = 3), the likelihood of the bolus-injected [1-13 C]pyruvate being converted to [1-13 C]lactate prior to decarboxylation was investigated by measuring [13 C]bicarbonate production with and without [1-13 C]lactate saturation. RESULTS: In the course of visual stimulation, the measured [13 C]bicarbonate signal normalized to the total 13 C signal in the visual cortex increased by 17.1% ± 15.9% (p = 0.017), whereas no significant change was detected in [1-13 C]lactate. Proton BOLD fMRI confirmed the regional activation in the visual cortex with the stimuli. Lactate saturation decreased bicarbonate-to-pyruvate ratio by 44.4% ± 9.3% (p < 0.01). CONCLUSION: We demonstrated the utility of 13 C MRS with hyperpolarized [1-13 C]pyruvate for assessing the activation of cerebral PDH flux via the detection of [13 C]bicarbonate production.

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 hyperpolarized 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 & 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.

Hyperpolarized 13 C†NMR Spectroscopy of Urine Samples at Natural Abundance by Quantitative Dissolution Dynamic Nuclear Polarization.

Hyperpolarized nuclear magnetic resonance (NMR) offers an ensemble of methods that remarkably address the sensitivity issues of conventional NMR. Dissolution Dynamic Nuclear Polarization (d-DNP) provides a unique and general way to detect 13 C NMR signals with a sensitivity enhanced by several orders of magnitude. The expanding application scope of d-DNP now encompasses the analysis of complex mixtures at natural 13 C abundance. However, the application of d-DNP in this area has been limited to metabolite extracts. Here, we report the first d-DNP-enhanced 13 C NMR analysis of a biofluid -urine- at natural abundance, offering unprecedented resolution and sensitivity for this challenging type of sample. We also show that accurate quantitative information on multiple targeted metabolites can be retrieved through a standard addition procedure.

Hyperpolarized NMR metabolomics.

Hyperpolarized NMR is a promising approach to address the sensitivity limits of conventional NMR metabolomics approaches, which currently fails to detect minute metabolite concentrations in biological samples. This review describes how tremendous signal enhancement offered by dissolution-dynamic nuclear polarization and parahydrogen-based techniques can be fully exploited for molecular omics sciences. Recent developments, including the combination of hyperpolarization techniques with fast multi-dimensional NMR implementation and quantitative workflows are described, and a comprehensive comparison of existing hyperpolarization techniques is proposed. High-throughput, sensitivity, resolution and other relevant challenges that should be tackled for a general application of hyperpolarized NMR in metabolomics are discussed.

A Toolbox for Glutamine Use in Dissolution Dynamic Nuclear Polarization: from Enzymatic Reaction Monitoring to the Study of Cellular Metabolic Pathways and Imaging.

Glutamine is under scrutiny regarding its metabolic deregulation linked to energetic reprogramming in cancer cells. Many analytical techniques have been used to better understand the impact of the metabolism of amino acids on biological processes, however only a few are suited to work with complex samples. Here, we report the use of a general dissolution dynamic nuclear polarization (D-DNP) formulation using an unexpensive radical as a multipurpose tool to study glutamine, with insights from enzymatic modelling to complex metabolic networks and fast imaging. First, hyperpolarized [5-13 C] glutamine is used as molecular probe to study the kinetic action of two enzymes: L-asparaginase that has been used as an anti-metabolic treatment for cancer, and glutaminase. These results are also compared with those acquired with another hyperpolarized amino acid, [1,4-13 C] asparagine. Second, we explored the use of hyperpolarized (HP) substrates to probe metabolic pathways by monitoring metabolic profiles arising from hyperpolarized glutamine in E. coli extracts. Finally, a highly concentrated sample formulation is proposed for the purpose of fast imaging applications. We think that this approach can be extended to formulate other amino acids as well as other metabolites and provide complementary insights into the analysis of metabolic networks.

Short-chain fatty acids reprogram metabolic profiles with the induction of reactive oxygen species production in human colorectal adenocarcinoma cells.

Short-chain fatty acids (SCFAs) exhibit anticancer activity in cellular and animal models of colon cancer. Acetate, propionate, and butyrate are the three major SCFAs produced from dietary fiber by gut microbiota fermentation and have beneficial effects on human health. Most previous studies on the antitumor mechanisms of SCFAs have focused on specific metabolites or genes involved in antitumor pathways, such as reactive oxygen species (ROS) biosynthesis. In this study, we performed a systematic and unbiased analysis of the effects of acetate, propionate, and butyrate on ROS levels and metabolic and transcriptomic signatures at physiological concentrations in human colorectal adenocarcinoma cells. We observed significantly elevated levels of ROS in the treated cells. Furthermore, significantly regulated signatures were involved in overlapping pathways at metabolic and transcriptomic levels, including ROS response and metabolism, fatty acid transport and metabolism, glucose response and metabolism, mitochondrial transport and respiratory chain complex, one-carbon metabolism, amino acid transport and metabolism, and glutaminolysis, which are directly or indirectly linked to ROS production. Additionally, metabolic and transcriptomic regulation occurred in a SCFAs types-dependent manner, with an increasing degree from acetate to propionate and then to butyrate. This study provides a comprehensive analysis of how SCFAs induce ROS production and modulate metabolic and transcriptomic levels in colon cancer cells, which is vital for understanding the mechanisms of the effects of SCFAs on antitumor activity in colon cancer.

Missing Pieces in Structure Puzzles: How Hyperpolarized NMR Spectroscopy Can Complement Structural Biology and Biochemistry.

Structure determination lies at the heart of many biochemical research programs. However, the "giants": X-ray diffraction, electron microscopy, molecular dynamics simulations, and nuclear magnetic resonance, among others, leave quite a few dark spots on the structural pictures drawn of proteins, nucleic acids, membranes, and other biomacromolecules. For example, structural models under physiological conditions or of short-lived intermediates often remain out of reach of the established experimental methods. This account frames the possibility of including hyperpolarized, that is, dramatically signal-enhanced NMR in existing workflows to fill these spots with detailed depictions. We highlight how integrating methods based on dissolution dynamic nuclear polarization can provide valuable complementary information about formerly inaccessible conformational spaces for many systems. A particular focus will be on hyperpolarized buffers to facilitate the NMR structure determination of challenging systems.

Signal enhancement of hyperpolarized 15 N sites in solution-increase in solid-state polarization at 3.35 T and prolongation of relaxation in deuterated water mixtures.

Hyperpolarized 15 N sites have been found to be promising for generating long-lived hyperpolarized states in solution, and present a promising approach for utilizing dissolution-dynamic nuclear polarization (dDNP)-driven hyperpolarized MRI for imaging in biology and medicine. Specifically, 15 N sites with directly bound protons were shown to be useful when dissolved in D2 O. The purpose of the current study was to further characterize and increase the visibility of such 15 N sites in solutions that mimic an intravenous injection during the first cardiac pass in terms of their H2 O:D2 O composition. The T1 values of hyperpolarized 15 N in [15 N2 ]urea and [15 N]NH4 Cl demonstrated similar dependences on the H2 O:D2 O composition of the solution, with a T1 of about 140 s in 100% D2 O, about twofold shortening in 90% and 80% D2 O, and about threefold shortening in 50% D2 O. [13 C]urea was found to be a useful solid-state 13 C marker for qualitative monitoring of the 15 N polarization process in a commercial pre-clinical dDNP device. Adding trace amounts of Gd3+ to the polarization formulation led to higher solid-state polarization of [13 C]urea and to higher polarization levels of [15 N2 ]urea in solution.

Interfacing Liquid State Hyperpolarization Methods with NMR Instrumentation.

Advances in liquid state hyperpolarization methods have enabled new applications of high-resolution NMR spectroscopy. Utilizing strong signal enhancements from hyperpolarization allows performing NMR spectroscopy at low concentration, or with high time resolution. Making use of the high, but rapidly decaying hyperpolarization in the liquid state requires new techniques to interface hyperpolarization equipment with liquid state NMR spectrometers. This article highlights rapid injection, high resolution NMR spectroscopy with hyperpolarization produced by the techniques of dissolution dynamic nuclear polarization (D-DNP) and para-hydrogen induced polarization (PHIP). These are popular, albeit not the only methods to produce high polarization levels for liquid samples. Gas and liquid driven sample injection techniques are compatible with both of these hyperpolarization methods. The rapid sample injection techniques are combined with adapted NMR experiments working in a single, or small number of scans. They expand the application of liquid state hyperpolarization to spins with comparably short relaxation times, provide enhanced control over sample conditions, and allow for mixing experiments to study reactions in real time.

Classification and biomarker identification of prostate tissue from TRAMP mice with hyperpolarized 13C-SIRA.

Hyperpolarized 13C isotope resolved spectroscopy boosts NMR signal intensity, which improves signal detection and allows metabolic fluxes to be analyzed. Such hyperpolarized flux data may offer new approaches to tissue classification and biomarker identification that could be translated in vivo. Here we used hyperpolarized stable isotope resolved analysis (SIRA) to measure metabolite specific 13C isotopic enrichments in the central carbon metabolism of mouse prostate. Prostate and tumor tissue samples were acquired from transgenic adenocarcinomas of the mouse prostate (TRAMP) mice. Before euthanasia, mice were injected with [U-13C]glucose intraperitoneally (i.p.). Polar metabolite extracts were prepared, and hyperpolarized 1D-13C NMR spectra were obtained from normal prostate (n = 19) and cancer tissue (n = 19) samples. Binary classification and feature analysis was performed to make a separation model and to investigate differences between samples originating from normal and cancerous prostate tissue, respectively. Hyperpolarized experiments were carried out according to a standardized protocol, which showed a high repeatability (CV = 15%) and an average linewidth in the 1D-13C NMR spectra of 2 ± 0.5 Hz. The resolution of the hyperpolarized 1D-13C spectra was high with little signal overlap in the carbonyl region and metabolite identification was easily accomplished. A discrimination with 95% success rate could be made between samples originating from TRAMP mice prostate and tumor tissue based on isotopomers from uniquely identified metabolites. Hyperpolarized 13C-SIRA allowed detailed metabolic information to be obtained from tissue specimens. The positional information of 13C isotopic enrichments lead to easily interpreted features responsible for high predictive classification of tissue types. This analytical approach has matured, and the robust experimental protocols currently available allow systematic tracking of metabolite flux ex vivo.

Real-Time Non-Invasive and Direct Determination of Lactate Dehydrogenase Activity in Cerebral Organoids-A New Method to Characterize the Metabolism of Brain Organoids?

Organoids are a powerful tool in the quest to understand human diseases. As the developing brain is extremely inaccessible in mammals, cerebral organoids (COs) provide a unique way to investigate neural development and related disorders. The aim of this study was to utilize hyperpolarized 13C NMR to investigate the metabolism of COs in real-time, in a non-destructive manner. The enzymatic activity of lactate dehydrogenase (LDH) was determined by quantifying the rate of [1-13C]lactate production from hyperpolarized [1-13C]pyruvate. Organoid development was assessed by immunofluorescence imaging. Organoid viability was confirmed using 31P NMR spectroscopy. A total of 15 organoids collated into 3 groups with a group total weight of 20-77 mg were used in this study. Two groups were at the age of 10 weeks and one was at the age of 33 weeks. The feasibility of this approach was demonstrated in both age groups, and the LDH activity rate was found to be 1.32 ± 0.75 nmol/s (n = 3 organoid batches). These results suggest that hyperpolarized NMR can be used to characterize the metabolism of brain organoids with a total tissue wet weight of as low as 20 mg (<3 mm3) and a diameter ranging from 3 to 6 mm.

LDH and PDH Activities in the Ischemic Brain and the Effect of Reperfusion-An Ex Vivo MR Study in Rat Brain Slices Using Hyperpolarized [1-13C]Pyruvate.

Ischemic stroke is a leading cause for neurologic disability worldwide, for which reperfusion is the only available treatment. Neuroimaging in stroke guides treatment, and therefore determines the clinical outcome. However, there are currently no imaging biomarkers for the status of the ischemic brain tissue. Such biomarkers could potentially be useful for guiding treatment in patients presenting with ischemic stroke. Hyperpolarized 13C MR of [1-13C]pyruvate is a clinically translatable method used to characterize tissue metabolism non-invasively in a relevant timescale. The aim of this study was to utilize hyperpolarized [1-13C]pyruvate to investigate the metabolic consequences of an ischemic insult immediately during reperfusion and upon recovery of the brain tissue. The rates of lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH) were quantified by monitoring the rates of [1-13C]lactate and [13C]bicarbonate production from hyperpolarized [1-13C]pyruvate. 31P NMR of the perfused brain slices showed that this system is suitable for studying ischemia and recovery following reperfusion. This was indicated by the levels of the high-energy phosphates (tissue viability) and the chemical shift of the inorganic phosphate signal (tissue pH). Acidification, which was observed during the ischemic insult, has returned to baseline level following reperfusion. The LDH/PDH activity ratio increased following ischemia, from 47.0 ± 12.7 in the control group (n = 6) to 217.4 ± 121.3 in the ischemia-reperfusion group (n = 6). Following the recovery period (ca. 1.5 h), this value had returned to its pre-ischemia (baseline) level, suggesting the LDH/PDH enzyme activity ratio may be used as a potential indicator for the status of the ischemic and recovering brain.

Stable isotope resolved metabolomics classification of prostate cancer cells using hyperpolarized NMR data.

Metabolic fingerprinting is a strong tool for characterization of biological phenotypes. Classification with machine learning is a critical component in the discrimination of molecular determinants. Cellular activity can be traced using stable isotope labelling of metabolites from which information on cellular pathways may be obtained. Nuclear magnetic resonance (NMR) spectroscopy is, due to its ability to trace labelling in specific atom positions, a method of choice for such metabolic activity measurements. In this study, we used hyperpolarization in the form of dissolution Dynamic Nuclear Polarization (dDNP) NMR to measure signal enhanced isotope labelled metabolites reporting on pathway activity from four different prostate cancer cell lines. The spectra have a high signal-to-noise, with less than 30 signals reporting on 10 metabolic reactions. This allows easy extraction and straightforward interpretation of spectral data. Four metabolite signals selected using a Random Forest algorithm allowed a classification with Support Vector Machines between aggressive and indolent cancer cells with 96.9% accuracy, -corresponding to 31 out of 32 samples. This demonstrates that the information contained in the few features measured with dDNP NMR, is sufficient and robust for performing binary classification based on the metabolic activity of cultured prostate cancer cells.

Sensitivity-enhanced three-dimensional and carbon-detected two-dimensional NMR of proteins using hyperpolarized water.

Signal enhancements of up to two orders of magnitude in protein NMR can be achieved by employing HDO as a vector to introduce hyperpolarization into folded or intrinsically disordered proteins. In this approach, hyperpolarized HDO produced by dissolution-dynamic nuclear polarization (D-DNP) is mixed with a protein solution waiting in a high-field NMR spectrometer, whereupon amide proton exchange and nuclear Overhauser effects (NOE) transfer hyperpolarization to the protein and enable acquisition of a signal-enhanced high-resolution spectrum. To date, the use of this strategy has been limited to 1D and 1H-15N 2D correlation experiments. Here we introduce 2D 13C-detected D-DNP, to reduce exchange-induced broadening and other relaxation penalties that can adversely affect proton-detected D-DNP experiments. We also introduce hyperpolarized 3D spectroscopy, opening the possibility of D-DNP studies of larger proteins and IDPs, where assignment and residue-specific investigation may be impeded by spectral crowding. The signal enhancements obtained depend in particular on the rates of chemical and magnetic exchange of the observed residues, thus resulting in non-uniform 'hyperpolarization-selective' signal enhancements. The resulting spectral sparsity, however, makes it possible to resolve and monitor individual amino acids in IDPs of over 200 residues at acquisition times of just over a minute. We apply the proposed experiments to two model systems: the compactly folded protein ubiquitin, and the intrinsically disordered protein (IDP) osteopontin (OPN).

Detection of myocardial medium-chain fatty acid oxidation and tricarboxylic acid cycle activity with hyperpolarized [1-13 C]octanoate.

Under normal conditions, the heart mainly relies on fatty acid oxidation to meet its energy needs. Changes in myocardial fuel preference are noted in the diseased and failing heart. The magnetic resonance signal enhancement provided by spin hyperpolarization allows the metabolism of substrates labeled with carbon-13 to be followed in real time in vivo. Although the low water solubility of long-chain fatty acids abrogates their hyperpolarization by dissolution dynamic nuclear polarization, medium-chain fatty acids have sufficient solubility to be efficiently polarized and dissolved. In this study, we investigated the applicability of hyperpolarized [1-13 C]octanoate to measure myocardial medium-chain fatty acid metabolism in vivo. Scanning rats infused with a bolus of hyperpolarized [1-13 C]octanoate, the primary metabolite observed in the heart was identified as [1-13 C]acetylcarnitine. Additionally, [5-13 C]glutamate and [5-13 C]citrate could be respectively resolved in seven and five of 31 experiments, demonstrating the incorporation of oxidation products of octanoate into the tricarboxylic acid cycle. A variable drop in blood pressure was observed immediately following the bolus injection, and this drop correlated with a decrease in normalized acetylcarnitine signal (acetylcarnitine/octanoate). Increasing the delay before infusion moderated the decrease in blood pressure, which was attributed to the presence of residual gas bubbles in the octanoate solution. No significant difference in normalized acetylcarnitine signal was apparent between fed and 12-hour fasted rats. Compared with a solution in buffer, the longitudinal relaxation of [1-13 C]octanoate was accelerated ~3-fold in blood and by the addition of serum albumin. These results demonstrate the potential of hyperpolarized [1-13 C]octanoate to probe myocardial medium-chain fatty acid metabolism as well as some of the limitations that may accompany its use.

Applications of Dissolution-DNP for NMR Screening.

Experimental screening for protein-ligand interactions is a central task in drug discovery. Nuclear magnetic resonance (NMR) spectroscopy enables the determination of binding affinities, as well as the measurement of structural and dynamic parameters governing the interaction. With traditional liquid-state NMR relying on a nuclear spin polarization on the order of 10-5, hyperpolarization methods such as dissolution dynamic nuclear polarization (D-DNP) can increase signals by several orders of magnitude. The resulting increase in sensitivity has the potential to reduce requirements for the concentration of protein and ligands, improve the accuracy of the detection of interaction by allowing the use of near-stoichiometric conditions, and increase throughput. This chapter introduces a selection of basic techniques for the application of D-DNP to screening. Procedures for hyperpolarization are briefly reviewed, followed by the description of NMR methods for detection of binding through changes in chemical shift and relaxation parameters. Experiments employing competitive binding with a known ligand are shown, which can be used to determine binding affinity or yield structural information on the pharmacophore. The specific challenges of working with nonrenewable hyperpolarization are reviewed, and solutions including the use of multiplexed NMR detection are described. Altogether, the methods summarized in this chapter are intended to allow for the efficient detection of binding affinity, structure, and dynamics facilitated through substantial signal enhancements provided by hyperpolarization.

Hyperpolarized [15N]nitrate as a potential long lived hyperpolarized contrast agent for MRI.

Reports on gadolinium deposits in the body and brains of adults and children who underwent contrast-enhanced MRI examinations warrant development of new, metal free, contrast agents for MRI. Nitrate is an abundant ion in mammalian biochemistry and sodium nitrate can be safely injected intravenously. We show that hyperpolarized [15N]nitrate can potentially be used as an MR tracer. The 15N site of hyperpolarized [15N]nitrate showed a T1 of more than 100 s in aqueous solutions, which was prolonged to more than 170 s below 20 °C. Capitalizing on this effect for polarization storage we obtained a visibility window of 9 min in blood. Conversion to [15N]nitrite, the bioactive reduced form of nitrate, was not observed in human blood and human saliva in this time frame. Thus, [15N]nitrate may serve as a long-lived hyperpolarized tracer for MR. Due to its ionic nature, the immediate applications appear to be perfusion and tissue retention imaging.

Real-time ALT and LDH activities determined in viable precision-cut mouse liver slices using hyperpolarized [1-13 C]pyruvate-Implications for studies on biopsied liver tissues.

Precision-cut liver slices (PCLS) are widely used in liver research as they provide a liver model with all liver cell types in their natural architecture. The purpose of this study was to demonstrate the use of PCLS for hyperpolarized metabolic investigation in a mouse model, for potential future application in liver biopsy cores. Fresh normal liver was harvested from six mice. 500 µm PCLS were prepared and placed in a 10 mm NMR tube in an NMR spectrometer and perfused continuously. 31 P spectra were acquired to evaluate the presence of adenosine triphosphate (ATP) and validate viability in all samples. Hyperpolarized [1-13 C]pyruvate was flushed into the NMR tube in the spectrometer. Consecutive 13 C NMR spectra were acquired immediately after the injection using both non-selective (five injections, two livers) and selective RF excitation (six injections, three livers). The 31 P spectra showed the characteristic signals of ATP, confirming the viability of the PCLS for more than 2.5 h in the spectrometer. After each of the [1-13 C]pyruvate injections, both [1-13 C]lactate and [1-13 C]alanine signals were detected. Selective RF excitation aimed at both [1-13 C]lactate and [1-13 C]alanine enabled better visualization and quantification of the metabolic activity. Using this acquisition approach only the newly formed metabolites are observed upon excitation, and their intensities relative to those of hyperpolarized pyruvate enable quantification of metabolite production rates. This rate of lactate and alanine production appeared to be constant throughout the measurement time, with alanine production about 2.3 times higher than lactate. In summary, the viability of PCLS in an NMR spectrometer was demonstrated and hyperpolarized [1-13 C]pyruvate metabolism was recorded. This study opens up the possibility of evaluating alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) activities in human liver biopsies, while preserving the tissue architecture and viability. In healthy, well-perfused liver slices the ratio of ALT to LDH activity is about 2.3.

Cryogen-free dissolution dynamic nuclear polarization polarizer operating at 3.35 T, 6.70 T, and 10.1 T.

PURPOSE: A novel dissolution dynamic nuclear polarization (dDNP) polarizer platform is presented. The polarizer meets a number of key requirements for in vitro, preclinical, and clinical applications. METHOD: It uses no liquid cryogens, operates in continuous mode, accommodates a wide range of sample sizes up to and including those required for human studies, and is fully automated. RESULTS: It offers a wide operational window both in terms of magnetic field, up to 10.1 T, and temperature, from room temperature down to 1.3 K. The polarizer delivers a 13 C liquid state polarization for [1-13 C]pyruvate of 70%. The build-up time constant in the solid state is approximately 1200 s (20 minutes), allowing a sample throughput of at least one sample per hour including sample loading and dissolution. CONCLUSION: We confirm the previously reported strong field dependence in the range 3.35 to 6.7 T, but see no further increase in polarization when increasing the magnetic field strength to 10.1 T for [1-13 C]pyruvate and trityl. Using a custom dry magnet, cold head and recondensing, closed-cycle cooling system, combined with a modular DNP probe, and automation and fluid handling systems, we have designed a unique dDNP system with unrivalled flexibility and performance.