Enzymatic Reactions Observed with Zero- and Low-Field Nuclear Magnetic Resonance.

We demonstrate that enzyme-catalyzed reactions can be observed in zero- and low-field NMR experiments by combining recent advances in parahydrogen-based hyperpolarization methods with state-of-the-art magnetometry. Specifically, we investigated two model biological processes: the conversion of fumarate into malate, which is used in vivo as a marker of cell necrosis, and the conversion of pyruvate into lactate, which is the most widely studied metabolic process in hyperpolarization-enhanced imaging. In addition to this, we constructed a microfluidic zero-field NMR setup to perform experiments on microliter-scale samples of [1-13C]fumarate in a lab-on-a-chip device. Zero- to ultralow-field (ZULF) NMR has two key advantages over high-field NMR: the signals can pass through conductive materials (e.g., metals), and line broadening from sample heterogeneity is negligible. To date, the use of ZULF NMR for process monitoring has been limited to studying hydrogenation reactions. In this work, we demonstrate this emerging analytical technique for more general reaction monitoring and compare zero- vs low-field detection.

New tools to investigate tumor metabolism by NMR/MRI.

Changes in metabolism is an hallmark that characterizes tumour cells from healthy ones. Their detection can be highly relevant for staging the tumor and for monitoring the response to therapeutic treatments. Herein it is shown the readout of these changes can be achieved either by assessing the pH of the extracellular space in the tumour region and by monitoring real time transformations of hyperpolarized C-13 labelled substrates. Mapping pH in a MR image is possible by measuring the CEST response of an administered contrast agent such as Iopamidol that can provide accurate measurements of the heterogeneity of tumour acidosis. Direct detection of relevant enzymatic activities have been acquired by using Pyruvate and Fumarate hyperpolarized by the incorporation of a molecule of para-H2. Finally, it has been found that the tumour transformation involves an increase in the water exchange rate between the intra- and the extra-cellular compartments. A quantitative estimation of these changes can be obtained by acquiring the longitudinal relaxation times of tissue water protons at low magnetic field strength on Fast Field Cycling Relaxometers. This finding has been exploited in an application devoted to the assessment of the presence of residual tumour tissue in the margins of the resected mass in breast conservative surgery.

Rapid hyperpolarization and purification of the metabolite fumarate in aqueous solution.

Hyperpolarized fumarate is a promising biosensor for carbon-13 magnetic resonance metabolic imaging. Such molecular imaging applications require nuclear hyperpolarization to attain sufficient signal strength. Dissolution dynamic nuclear polarization is the current state-of-the-art methodology for hyperpolarizing fumarate, but this is expensive and relatively slow. Alternatively, this important biomolecule can be hyperpolarized in a cheap and convenient manner using parahydrogen-induced polarization. However, this process requires a chemical reaction, and the resulting solutions are contaminated with the catalyst, unreacted reagents, and reaction side-product molecules, and are hence unsuitable for use in vivo. In this work we show that the hyperpolarized fumarate can be purified from these contaminants by acid precipitation as a pure solid, and later redissolved to a desired concentration in a clean aqueous solvent. Significant advances in the reaction conditions and reactor equipment allow for formation of hyperpolarized fumarate at 13C polarization levels of 30-45%.

ParaHydrogen Polarized Ethyl-[1-13 C]pyruvate in Water, a Key Substrate for Fostering the PHIP-SAH Approach to Metabolic Imaging.

An efficient synthesis of vinyl-[1-13 C]pyruvate has been reported, from which 13 C hyperpolarized (HP) ethyl-[1-13 C]pyruvate has been obtained by means of ParaHydrogen Induced Polarization (PHIP). Due to the intrinsic lability of pyruvate, which leads quickly to degradation of the reaction mixture even under mild reaction conditions, the vinyl-ester has been synthesized through the intermediacy of a more stable ketal derivative. 13 C and 1 H hyperpolarizations of ethyl-[1-13 C]pyruvate, hydrogenated using ParaHydrogen, have been compared to those observed on the more widely used allyl-derivative. It has been demonstrated that the spin order transfer from ParaHydrogen protons to 13 C, is more efficient on the ethyl than on the allyl-esterdue to the larger J-couplings involved. The main requirements needed for the biological application of this HP product have been met, i. e. an aqueous solution of the product at high concentration (40 mM) with a good 13 C polarization level (4.8 %) has been obtained. The in vitro metabolic transformation of the HP ethyl-[1-13 C]pyruvate, catalyzed by an esterase, has been observed. This substrate appears to be a good candidate for in vivo metabolic investigations using PHIP hyperpolarized probes.

Hydrogenative-PHIP polarized metabolites for biological studies.

ParaHydrogen induced polarization (PHIP) is an efficient and cost-effective hyperpolarization method, but its application to biological investigations has been hampered, so far, due to chemical challenges. PHIP is obtained by means of the addition of hydrogen, enriched in the para-spin isomer, to an unsaturated substrate. Both hydrogen atoms must be transferred to the same substrate, in a pairwise manner, by a suitable hydrogenation catalyst; therefore, a de-hydrogenated precursor of the target molecule is necessary. This has strongly limited the number of parahydrogen polarized substrates. The non-hydrogenative approach brilliantly circumvents this central issue, but has not been translated to in-vivo yet. Recent advancements in hydrogenative PHIP (h-PHIP) considerably widened the possibility to hyperpolarize metabolites and, in this review, we will focus on substrates that have been obtained by means of this method and used in vivo. Attention will also be paid to the requirements that must be met and on the issues that have still to be tackled to obtain further improvements and to push PHIP substrates in biological applications.

In-vitro NMR Studies of Prostate Tumor Cell Metabolism by Means of Hyperpolarized [1-13C]Pyruvate Obtained Using the PHIP-SAH Method.

Nuclear Magnetic Resonance allows the non-invasive detection and quantitation of metabolites to be carried out in cells and tissues. This means that that metabolic changes can be revealed without the need for sample processing and the destruction of the biological matrix. The main limitation to the application of this method to biological studies is its intrinsic low sensitivity. The introduction of hyperpolarization techniques and, in particular, of dissolution-Dynamic Nuclear Polarization (d-DNP) and ParaHydrogen Induced Polarization (PHIP) is a significant breakthrough for the field as the MR signals of molecules and, most importantly, metabolites, can be increased by some orders of magnitude. Hyperpolarized pyruvate is the metabolite that has been most widely used for the investigation of metabolic alterations in cancer and other diseases. Although d-DNP is currently the gold-standard hyperpolarization method, its high costs and intrinsically slow hyperpolarization procedure are a hurdle to the application of this tool. However, PHIP is cost effective and fast and hyperpolarized pyruvate can be obtained using the so-called Side Arm Hydrogenation approach (PHIP-SAH). The potential toxicity of a solution of the hyperpolarized metabolite that is obtained in this way is presented herein. HP pyruvate has then been used for metabolic studies on different prostate cancer cells lines (DU145, PC3, and LnCap). The results obtained using the HP metabolite have been compared with those from conventional biochemical assays.

Real-Time Nuclear Magnetic Resonance Detection of Fumarase Activity Using Parahydrogen-Hyperpolarized [1-13C]Fumarate.

Hyperpolarized fumarate can be used as a probe of real-time metabolism in vivo, using carbon-13 magnetic resonance imaging. Dissolution dynamic nuclear polarization is commonly used to produce hyperpolarized fumarate, but a cheaper and faster alternative is to produce hyperpolarized fumarate via PHIP (parahydrogen-induced polarization). In this work, we trans-hydrogenate [1-13C]acetylene dicarboxylate with para-enriched hydrogen using a commercially available Ru catalyst in water to produce hyperpolarized [1-13C]fumarate. We show that fumarate is produced in 89% yield, with succinate as a side product in 11% yield. The proton polarization is converted into 13C magnetization using a constant adiabaticity field cycle, and a polarization level of 24% is achieved using 86% para-enriched hydrogen gas. We inject the hyperpolarized [1-13C]fumarate into cell suspensions and track the metabolism. This work opens the path to greatly accelerated preclinical studies using fumarate as a biomarker.

Metabolic Studies of Tumor Cells Using [1-13 C] Pyruvate Hyperpolarized by Means of PHIP-Side Arm Hydrogenation.

The kinetics of metabolic processes can be assessed, in real time by means of MR hyperpolarized (HP) metabolites. [1-13 C]pyruvate, hyperpolarized by means of d-DNP, is, by far, the substrate most widely applied to the investigation of several pathologies characterized by deregulated glycolytic metabolic networks, including cancer. Hyperpolarization of [1-13 C]pyruvate by means of the cost effective, fast and easy to handle PHIP-SAH (para-hydrogen induced polarization-side arm hydrogenation) method opens-up a pathway for the application of HP metabolites to a wide range of cancer-related studies. Herein, we report the first application of PHIP-SAH hyperpolarized [1-13 C]pyruvate in the investigation of upregulated glycolysis in two murine breast cancer cell lines (168FARN and 4T1). The results obtained using HP pyruvate have been validated with a conventional biochemical assay and are coherent with previously-reported lactate dehydrogenase activity measured in those cells.

The 13C hyperpolarized pyruvate generated by ParaHydrogen detects the response of the heart to altered metabolism in real time.

Many imaging methods have been proposed to act as surrogate markers of organ damage, yet for many candidates the essential biomarkers characteristics of the injured organ have not yet been described. Hyperpolarized [1-13C]pyruvate allows real time monitoring of metabolism in vivo. ParaHydrogen Induced Polarization (PHIP) is a portable, cost effective technique able to generate 13C MR hyperpolarized molecules within seconds. The introduction of the Side Arm Hydrogenation (SAH) strategy offered a way to widen the field of PHIP generated systems and to make this approach competitive with the currently applied dissolution-DNP (Dynamic Nuclear Polarization) method. Herein, we describe the first in vivo metabolic imaging study using the PHIP-SAH hyperpolarized [1-13C]pyruvate. In vivo maps of pyruvate and of its metabolic product lactate have been acquired on a 1 T MRI scanner. By comparing pyruvate/lactate 13C label exchange rate in a mouse model of dilated cardiomyopathy, it has been found that the metabolic dysfunction occurring in the cardiac muscle of the diseased mice can be detected well before the disease can be assessed by echocardiographic investigations.

Parahydrogen-Based Hyperpolarization for Biomedicine.

Magnetic resonance (MR) is one of the most versatile and useful physical effects used for human imaging, chemical analysis, and the elucidation of molecular structures. However, its full potential is rarely used, because only a small fraction of the nuclear spin ensemble is polarized, that is, aligned with the applied static magnetic field. Hyperpolarization methods seek other means to increase the polarization and thus the MR signal. A unique source of pure spin order is the entangled singlet spin state of dihydrogen, parahydrogen (pH2 ), which is inherently stable and long-lived. When brought into contact with another molecule, this "spin order on demand" allows the MR signal to be enhanced by several orders of magnitude. Considerable progress has been made in the past decade in the area of pH2 -based hyperpolarization techniques for biomedical applications. It is the goal of this Review to provide a selective overview of these developments, covering the areas of spin physics, catalysis, instrumentation, preparation of the contrast agents, and applications.

Studies to enhance the hyperpolarization level in PHIP-SAH-produced C13-pyruvate.

The use of [1-13C]pyruvate, hyperpolarized by dissolution-Dynamic Nuclear Polarization (d-DNP), in in vivo metabolic studies has developed quickly, thanks to the imaging probe's diagnostic relevance. Nevertheless, the cost of a d-DNP polarizer is quite high and the speed of hyperpolarization process is relatively slow, meaning that its use is limited to few research laboratories. ParaHydrogen Induced Polarization Side Arm Hydrogenation (PHIP-SAH) (Reineri et al., 2015) is a cost effective and easy-to-handle method that produces 13C-MR hyperpolarization in [1-13C]pyruvate and other metabolites. This work aims to identify the main determinants of the hyperpolarization levels observed in C13-pyruvate using this method. By dissecting the various steps of the PHIP-SAH procedure, it has been possible to assess the role of several experimental parameters whose optimization must be pursued if this method is to be made suitable for future translational steps. The search for possible solutions has led to improvements in the polarization of sodium [1-13C]pyruvate from 2% to 5%. Moreover, these results suggest that observed polarization levels could be increased considerably by an automatized procedure which would reduce the time required for the work-up passages that are currently carried out manually. The results reported herein mean that the attainment of polarization levels suitable for the metabolic imaging applications of these hyperpolarized substrates show significant promise.

In vivo evaluation of tumour acidosis for assessing the early metabolic response and onset of resistance to dichloroacetate by using magnetic resonance pH imaging.

Dichloroacetate (DCA) can reverse the glycolytic phenotype that is responsible of increased lactate production and extracellular pH acidification in cancer cells. Magnetic resonance imaging-chemical exchange saturation transfer (MRI-CEST) pH mapping is a novel non-invasive imaging approach that can measure in vivo extracellular tumour pH. We examined whether MRI-CEST pH mapping can monitor in vivo changes in tumour acidosis for assessing treatment response to DCA. Cell viability and extracellular pH were assessed in TS/A breast cancer cells treated with 1-10 mM DCA for 24 h in normoxia or hypoxia (1% O2) conditions. Extracellular tumour pH values were measured in vivo by MRI-CEST pH mapping of TS/A tumour-bearing mice before, three days and fifteen days after DCA or saline treatment. Reduced extracellular acidification and vitality were observed in DCA-treated TS/A cells. Tumour-bearing mice showed a marked and significant increase of tumour extracellular pH at 3 days post-DCA treatment, reflecting DCA-induced glycolysis inhibition, as confirmed by reduced lactate production. After 15 days of DCA treatment, the onset of resistance to DCA was observed, with recover of tumour extracellular acidification and lactate levels that returned to baseline values. A significant correlation was observed between tumour extracellular pH values and lactate levels (r= -0.97, P<0.05). These results suggest that MRI-CEST pH imaging is a promising tool to monitor the early response and efficacy of cancer metabolic targeting drugs.

13 C†MR Hyperpolarization of Lactate by Using ParaHydrogen and Metabolic Transformation in Vitro.

Hyperpolarization of the 13 C magnetic resonance signal of l-[1-13 C]lactate has been obtained using the chemically based, cost-effective method called parahydrogen-induced polarization by means of side-arm hydrogenation (PHIP-SAH). Two ester derivatives of lactate were tested and the factors that determine the polarization level on the product have been investigated in detail. The metabolic conversion of hyperpolarized l-[1-13 C]lactate into pyruvate has been observed in vitro using lactate dehydrogenase (LDH) and in a cells lysate. From the acquisition of a series of 13 C NMR spectra, the metabolic build-up of the [1-13 C]pyruvate signal has been observed. These studies demonstrate that, even if the experimental set-up used for these PHIP-SAH hyperpolarization studies is still far from optimal, the attained polarization level is already sufficient to carry out in vitro metabolic studies.

Assessing the transport rate of hyperpolarized pyruvate and lactate from the intra- to the extracellular space.

The use of [1-(13) C]pyruvate hyperpolarized by means of dynamic nuclear polarization provides a direct way to track the metabolic transformations of this metabolite in vivo and in cell cultures. The identification of the intra- and extracellular contributions to the (13) C NMR resonances is not straightforward. In order to obtain information about the rate of pyruvate and lactate transport through the cellular membrane, we set up a method that relies on the sudden 'quenching' of the extracellular metabolites' signal. The paramagnetic Gd-tetraazacyclododecane triacetic acid (Gd-DO3A) complex was used to dramatically decrease the longitudinal relaxation time constants of the (13) C-carboxylate resonances of both pyruvate and lactate. When Gd-DO3A was added to an MCF-7 cellular culture, which had previously received a dose of hyperpolarized [1-(13) C]pyruvate, the contributions of the extracellular pyruvate and lactate signals were deleted. From the analysis of the decay curves of the (13) C-carboxylate resonances of pyruvate and lactate it was possible to extract information about the exchange rate of the two metabolites across the cellular membrane. In particular, it was found that, in the reported experimental conditions, the lactate transport from the intra- to the extracellular space is not much lower than the rate of lactate formation. The method reported herein is non-destructive and it could be translated to in vivo studies. It opens a route for the use of hyperpolarized pyruvate to assess altered activity of carboxylate transporter proteins that may occur in pathological conditions. Copyright © 2016 John Wiley & Sons, Ltd.

Probing lactate secretion in tumours with hyperpolarised NMR.

Most tumours exhibit a high rate of glycolysis and predominantly produce energy by lactic acid fermentation. To maintain energy production and prevent toxicity, the lactate generated needs to be rapidly transported out of the cell. This is achieved by monocarboxylate transporters (MCTs), which therefore play an essential role in cancer metabolism and development. In vivo experiments were performed on eight male Fisher F344 rats bearing a subcutaneous mammary carcinoma after injection of hyperpolarised [1-(13) C]pyruvate. A Gd(III)DO3A complex that binds to pyruvate and its metabolites was used to efficiently destroy the extracellular magnetisation after hyperpolarised lactate had been formed. Moreover, a pulse sequence including a frequency-selective saturation pulse was designed so that the pyruvate magnetisation could be destroyed to exclude effects arising from further conversion. Given this preparation, metabolite transport out of the cell manifested as additional decay and apparent cell membrane transporter rates could thus be obtained using a reference measurement without a relaxation agent. In addition to slice-selective spectra, spatially resolved maps of apparent membrane transporter activity were acquired using a single-shot spiral gradient readout. A considerable increase in decay rate was detected for lactate, indicating rapid transport out of the cell. The alanine signal was unaltered, which corresponds to a slower efflux rate. This technique could allow for better understanding of tumour metabolism and progression, and enable treatment response measurements for MCT-targeted cancer therapies. Moreover, it provides vital insights into the signal kinetics of hyperpolarised [1-(13) C]pyruvate examinations. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of Magnetic Field Cycle on the Polarization Transfer from Parahydrogen to Heteronuclei through Long-Range J-Couplings.

Hyperpolarization of (13)C carboxylate signals of metabolically relevant molecules, such as acetate and pyruvate, was recently obtained by means of ParaHydrogen Induced Polarization by Side Arm Hydrogenation (PHIP-SAH). This method relies on functionalization of the carboxylic acid with an unsaturated alcohol (side arm), hydrogenation of the unsaturated alcohol using parahydrogen, and polarization transfer to the target (13)C signal. In this case, parahydrogen protons are added three to four bonds away from the target (13)C nucleus, while biologically relevant molecules had been hyperpolarized, using parahydrogen, through hydrogenation of an unsaturated bond adjacent to the target (13)C signal. The herein reported results show that the same polarization level can be obtained on the (13)C carboxylate signal of an ester by means of addition of parahydrogen to the acidic or to the alcoholic moiety and successive application of magnetic field cycle (MFC). Experimental results are supported by calculations that allow one to predict that, upon accurate control of magnetic field strength and speed of the passages, more than 20% polarization can be achieved on the (13)C-carboxylate resonance of the esters by means of side arm hydrogenation and MFC.

ParaHydrogen Induced Polarization of 13C carboxylate resonance in acetate and pyruvate.

The advent of nuclear spins hyperpolarization techniques represents a breakthrough in the field of medical diagnoses by magnetic resonance imaging. Dynamic nuclear polarization (DNP) is the most widely used method, and hyperpolarized metabolites such as [1-(13)C]-pyruvate are shown to report on status of tumours. Parahydrogen-induced polarization (PHIP) is a chemistry-based technique, easier to handle and much less expensive in respect to DNP, with significantly shorter polarization times. Its main limitation is the availability of unsaturated precursors for the target substrates; for instance, acetate and pyruvate cannot be obtained by direct incorporation of the parahydrogen molecule. Herein we report a method that allows us to achieve hyperpolarization in this kind of molecule by means of a tailored precursor containing a hydrogenable functionality that, after polarization transfer to the target (13)C moiety, is cleaved to obtain the metabolite of interest. The reported procedure can be extended to a number of other biologically relevant substrates.

15N magnetic resonance hyperpolarization via the reaction of parahydrogen with 15N-propargylcholine.

(15)N-Propargylcholine has been synthesized and hydrogenated with para-H(2). Through the application of a field cycling procedure, parahydrogen spin order is transferred to the (15)N resonance. Among the different isomers formed upon hydrogenation of (15)N-propargylcholine, only the nontransposed derivative contributes to the observed N-15 enhanced emission signal. The parahydrogen-induced polarization factor is about 3000. The precise identification of the isomer responsible for the observed (15)N enhancement has been attained through a retro-INEPT ((15)N-(1)H) experiment. T(1) of the hyperpolarized (15)N resonance has been estimated to be ca. 150 s, i.e., similar to that reported for the parent propargylcholine (144 s). Experimental results are accompanied by theoretical calculations that stress the role of scalar coupling constants (J(HN) and J(HH)) and of the field dependence in the formation of the observed (15)N polarized signal. Insights into the good cellular uptake of the compound have been gained.

Hyperpolarized (13)C-pyruvate magnetic resonance imaging in cancer diagnostics.

INTRODUCTION: The use of hyperpolarized molecules allows one to obtain information about metabolism in both cells and animals; such a task represents a tremendous advancement with respect to the results achieved so far with in vivo NMR techniques. Pyruvate appears an excellent tumor biomarker as it allows the attainment of early diagnosis, stadiation and monitoring of response to therapy. AREAS COVERED: As pyruvate conversion to lactate in the glycolytic pathway is highly enhanced in tumor cells, the 1-(13)C-lactate levels after administration of hyperpolarized 1-(13)C-pyruvate are markedly higher in tumor tissues and depend on the type and grade of the tumor. This review covers the most recent research results (both in vitro and in vivo) about the use of hyperpolarized 1-(13)C-pyruvate for tumor localization, stadiation and for monitoring the response to therapy. The technique may find application in clinics, especially when other imaging modalities are of difficult applicability. EXPERT OPINION: While (13)C-pyruvate has been shown to be the candidate of choice for metabolic imaging, high expectations are present in the scientific community to see if other hyperpolarized substrates could provide more specific and sensitive biomarkers. The use of hyperpolarized molecules will have a tremendous impact in the armory of diagnostic tools.