Jessica Bastiaansen

Automated transfer and injection of hyperpolarized molecules with polarization measurement prior to in vivo NMR

Hyperpolarized magnetic resonance via dissolution dynamic nuclear polarization necessitates the transfer of the hyperpolarized molecules from the polarizer to the imager prior to in vivo measurements. This process leads to unavoidable losses in nuclear polarization, which are difficult to evaluate once the solution has been injected into an animal. We propose a method to measure the polarization of the hyperpolarized molecules inside the imager bore, 3 s following dissolution, at the time of the injection, using a precise quantification of the infusate concentration. This in situ quantification allows for distinguishing between signal modulations related to variations in the nuclear polarization at the time of the injection and signal modulations related to physiological processes such as tissue perfusion. In addition, our method includes a radical scavenging process that leads to a minor reduction in sample concentration and takes place within a couple of seconds following the dissolution in order to minimize the losses due to the presence of paramagnetic polarizing agent in the infusate. We showed that proton exchange between vitamin C, the scavenging molecule and the deuterated solvent shortens the long carboxyl 13C longitudinal relaxation time in [1‐13C]acetate. This additional source of dipolar relaxation can be avoided by using deuterated ascorbate. Overall, the method allows for a substantial gain in polarization and also leads to an extension of the time window available for in vivo measurements. Copyright

In vivo enzymatic activity of acetylCoA synthetase in skeletal muscle revealed by 13C turnover from hyperpolarized [1-13C]acetate to [1-13C]acetylcarnitine

BACKGROUND Acetate metabolism in skeletal muscle is regulated by acetylCoA synthetase (ACS). The main function of ACS is to provide cells with acetylCoA, a key molecule for numerous metabolic pathways including fatty acid and cholesterol synthesis and the Krebs cycle. METHODS Hyperpolarized [1-(13)C]acetate prepared via dissolution dynamic nuclear polarization was injected intravenously at different concentrations into rats. The (13)C magnetic resonance signals of [1-(13)C]acetate and [1-(13)C]acetylcarnitine were recorded in vivo for 1min. The kinetic rate constants related to the transformation of acetate into acetylcarnitine were deduced from the 3s time resolution measurements using two approaches, either mathematical modeling or relative metabolite ratios. RESULTS Although separated by two biochemical transformations, a kinetic analysis of the (13)C label flow from [1-(13)C]acetate to [1-(13)C]acetylcarnitine led to a unique determination of the activity of ACS. The in vivo Michaelis constants for ACS were KM=0.35±0.13mM and Vmax=0.199±0.031μmol/g/min. CONCLUSIONS The conversion rates from hyperpolarized acetate into acetylcarnitine were quantified in vivo and, although separated by two enzymatic reactions, these rates uniquely defined the activity of ACS. The conversion rates associated with ACS were obtained using two analytical approaches, both methods yielding similar results. GENERAL SIGNIFICANCE This study demonstrates the feasibility of directly measuring ACS activity in vivo and, since the activity of ACS can be affected by various pathological states such as cancer or diabetes, the proposed method could be used to non-invasively probe metabolic signatures of ACS in diseased tissue.

An Intact Small Animal Model of Myocardial Ischemia-Reperfusion: Characterization of Metabolic Changes by Hyperpolarized 13C MR Spectroscopy

Hyperpolarized carbon-13 magnetic resonance spectroscopy ((13)C MRS) enables the sensitive and noninvasive assessment of the metabolic changes occurring during myocardial ischemia-reperfusion. Ischemia-reperfusion models using hyperpolarized (13)C MRS are established in heart preparations ex vivo and in large animals in vivo, but an in vivo model in small animals would be advantageous to allow the study of reperfusion metabolism with neuroendocrine and inflammatory responses intact with the option to perform a greater number of experiments. A novel intact rat model of ischemia-reperfusion is presented that incorporates hyperpolarized (13)C MRS to characterize reperfusion metabolism. Typically, in an in vivo model, a tissue input function (TIF) is required to account for apparent changes in the metabolism of injected hyperpolarized [1-(13)C]pyruvate resulting from changes in perfusion. Whereas the measurement of a TIF by metabolic imaging is particularly challenging in small animals, the ratios of downstream metabolites can be used as an alternative. The ratio of [(13)C]bicarbonate:[1-(13)C]lactate (RatioBic/Lac) measured within 1-2 min after coronary release decreased vs. baseline in ischemic rats (n = 10, 15-min occlusion, controls: n = 10; P = 0.017 for interaction, 2-way ANOVA). The decrease in oxidative pyruvate metabolism [RatioBic/Lac(Ischemia)/RatioBic/Lac(Baseline)] modestly correlated with area at risk (r = 0.66; P = 0.002). Hyperpolarized (13)C MRS was also used to examine alanine production during ischemia, which is observed in ex vivo models, but no significant change was noted; metrics incorporating [1-(13)C]alanine did not substantially improve the discrimination of ischemic-reperfused myocardium from nonischemic myocardium. This intact rat model, which mimics the human situation of reperfused myocardial infarction, could be highly valuable for the testing of new drugs to treat reperfusion injury, thereby facilitating translational research.

Direct noninvasive estimation of myocardial tricarboxylic acid cycle flux in vivo using hyperpolarized 13C magnetic resonance

BACKGROUND The heart relies on continuous energy production and imbalances herein impair cardiac function directly. The tricarboxylic acid (TCA) cycle is the primary means of energy generation in the healthy myocardium, but direct noninvasive quantification of metabolic fluxes is challenging due to the low concentration of most metabolites. Hyperpolarized (13)C magnetic resonance spectroscopy (MRS) provides the opportunity to measure cellular metabolism in real time in vivo. The aim of this work was to noninvasively measure myocardial TCA cycle flux (VTCA) in vivo within a single minute. METHODS AND RESULTS Hyperpolarized [1-(13)C]acetate was administered at different concentrations in healthy rats. (13)C incorporation into [1-(13)C]acetylcarnitine and the TCA cycle intermediate [5-(13)C]citrate was dynamically detected in vivo with a time resolution of 3s. Different kinetic models were established and evaluated to determine the metabolic fluxes by simultaneously fitting the evolution of the (13)C labeling in acetate, acetylcarnitine, and citrate. VTCA was estimated to be 6.7±1.7 μmol·g(-1)·min(-1) (dry weight), and was best estimated with a model using only the labeling in citrate and acetylcarnitine, independent of the precursor. The TCA cycle rate was not linear with the citrate-to-acetate metabolite ratio, and could thus not be quantified using a ratiometric approach. The (13)C signal evolution of citrate, i.e. citrate formation was independent of the amount of injected acetate, while the (13)C signal evolution of acetylcarnitine revealed a dose dependency with the injected acetate. The (13)C labeling of citrate did not correlate to that of acetylcarnitine, leading to the hypothesis that acetylcarnitine formation is not an indication of mitochondrial TCA cycle activity in the heart. CONCLUSIONS Hyperpolarized [1-(13)C]acetate is a metabolic probe independent of pyruvate dehydrogenase (PDH) activity. It allows the direct estimation of VTCA in vivo, which was shown to be neither dependent on the administered acetate dose nor on the (13)C labeling of acetylcarnitine. Dynamic (13)C MRS coupled to the injection of hyperpolarized [1-(13)C]acetate can enable the measurement of metabolic changes during impaired heart function.

Measuring changes in substrate utilization in the myocardium in response to fasting using hyperpolarized [1- 13 C]butyrate and [1- 13 C]pyruvate

Cardiac dysfunction is often associated with a shift in substrate preference for ATP production. Hyperpolarized (HP) 13C magnetic resonance spectroscopy (MRS) has the unique ability to detect real-time metabolic changes in vivo due to its high sensitivity and specificity. Here a protocol using HP [1-13C]pyruvate and [1-13C]butyrate is used to measure carbohydrate versus fatty acid metabolism in vivo. Metabolic changes in fed and fasted Sprague Dawley rats (n = 36) were studied at 9.4 T after tail vein injections. Pyruvate and butyrate competed for acetyl-CoA production, as evidenced by significant changes in [13C]bicarbonate (−48%), [1-13C]acetylcarnitine (+113%), and [5-13C]glutamate (−63%), following fasting. Butyrate uptake was unaffected by fasting, as indicated by [1-13C]butyrylcarnitine. Mitochondrial pseudoketogenesis facilitated the labeling of the ketone bodies [1-13C]acetoacetate and [1-13C]β-hydroxybutyryate, without evidence of true ketogenesis. HP [1-13C]acetoacetate was increased in fasting (250%) but decreased during pyruvate co-injection (−82%). Combining HP 13C technology and co-administration of separate imaging agents enables noninvasive and simultaneous monitoring of both fatty acid and carbohydrate oxidation. This protocol illustrates a novel method for assessing metabolic flux through different enzymatic pathways simultaneously and enables mechanistic studies of the changing myocardial energetics often associated with disease.

Direct Monitoring of γ-Glutamyl Transpeptidase Activity In Vivo Using a Hyperpolarized (13) C-Labeled Molecular Probe.

The γ-glutamyl transpeptidase (GGT) enzyme plays a central role in glutathione homeostasis. Direct detection of GGT activity could provide critical information for the diagnosis of several pathologies. We propose a new molecular probe, γ-Glu-[1-(13) C]Gly, for monitoring GGT activity in vivo by hyperpolarized (HP) (13) C magnetic resonance (MR). The properties of γ-Glu-[1-(13) C]Gly are suitable for in vivo HP (13) C metabolic analysis since the chemical shift between γ-Glu-[1-(13) C]Gly and its metabolic product, [1-(13) C]Gly, is large (4.3 ppm) and the T1 of both compounds is relatively long (30 s and 45 s, respectively, in H2 O at 9.4 T). We also demonstrate that γ-Glu-[1-(13) C]Gly is highly sensitive to in vivo modulation of GGT activity induced by the inhibitor acivicin.

Tuning Properties of Iron Oxide Nanoparticles in Aqueous Synthesis without Ligands to Improve MRI Relaxivity and SAR

Aqueous synthesis without ligands of iron oxide nanoparticles (IONPs) with exceptional properties still remains an open issue, because of the challenge to control simultaneously numerous properties of the IONPs in these rigorous settings. To solve this, it is necessary to correlate the synthesis process with their properties, but this correlation is until now not well understood. Here, we study and correlate the structure, crystallinity, morphology, as well as magnetic, relaxometric and heating properties of IONPs obtained for different durations of the hydrothermal treatment that correspond to the different growth stages of IONPs upon initial co-precipitation in aqueous environment without ligands. We find that their properties were different for IONPs with comparable diameters. Specifically, by controlling the growth of IONPs from primary to secondary particles firstly by colloidal and then also by magnetic interactions, we control their crystallinity from monocrystalline to polycrystalline IONPs, respectively. Surface energy minimization in the aqueous environment along with low temperature treatment is used to favor nearly defect-free IONPs featuring superior properties, such as high saturation magnetization, magnetic volume, surface crystallinity, the transversal magnetic resonance imaging (MRI) relaxivity (up to r2 = 1189 mM−1·s−1 and r2/r1 = 195) and specific absorption rate, SAR (up to 1225.1 W·gFe−1).

Real time measurement of myocardial substrate selection in vivo using hyperpolarized 13C magnetic resonance

Background Cardiac dysfunction is often associated with a shift in substrate preference and metabolism, but current in vivo techniques only provide information on substrate uptake. Hyperpolarized (HP) MR has the unique ability to detect metabolism in vivo and is highly specific. To evaluate the prospects for measuring myocardial substrate selection in vivo ,H P [1- 13 C]pyruvate and [1- 13 C] butyrate were co-infused into rats. Methods Pyruvate and butyrate were hyperpolarized by DNP. After dissolution the samples were automatically infused into fed or fasted animals. Myocardial metabolism was detected using a train of cardiac triggered 30 o adiabatic inspection pulses applied every 3 s at 9.4T with 1 H decoupling. Metabolite ratios were calculated to assess overall consumption of the imaging agents. Results

Versatility of Pyridoxal Phosphate as a Coating of Iron Oxide Nanoparticles

Pyridoxal 5′-phosphate (PLP) is the most important cofactor of vitamin B6-dependent enzymes, which catalyses a wide range of essential body functions (e.g., metabolism) that could be exploited to specifically target highly metabolic cells, such as tumour metastatic cells. However, the use of PLP as a simultaneous coating and targeting molecule, which at once provides colloidal stability and specific biological effects has not been exploited so far. Therefore, in this work iron oxide nanoparticles (IONPs) were coated by PLP at two different pH values to tune PLP bonding (e.g., orientation) at the IONP surface. The surface study, as well as calculations, confirmed different PLP bonding to the IONP surface at these two pH values. Moreover, the obtained PLP-IONPs showed different zeta potential, hydrodynamic radius and agglomeration state, and consequently different uptake by two metastatic-prostate-cancer cell lines (LnCaP and PC3). In LnCaP cells, PLP modified the morphology of IONP-containing intracellular vesicles, while in PC3 cells PLP impacted the amount of IONPs taken up by cells. Moreover, PLP-IONPs displayed high magnetic resonance imaging (MRI) r2 relaxivity and were not toxic for the two studied cell lines, rendering PLP promising for biomedical applications. We here report the use of PLP simultaneously as a coating and targeting molecule, directly bound to the IONP surface, with the additional high potential for MRI detection.

Characterization of perfluorocarbon relaxation times and their influence on the optimization of fluorine-19 MRI at 3 tesla.

To characterize and optimize 19F MRI for different perfluorocarbons (PFCs) at 3T and quantify the loss of acquisition efficiency as a function of different temperature and cellular conditions.