hyperpolarized-13c-metabolic-magnetic-resonance-spectroscopy

Hyperpolarized <sup>13</sup>C Metabolic Magnetic Resonance Spectroscopy and Imaging

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have ...

Technische Universität München

Helmholtz Zentrum München

Human serum albumin (HSA) is known for its exceptional ligand-binding capacity; indeed, its modular domain organization provides a variety of ligand-binding sites. Its flexible modular structure involves more than the immediate vicinity of the binding site(s), affecting the ligand-binding properties of the whole protein. Here, biochemical characterization by (1)H-NMR relaxometry and optical spectroscopy of a truncated form of HSA (tHSA) encompassing domains I and II (Asp1-Glu382) is reported. Removal of the C-terminal domain III results in a number of contacts that involve domain I (containing the heme site) and domain II (containing the warfarin site) being lost; however, the allosteric linkage between heme and warfarin sites is maintained. tHSA shows a nuclear magnetic relaxation dispersion profile similar to that of HSA, and displays increased affinity for ibuprofen, warfarin, and heme, suggesting that the fold is preserved. Moreover, the allosteric properties that make HSA a peculiar monomeric protein and account for the regulation of ligand-binding modes by heterotropic interactions are maintained after removal of domain III. Therefore, tHSA is a valuable model with which to investigate allosteric properties of HSA, allowing independent analysis of the linkages between different drug-binding sites.

Allosteric and binding properties of Asp1-Glu382 truncated recombinant human serum albumin--an optical and NMR spectroscopic investigation.

Ferric human serum heme-albumin (heme-HSA) shows a peculiar nuclear magnetic relaxation dispersion (NMRD) behavior that allows to investigate structural and functional properties. Here, we report a thermodynamic analysis of NMRD profiles of heme-HSA between 20 and 60 degrees C to characterize its hydration. NMRD profiles, all showing two Lorentzian dispersions at 0.3 and 60 MHz, were analyzed in terms of modulation of the zero field splitting tensor for the S=5/2 manifold. Values of correlation times for tensor fluctuation (tau(v)) and chemical exchange of water molecules (tau(M)) show the expected temperature dependence, with activation enthalpies of -1.94 and -2.46+/-0.2 kJ mol(-1), respectively. The cluster of water molecules located in the close proximity of the heme is progressively reduced in size by increasing the temperature, with DeltaH=68+/-28 kJ mol(-1) and DeltaS=200+/-80 J mol(-1) K(-1). These results highlight the role of the water solvent in heme-HSA structure-function relationships.

Thermodynamic analysis of hydration in human serum heme-albumin.

The development of photochromic Computer Generated Holograms (CGHs) to test any complex optics, such as aspheres and free-form optics, is described. A thermally irreversible photochromic polyurethane has been synthesized to give good thin films with a strong modulation of the optical transmission. The photochromic CGH has been tested with a simple interferometrical configuration showing promising results. The use of photochromic CGHs provides advantages over standard technologies, as rewritability and self developing.

Photochromic polyurethanes for rewritable CGHs in optical testing.

Quantum yield is one of the most important properties of photochromic systems. Unfortunately, a lack of data at the solid state exists, because measurements are intrinsically not straightforward. A kinetic model describing the conversion of the photoactive species is reported and both analytic and numeric solutions are provided according to relevant cases. The model is then applied to measure the quantum yield of dithienylethene-based polymers; the ring-opening quantum yield is measured for different laser beam profiles (i.e., Gaussian and uniform) and at different wavelengths, showing an increased value with increasing photon energy.

Kinetics of photochromic conversion at the solid state: quantum yield of dithienylethene-based films.

The unambiguous detection of specific neuronal subtypes is up to now only possible with invasive techniques or optical imaging after genetic modification. High field magnetic resonance imaging (MRI) has the ability to visualize the brain structure and anatomy noninvasively, with high resolution--but missing the cell specific and functional information. Here we present a new tool for neuroimaging with MRI, enabling the selective detection of GABAergic neurons under in vivo conditions. The specific imaging contrast is achieved by a novel paramagnetic contrast agent, which responds to the activity of the enzyme glutamic acid decarboxylase--expressed solely by inhibitory neurons. The relaxivity of the complex is increased upon decarboxylation of two glutamic acid moieties, thus allowing increased water access to the inner and outer coordination spheres of the paramagnetic ion. The mechanism and specificity of activation were proven with tissue lysates and further applied to a differentiation protocol for murine embryonic stem cells. The relaxation enhancement was studied quantitatively and revealed decreased longitudinal relaxation times in the inhibitory neuron samples compared to the naïve stem cells in vitro and in vivo. Furthermore, this approach offers not only the discrimination of inhibitory, GABAergic neurons in the brain but also may expand the usefulness of MRI for functional imaging on a cellular level.

In vivo imaging of inhibitory, GABAergic neurons by MRI.

Novel contrast agent based systems, which selectively visualize specific cells, e.g., neurons in the brain, would be of substantial importance for the fast developing field of molecular magnetic resonance imaging (MRI). We report here the synthesis and in vitro validation of a Gd(III)-based contrast agent designed to act as an MRI responsive probe for imaging the activity of the enzyme glutamic acid decarboxylase (GAD) present in neurons. Upon the action of the enzyme, the Gd(III) complex increases its hydration sphere and takes on a residual positive charge that promotes its binding to endogenous macromolecules. Both effects contribute in a synergic way to generate a marked relaxation enhancement, which directly reports enzyme activity and will allow activity detection of GAD positive cells in vitro and in vivo selectively.

Synthesis and relaxometric characterization of a MRI Gd-based probe responsive to glutamic acid decarboxylase enzymatic activity.

We report the synthesis and characterization of a water-soluble, star-shaped macromolecular platform consisting of eight β-cyclodextrin (β-CD) units anchored to the narrower rim of a γ-CD core through bis(triazolyl)alkyl spacers. The efficient synthetic protocol is based on the microwave (MW)-promoted Cu-catalyzed 1,3-dipolar cycloaddition of CD monoazides to CD monoacetylenes. The ligand-hosting capability of the construct has been assessed by relaxometric titration and nuclear magnetic relaxation dispersion (NMRD) profiling, which showed it to be good, and this was supported by molecular dynamics simulations. To demonstrate the feasibility of obtaining supramolecular structures with high hosting ability, we designed a dimeric platform, formed by joining two nonamers through the γ-CD cores through a bis(lithocholic acid) linker. With a view to the potential biological applications, cytotoxicity and extent of binding to human serum albumin were assessed. The properties of this dendrimeric multicarrier make it suitable for pharmaceutical and diagnostic purposes, ranging from targeted drug delivery to molecular imaging.

Design and Synthesis of a γ(1)β(8)-Cyclodextrin Oligomer: A New Platform with Potential Application as a Dendrimeric Multicarrier.

A kinetic model describing the conversion of a photochromic layer under complex illumination conditions is applied to absorbance-modulation optical lithography to determine the influence of the material characteristics on the confinement to subdiffraction dimensions of the transmitted dose. We show that the most important parameters are the intensity ratio between the confining and writing beams, the overall absorption at the writing wavelength, the relative absorption coefficients, and the photoreaction quantum yields at the two wavelengths. As the confining beam ultimately determines the transferred dose pattern, we conclude that the modulation of the writing beam is not strictly necessary to produce subwavelength apertures.

Modeling absorbance-modulation optical lithography in photochromic films.

In this paper, a photochromic electret for light energy harvesting is proposed and discussed. Such electret directly converts the photon energy into electric energy thanks to a polarization modulation caused by the photochromic reaction, which leads to a change in dipole moment. Theoretical concepts on which the photochromic electret is based are considered with an estimation of the effectiveness as a function of material properties. Finally, an electret based on a photochromic diarylethene is shown with the photoelectric characterization as a proof of concept device.

Photochromic Electret: A New Tool for Light Energy Harvesting.

We introduce hyperpolarizable (13)C-labeled probes that identify multiple biologically important divalent metals via metal-specific chemical shifts. These features enable NMR measurements of calcium concentrations in human serum in the presence of magnesium. In addition, signal enhancement through dynamic nuclear polarization (DNP) increases the sensitivity of metal detection to afford measuring micromolar concentrations of calcium as well as simultaneous multi-metal detection by chemical shift imaging. The hyperpolarizable (13)C-MRI sensors presented here enable sensitive NMR measurements and MR imaging of multiple divalent metals in opaque biological samples.

Hyperpolarized Multi-Metal (13)C-Sensors for Magnetic Resonance Imaging.

The dipole moment of photochromic diarylethenes is determined in solution for both the coloured and uncoloured forms by measuring the capacitance of a capacitor filled with a photochromic solution as a dielectric material. Diarylethenes with different substituents are investigated and the modulation of the dipole moment is related to their chemical structures. We determine a modulation of the dipole moment up to 4 Debye. We discuss the model used to obtain the dipole moment from the capacitance measurements and we compare the experimental results with the outcomes from DFT calculations. The results highlight the importance of conformational effects in the description of the dipole moment of diarylethenes.

Article	Year
Allosteric and binding properties of Asp1-Glu382 truncated recombinant human serum albumin--an optical and NMR spectroscopic investigation. The FEBS journal\| PubMed ID: 19298387	2009
Thermodynamic analysis of hydration in human serum heme-albumin. Biochemical and biophysical research communications\| PubMed ID: 19464261	2009
Photochromic polyurethanes for rewritable CGHs in optical testing. Optics express\| PubMed ID: 21369285	2011
Kinetics of photochromic conversion at the solid state: quantum yield of dithienylethene-based films. The journal of physical chemistry. A\| PubMed ID: 21939221	2011
In vivo imaging of inhibitory, GABAergic neurons by MRI. NeuroImage\| PubMed ID: 22609794	2012
Synthesis and relaxometric characterization of a MRI Gd-based probe responsive to glutamic acid decarboxylase enzymatic activity. Journal of medicinal chemistry\| PubMed ID: 23469759	2013
Design and Synthesis of a γ(1)β(8)-Cyclodextrin Oligomer: A New Platform with Potential Application as a Dendrimeric Multicarrier. Chemistry (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 23873698	2013
Modeling absorbance-modulation optical lithography in photochromic films. Optics letters\| PubMed ID: 24104638	2013
Photochromic Electret: A New Tool for Light Energy Harvesting. The journal of physical chemistry letters\| PubMed ID: 26697774	2012
Hyperpolarized Multi-Metal (13)C-Sensors for Magnetic Resonance Imaging. Analytical chemistry\| PubMed ID: 27766840	2016
Light-induced dipole moment modulation in diarylethenes: a fundamental study. Physical chemistry chemical physics : PCCP\| PubMed ID: 27812576	2016

Giorgio Pariani

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