Characterization of Brain Metabolism by Nuclear Magnetic Resonance.

The noninvasive, quantitative ability of nuclear magnetic resonance (NMR) spectroscopy to characterize small molecule metabolites has long been recognized as a major strength of its application in biology. Numerous techniques exist for characterizing metabolism in living, excised, or extracted tissue, with a particular focus on 1 H-based methods due to the high sensitivity and natural abundance of protons. With the increasing use of high magnetic fields, the utility of in vivo 1 H magnetic resonance spectroscopy (MRS) has markedly improved for measuring specific metabolite concentrations in biological tissues. Higher fields, coupled with recent developments in hyperpolarization, also enable techniques for complimenting 1 H measurements with spectroscopy of other nuclei, such as 31 P and 13 C, and for combining measurements of metabolite pools with metabolic flux measurements. We compare ex vivo and in vivo methods for studying metabolism in the brain using NMR and highlight insights gained through using higher magnetic fields, the advent of dissolution dynamic nuclear polarization, and combining in vivo MRS and ex vivo NMR approaches.

Ratiometric pH Imaging with a CoII2 MRI Probe via CEST Effects of Opposing pH Dependences.

We report a Co2-based magnetic resonance (MR) probe that enables the ratiometric quantitation and imaging of pH through chemical exchange saturation transfer (CEST). This approach is illustrated in a series of air- and water-stable CoII2 complexes featuring CEST-active tetra(carboxamide) and/or hydroxyl-substituted bisphosphonate ligands. For the complex bearing both ligands, variable-pH CEST and NMR analyses reveal highly shifted carboxamide and hydroxyl peaks with intensities that increase and decrease with increasing pH, respectively. The ratios of CEST peak intensities at 104 and 64 ppm are correlated with solution pH in the physiological range 6.5-7.6 to construct a linear calibration curve of log(CEST104 ppm/CEST64 ppm) versus pH, which exhibits a remarkably high pH sensitivity of 0.99(7) pH unit-1 at 37 °C. In contrast, the analogous CoII2 complex with a CEST-inactive bisphosphonate ligand exhibits no such pH response, confirming that the pH sensitivity stems from the integration of amide and hydroxyl CEST effects that show base- and acid-catalyzed proton exchange, respectively. Importantly, the pH calibration curve is independent of the probe concentration and is identical in aqueous buffer and fetal bovine serum. Furthermore, phantom images reveal analogous linear pH behavior. The CoII2 probe is stable toward millimolar concentrations of H2PO4-/HPO42-, CO32-, SO42-, CH3COO-, and Ca2+ ions, and more than 50% of melanoma cells remain viable in the presence of millimolar concentrations of the complex. The stability of the probe in physiological environments suggests that it may be suitable for in vivo studies. Together, these results highlight the ability of dinuclear transition metal PARACEST probes to provide a concentration-independent measure of pH, and they provide a potential design strategy toward the development of MR probes for ratiometric pH imaging.

Expeditious dissolution dynamic nuclear polarization without glassing agents.

The hyperpolarization of metabolic substrates at low temperature using dynamic nuclear polarization (DNP), followed by rapid dissolution and injection into an MRSI or NMR system, allows in vitro or in vivo observation and tracking of biochemical reactions and metabolites in real time. This article describes an elegant approach to sample preparation which is broadly applicable for the rapid polarization of aqueous small-molecule substrate solutions and obviates the need for glassing agents. We demonstrate its utility for solutions of sodium acetate, pyruvate and butyrate. The polarization behavior of substrates prepared using rapid freezing without glassing agents enabled a 1.5-3-fold time savings in polarization buildup, whilst removing the need for toxic glassing agents used as standard for dissolution DNP. The achievable polarization with fully aqueous substrate solutions was equal to that observed using standard approaches and glassing agents. Copyright © 2016 John Wiley & Sons, Ltd.

Structure and dynamics of aqueous 2-propanol: a THz-TDS, NMR and neutron diffraction study.

Aqueous liquid mixtures, in particular, those involving amphiphilic species, play an important role in many physical, chemical and biological processes. Of particular interest are alcohol/water mixtures; however, the structural dynamics of such systems are still not fully understood. Herein, a combination of terahertz time-domain spectroscopy (THz-TDS) and NMR relaxation time analysis has been applied to investigate 2-propanol/water mixtures across the entire composition range; while neutron diffraction studies have been carried out at two specific concentrations. Excellent agreement is seen between the techniques with a maximum in both the relative absorption coefficient and the activation energy to molecular motion occurring at ∼90 mol% H2O. Furthermore, this is the same value at which well-established excess thermodynamic functions exhibit a maximum/minimum. Additionally, both neutron diffraction and THz-TDS have been used to provide estimates of the size of the hydration shell around 2-propanol in solution. Both methods determine that between 4 and 5 H2O molecules per 2-propanol are found in the 2-propanol/water clusters at 90 mol% H2O. Based on the acquired data, a description of the structure of 2-propanol/water across the composition range is presented.

Type-1 transforming growth factor-beta differentially modulates tumoricidal activity of murine peritoneal macrophages against metastatic variants of the B16 murine melanoma.

Transforming growth factor-beta 1 (TGF-beta 1) renders mouse peritoneal macrophages tumoricidal against metastatic variants of the B16 mouse melanoma in vitro. Both direct cytotoxicity and indirect cytotoxicity were observed. A subthreshold concentration (10 U/ml) of recombinant murine interferon-gamma (rMuIFN-gamma) enhanced the direct tumoricidal activity of TGF-beta 1-activated macrophages from 29% to 88% but did not change their indirect tumoricidal profile. Data obtained from macrophages preincubated with either TGF-beta 1 or rMuIFN-gamma showed that TGF-b1 can initiate tumoricidal activity better than rMuIFN-gamma. These effects were plasma-membrane mediated because targeting macrophages with liposomal TGF-beta 1 was ineffective. The order of tumoricidal susceptibility of the B16 melanoma lines to activated macrophages was B16F1 > B16F10 > B16BL6, in inverse order of metastatic potential.