Lloyd Lumata

Glutamine oxidation maintains the TCA cycle and cell survival during impaired mitochondrial pyruvate transport.

Alternative modes of metabolism enable cells to resist metabolic stress. Inhibiting these compensatory pathways may produce synthetic lethality. We previously demonstrated that glucose deprivation stimulated a pathway in which acetyl-CoA was formed from glutamine downstream of glutamate dehydrogenase (GDH). Here we show that import of pyruvate into the mitochondria suppresses GDH and glutamine-dependent acetyl-CoA formation. Inhibiting the mitochondrial pyruvate carrier (MPC) activates GDH and reroutes glutamine metabolism to generate both oxaloacetate and acetyl-CoA, enabling persistent tricarboxylic acid (TCA) cycle function. Pharmacological blockade of GDH elicited largely cytostatic effects in culture, but these effects became cytotoxic when combined with MPC inhibition. Concomitant administration of MPC and GDH inhibitors significantly impaired tumor growth compared to either inhibitor used as a single agent. Together, the data define a mechanism to induce glutaminolysis and uncover a survival pathway engaged during compromised supply of pyruvate to the mitochondria.

DNP by Thermal Mixing under Optimized Conditions Yields >60 000-fold Enhancement of 89Y NMR Signal

Hyperpolarized (89)Y complexes are attractive NMR spectroscopy and MR imaging probes due to the exceptionally long spin-lattice relaxation time (T(1) ≈ 10 min) of the (89)Y nucleus. However, in vivo imaging of (89)Y has not yet been realized because of the low NMR signal enhancement levels previously achieved for this ultra low-γ(n) nucleus. Here, we report liquid-state (89)Y NMR signal enhancements over 60,000 times the thermal signal at 298 K in a 9.4 T magnet, achieved after the dynamic nuclear polarization (DNP) of Y(III) complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) samples at 3.35 T and 1.4 K. The (89)Y DNP was shown to proceed by thermal mixing and the liquid state (89)Y NMR signal enhancement was maximized by (i) establishing the optimal microwave irradiation frequency, (ii) optimizing the glassing matrix, (iii) choosing a radical with negligible inhomogeneous line broadening contribution to the ESR linewidth, and (iv) addition of an electron T(1e) relaxation agent. The highest enhancements were achieved using a trityl OX063 radical combined with a gadolinium relaxation agent in water-glycerol matrix. Co-polarization of (89)YDOTA and sodium [1-(13)C]pyruvate showed that both (89)Y and (13)C nuclear species acquired the same spin temperature, consistent with thermal mixing theory of DNP. This methodology may be applicable for the optimization of DNP of other low-γ(n) nuclei.

BDPA: an efficient polarizing agent for fast dissolution dynamic nuclear polarization NMR spectroscopy.

Keywords: dynamic nuclear polarization ; hyperpolarization ; NMR spectroscopy ; radicals ; relaxation ; Signal Reference EPFL-ARTICLE-169192doi:10.1002/chem.201102037View record in Web of Science Record created on 2011-10-03, modified on 2017-05-12

Electron spin resonance studies of trityl OX063 at a concentration optimal for DNP.

We have performed temperature-dependent electron spin resonance (ESR) measurements of the stable free radical trityl OX063, an efficient polarizing agent for dissolution dynamic nuclear polarization (DNP), at the optimum DNP concentration (15 mM). We have found that (i) when compared to the W-band electron spin-lattice relaxation rate T1e(-1) of other free radicals used in DNP at the same concentration, trityl OX063 has slower T1e(-1) than BDPA and 4-oxo-TEMPO. At T > 20 K, the T1e(-1)vs. T data of trityl OX063 appears to follow a power law dependence close to the Raman process prediction whereas at T < 10 K, electronic relaxation slows and approaches the direct process behaviour. (ii) Gd(3+) doping, a factor known to enhance DNP, of trityl OX063 samples measured at W-band resulted in monotonic increases of T1e(-1) especially at temperatures below 20-40 K while the ESR lineshapes remained essentially unchanged. (iii) The high frequency ESR spectrum can be fitted with an axial g-tensor with a slight g-anisotropy: g(x) = g(y) = 2.00319(3) and g(z) = 2.00258(3). Although the ESR linewidth D monotonically increases with field, the temperature-dependent T1e(-1) is almost unchanged as the ESR frequency is increased from 9.5 GHz to 95 GHz, but becomes faster at 240 GHz and 336 GHz. The ESR properties of trityl OX063 reported here may provide insights into the efficiency of DNP of low-γ nuclei performed at various magnetic fields, from 0.35 T to 12 T.

Hyperpolarized 15 N-pyridine Derivatives as pH-Sensitive MRI Agents

Highly sensitive MR imaging agents that can accurately and rapidly monitor changes in pH would have diagnostic and prognostic value for many diseases. Here, we report an investigation of hyperpolarized 15N-pyridine derivatives as ultrasensitive pH-sensitive imaging probes. These molecules are easily polarized to high levels using standard dynamic nuclear polarization (DNP) techniques and their 15N chemical shifts were found to be highly sensitive to pH. These probes displayed sharp 15N resonances and large differences in chemical shifts (Δδ >90u2005ppm) between their free base and protonated forms. These favorable features make these agents highly suitable candidates for the detection of small changes in tissue pH near physiological values.

Influence of deuteration in the glassing matrix on 13C dynamic nuclear polarization

Replacement of protons by deuterons in the glassing solvents led to 2-3-fold improvement of the (13)C dynamic nuclear polarization (DNP) solid-state NMR signal for samples doped with large electron spin resonance (ESR) linewidth free radicals galvinoxyl, DPPH, and 4-oxo-TEMPO. Meanwhile, the reverse effect is observed for (13)C DNP using small ESR linewidth free radicals BDPA and trityl OX063.

The effect of 13C enrichment in the glassing matrix on dynamic nuclear polarization of [1-13C]pyruvate.

Dimethyl sulfoxide (DMSO) can effectively form a glassy matrix necessary for dynamic nuclear polarization (DNP) experiments. We tested the effects of (13)C enrichment in DMSO on DNP of [1-(13)C]pyruvate doped with trityl radical OX063Me. We found that the polarization build-up time τ of pyruvate in (13)C-labeled DMSO glassing solution is twice as fast as the unenriched DMSO while the nuclear magnetic resonance enhancement was unchanged. This indicates that (13)C-(13)C spin diffusion is a limiting factor in the kinetics of DNP in this system, but it has a minimal effect on the absolute value of polarization achievable for the target.

Production and NMR Characterization of Hyperpolarized 107,109Ag Complexes

Both isotopes of silver, (107)Ag and (109)Ag, were simultaneously polarized by dynamic nuclear polarization (DNP), thus allowing large signal enhancements and the NMR characterization of Ag complexes in the millimolar concentration range. Since both isotopes have long relaxation times T(1), the hyperpolarized NMR signal of one isotope could still be observed even after the magnetization of the other isotope had been destroyed by radio-frequency pulses.

Dissolution DNP-NMR spectroscopy using galvinoxyl as a polarizing agent.

The goal of this work was to test feasibility of using galvinoxyl (2,6-di-tert-butyl-α-(3,5-di-tert-butyl-4-oxo-2,5-cyclohexadien-1-ylidene)-p-tolyloxy) as a polarizing agent for dissolution dynamic nuclear polarization (DNP) NMR spectroscopy. We have found that galvinoxyl is reasonably soluble in ethyl acetate, chloroform, or acetone and the solutions formed good glasses when mixed together or with other solvents such as dimethyl sulfoxide. W-band electron spin resonance (ESR) measurements revealed that galvinoxyl has an ESR linewidth D intermediate between that of carbon-centered free radical trityl OX063 and the nitroxide-based 4-oxo-TEMPO, thus the DNP with galvinoxyl for nuclei with low gyromagnetic ratio γ such as (13)C and (15)N is expected to proceed predominantly via the thermal mixing process. The optimum radical concentration that would afford the highest (13)C nuclear polarization (approximately 6% for [1-(13)C]ethyl acetate) at 3.35 T and 1.4 K was found to be around 40 mM. After dissolution, large liquid-state NMR enhancements were achieved for a number of (13)C and (15)N compounds with long spin-lattice relaxation time T(1). In addition, the hydrophobic galvinoxyl free radical can be easily filtered out from the dissolution liquid when water is used as the solvent. These results indicate that galvinoxyl can be considered as an easily available free radical polarizing agent for routine dissolution DNP-NMR spectroscopy.

Modulation of CEST Images in Vivo by T1 Relaxation: A New Approach in the Design of Responsive PARACEST Agents

A novel approach for the design of responsive paramagnetic chemical exchange saturation transfer (PARACEST) magnetic resonance imaging (MRI) agents has been developed where the signal is turned on by altering the longitudinal relaxation time (T1) of bulk water protons. To demonstrate this approach, a model Eu(DOTA-tetraamide) complex (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) containing two nitroxide free radical units was synthesized. The nitroxide groups substantially shortened the T1 of the bulk water protons which, in turn, resulted in quenching of the CEST signal. Reduction of paramagnetic nitroxide moieties to a diamagnetic species resulted in the appearance of CEST. The modulation of CEST by T1 relaxation provides a new platform for designing biologically responsive MRI agents.