Multiple quantum filtered nuclear magnetic resonance of 23Na+ in uniformly stretched and compressed hydrogels.

Stretching or compressing hydrogels creates anisotropic environments that lead to motionally averaged alignment of embedded guest quadrupolar nuclear spins such as 23Na+. These distorted hydrogels can elicit a residual quadrupolar coupling that gives an oscillation in the trajectories of single quantum coherences (SQCs) as a function of the evolution time during a spin-echo experiment. We present solutions to equations of motion derived with a Liouvillian superoperator approach, which encompass the coherent quadrupolar interaction in conjunction with relaxation, to give a full analytical description of the evolution trajectories of rank-1 (T^1±1), rank-2 (T^2±1), and rank-3 (T^3±1) SQCs. We performed simultaneous numerical fitting of the experimental 23Na nuclear magnetic resonance (NMR) spectra and rank-2 (T^2±1) and rank-3 (T^3±1) SQC evolution trajectories measured in double and triple quantum filtered experiments, respectively. We estimated values of the quadrupolar coupling constant CQ, rotational correlation time τC, and 3 × 3 Saupe order matrix. We performed simultaneous fitting of the analytical expressions to the experimental data to estimate values of the quadrupolar coupling frequency ωQ/2π, residual quadrupolar coupling ωQ/2π, and corresponding spherical order parameter S0*, which showed a linear dependence on the extent of uniform hydrogel stretching and compression. The analytical expressions were completely concordant with the numerical approach. The insights gained here can be extended to more complicated (biological) systems such as 23Na+ bound to proteins or located inside and outside living cells in high-field NMR experiments and, by extension, to the anisotropic environments found in vivo with 23Na magnetic resonance imaging.

Modeling non-linear kinetics of hyperpolarized [1-(13)C] pyruvate in the crystalloid-perfused rat heart.

Hyperpolarized (13)C MR measurements have the potential to display non-linear kinetics. We have developed an approach to describe possible non-first-order kinetics of hyperpolarized [1-(13)C] pyruvate employing a system of differential equations that agrees with the principle of conservation of mass of the hyperpolarized signal. Simultaneous fitting to a second-order model for conversion of [1-(13)C] pyruvate to bicarbonate, lactate and alanine was well described in the isolated rat heart perfused with Krebs buffer containing glucose as sole energy substrate, or glucose supplemented with pyruvate. Second-order modeling yielded significantly improved fits of pyruvate-bicarbonate kinetics compared with the more traditionally used first-order model and suggested time-dependent decreases in pyruvate-bicarbonate flux. Second-order modeling gave time-dependent changes in forward and reverse reaction kinetics of pyruvate-lactate exchange and pyruvate-alanine exchange in both groups of hearts during the infusion of pyruvate; however, the fits were not significantly improved with respect to a traditional first-order model. The mechanism giving rise to second-order pyruvate dehydrogenase (PDH) kinetics was explored experimentally using surface fluorescence measurements of nicotinamide adenine dinucleotide reduced form (NADH) performed under the same conditions, demonstrating a significant increase of NADH during pyruvate infusion. This suggests a simultaneous depletion of available mitochondrial NAD(+) (the cofactor for PDH), consistent with the non-linear nature of the kinetics. NADH levels returned to baseline following cessation of the pyruvate infusion, suggesting this to be a transient effect.

Dichloroacetate induces autophagy in colorectal cancer cells and tumours.

BACKGROUND: Dichloroacetate (DCA) has been found to have antitumour properties. METHODS: We investigated the cellular and metabolic responses to DCA treatment and recovery in human colorectal (HT29, HCT116 WT and HCT116 Bax-ko), prostate carcinoma cells (PC3) and HT29 xenografts by flow cytometry, western blotting, electron microscopy, (1)H and hyperpolarised (13)C-magnetic resonance spectroscopy. RESULTS: Increased expression of the autophagy markers LC3B II was observed following DCA treatment both in vitro and in vivo. We observed increased production of reactive oxygen species (ROS) and mTOR inhibition (decreased pS6 ribosomal protein and p4E-BP1 expression) as well as increased expression of MCT1 following DCA treatment. Steady-state lactate excretion and the apparent hyperpolarised [1-(13)C] pyruvate-to-lactate exchange rate (k(PL)) were decreased in DCA-treated cells, along with increased NAD(+)/NADH ratios and NAD(+). Steady-state lactate excretion and k(PL) returned to, or exceeded, control levels in cells recovered from DCA treatment, accompanied by increased NAD(+) and NADH. Reduced k(PL) with DCA treatment was found in HT29 tumour xenografts in vivo. CONCLUSIONS: DCA induces autophagy in cancer cells accompanied by ROS production and mTOR inhibition, reduced lactate excretion, reduced k(PL) and increased NAD(+)/NADH ratio. The observed cellular and metabolic changes recover on cessation of treatment.

Detection of cancer in cervical tissue biopsies using mobile lipid resonances measured with diffusion-weighted (1)H magnetic resonance spectroscopy.

The purpose of this study was to implement a diffusion-weighted sequence for visualisation of mobile lipid resonances (MLR) using high resolution magic angle spinning (HR-MAS) (1)H MRS and to evaluate its use in establishing differences between tissues from patients with cervical carcinoma that contain cancer from those that do not. A stimulated echo sequence with bipolar gradients was modified to allow T(1) and T(2) measurements and optimised by recording signal loss in HR-MAS spectra as a function of gradient strength in model lipids and tissues. Diffusion coefficients, T(1) and apparent T(2) relaxation times were measured in model lipid systems. MLR profiles were characterised in relation to T(1) and apparent T(2) relaxation in human cervical cancer tissue samples. Diffusion-weighted (DW) spectra of cervical biopsies were quantified and peak areas analysed using linear discriminant analysis (LDA). The optimised sequence reduced spectral overlap by suppressing signals originating from low molecular weight metabolites and non-lipid contributions. Significantly improved MLR visualisation allowed visualisation of peaks at 0.9, 1.3, 1.6, 2.0, 2.3, 2.8, 4.3 and 5.3 ppm. MLR analysis of DW spectra showed at least six peaks arising from saturated and unsaturated lipids and those arising from triglycerides. Significant differences in samples containing histologically confirmed cancer were seen for peaks at 0.9 (p < 0.006), 1.3 (p < 0.04), 2.0 (p < 0.03), 2.8 (p < 0.003) and 4.3 ppm (p < 0.0002). LDA analysis of MLR peaks from DW spectra almost completely separated two clusters of cervical biopsies (cancer, 'no-cancer'), reflecting underlying differences in MLR composition. Generated Receiver Operating Characteristic (ROC) curves and calculated area under the curve (0.962) validated high sensitivity and specificity of the technique. Diffusion-weighting of HR-MAS spectroscopic sequences is a useful method for characterising MLR in cancer tissues and displays an accumulation of lipids arising during tumourigenesis and an increase in the unsaturated lipid and triglyceride peaks with respect to saturated MLR.

Magnetic Resonance Spectroscopy to Study Glycolytic Metabolism During Autophagy.

Cancer cells undergoing starvation- and treatment-induced autophagy were found to exhibit reduced intracellular lactate, reduced rates of steady-state lactate excretion and reduced real-time pyruvate-lactate exchange rates, indicating that glycolytic metabolism was altered in autophagic cells. In this chapter, we describe the technical details of the use of 1H-magnetic resonance spectroscopy (MRS) to measure endogenous cellular concentrations of lactate and glucose in autophagic cells and tissues, how to measure the rate of steady-state lactate excretion and glucose uptake by 1H-MRS in autophagic cells, and details of the real-time measurement of [1-13C] pyruvate to lactate exchange in autophagic cells by 13C-MRS-DNP (dynamic nuclear polarization).

Single-transition coherence transfer by adiabatic cross polarization in NMR.

Spin-state selective coherence transfer between single-transition coherences in a scalar-coupled two-spin system may be achieved by employing highly selective, constant amplitude radio frequency fields. We extend this method by employing amplitude-modulated pulses to achieve an adiabatic transfer, as shown in the picture (I=intensity). Different limiting cases are considered. The imperfect reversibility of the transfer reveals deviations from adiabaticity.

Identification of spin diffusion pathways in isotopically labeled biomolecules.

One-dimensional NOE experiments applicable to labeled macromolecules are presented which allow the manipulation of specific spin diffusion pathways and thus unambiguously identify clandestine spins through which the direct NOE is mediated. A treatment of spin diffusion using average Liouvillian theory is shown to describe adequately these phenomena. Experiments are carried out on an 15N-labeled sample of human ubiquitin.

Offset profiles of selective pulses in isotopically labeled macromolecules.

The experimental verification of offset profiles and calibration of selective pulses in NMR is usually carried out with doped water samples but not under conditions typical of macromolecules with short T2, long T1, and possibly homo- and heteronuclear couplings. A new method for selective excitation in isotopically labeled macromolecules is shown to be particularly suited to this purpose. This is illustrated for a backbone amide resonance in a sample of 15N-labeled human ubiquitin.

Normalized one-dimensional NOE measurements in isotopically labeled macromolecules using two-way cross-polarization.

A novel one-dimensional NOE experiment is presented where a selected proton is excited by two-way heteronuclear cross- polarization between protons and nitrogen-15 or carbon-13. The utility of the method is demonstrated for a sample of 15N labeled human ubiquitin. Inter- and intra-residue NOEs are clearly observed in a very time-effective manner. The signal intensities can be easily normalized.