Quantitative Analysis of 2D EXSY NMR Spectra of Strongly Coupled Spin Systems in Transmembrane Exchange.

Solute translocation by membrane transport proteins is a vital biological process that can be tracked, on the sub-second timescale, using nuclear magnetic resonance (NMR). Fluorinated substrate analogues facilitate such studies because of high sensitivity of 19 F NMR and absence of background signals. Accurate extraction of translocation rate constants requires precise quantification of NMR signal intensities. This becomes complicated in the presence of J-couplings, cross-correlations, and nuclear Overhauser effects (NOE) that alter signal integrals through mechanisms unrelated to translocation. Geminal difluorinated motifs introduce strong and hard-to-quantify contributions from non-exchange effects, the nuanced nature of which makes them hard to integrate into data analysis methodologies. With analytical expressions not being available, numerical least squares fitting of theoretical models to 2D spectra emerges as the preferred quantification approach. For large spin systems with simultaneous coherent evolution, cross-relaxation, cross-correlation, conformational exchange, and membrane translocation between compartments with different viscosities, the only available simulation framework is Spinach. In this study, we demonstrate GLUT-1 dependent membrane transport of two model sugars featuring CF2 and CF2 CF2 fluorination motifs, with precise determination of translocation rate constants enabled by numerical fitting of 2D EXSY spectra. For spin systems and kinetic networks of this complexity, this was not previously tractable.

Taipan Natriuretic Peptides Are Potent and Selective Agonists for the Natriuretic Peptide Receptor A.

Cardiovascular ailments are a major cause of mortality where over 1.3 billion people suffer from hypertension leading to heart-disease related deaths. Snake venoms possess a broad repertoire of natriuretic peptides with therapeutic potential for treating hypertension, congestive heart failure, and related cardiovascular disease. We now describe several taipan (Oxyuranus microlepidotus) natriuretic peptides TNPa-e which stimulated cGMP production through the natriuretic peptide receptor A (NPR-A) with higher potencies for the rat NPR-A (rNPR-A) over human NPR-A (hNPR-A). TNPc and TNPd were the most potent, demonstrating 100- and 560-fold selectivity for rNPR-A over hNPR-A. In vivo studies found that TNPc decreased diastolic and systolic blood pressure (BP) and increased heart rate (HR) in conscious normotensive rabbits, to a level that was similar to that of human atrial natriuretic peptide (hANP). TNPc also enhanced the bradycardia due to cardiac afferent stimulation (Bezold-Jarisch reflex). This indicated that TNPc possesses the ability to lower blood pressure and facilitate cardiac vagal afferent reflexes but unlike hANP does not produce tachycardia. The 3-dimensional structure of TNPc was well defined within the pharmacophoric disulfide ring, displaying two turn-like regions (RMSD = 1.15 Å). Further, its much greater biological stability together with its selectivity and potency will enhance its usefulness as a biological tool.

Identification of beryllium fluoride complexes in mechanically distorted gels using quadrupolar split 9Be NMR spectra resolved with solution-state selective cross-polarization.

The uniformly anisotropic media afforded by hydrogels are being increasingly exploited in analytical (structure elucidation) nuclear magnetic resonance (NMR) spectroscopy, and in studies of mechanosensitive biophysical and biochemical properties of living cells. The 9Be NMR parameters of beryllium fluoride complexes formed in aqueous solutions are sensitive markers of the anisotropic molecular environments produced by gelatin gels. The electric quadrupole moment of the 9Be nucleus (spin I = 3/2) interacts with the electric field gradient tensor in a stretched (or compressed) gel, giving rise to the splitting of peaks in 9Be NMR spectra. These are in addition to those produced by scalar coupling to the 19F nuclei. Thus, an equilibrium mixture of beryllofluoride complexes (BeF2, BeF3-, and BeF42-) in mechanically distorted gels generates an envelope of overlapping 9Be NMR multiplets. In the present work, the multiplets were dissected apart by using selective excitation of 9Be-19F cross-polarization; and the spectral components were quantified with multi-parameter line-shape decomposition, coupled with SpinDynamica simulations. The effects of gel density and Bloom number (a measure of gelatin-gel rigidity under standard conditions of sample preparation) on residual quadrupolar splittings were examined. Cross-polarization experiments revealed a bimodal distribution of  residual quadrupolar coupling constants (RQC) of the BeF3- complexes. The average RQC of the dominant BeF3- population was ∼3 times larger than that of BeF42-. The secondary BeF3- population existed in a tetrahedral configuration. It was attributed to BeF3- complexes associated with negatively charged -COO- groups of the denatured collagen matrix.

Transmembrane Exchange of Fluorosugars: Characterization of Red Cell GLUT1 Kinetics Using 19F NMR.

We have developed a new approach, to our knowledge, to quantify the equilibrium exchange kinetics of carrier-mediated transmembrane transport of fluorinated substrates. The method is based on adapted kinetic theory that describes the concentration dependence of the transmembrane exchange rates of two competing, simultaneously transported species. Using the new approach, we quantified the kinetics of membrane transport of both anomers of three monofluorinated glucose analogs in human erythrocytes (red blood cells) using 19F NMR exchange spectroscopy. An inosine-based glucose-free medium was shown to promote survival and stable metabolism of red blood cells over the duration of the experiments (several hours). Earlier NMR studies only yielded the apparent rate constants and transmembrane fluxes of the anomeric species, whereas we could categorize the two anomers in terms of the catalytic activity (specificity constants) of the glucose transport protein GLUT1 toward them. Differences in the membrane permeability of the three glucose analogs were qualitatively interpreted in terms of local perturbations in the bonding of substrates to key amino acid residues in the active site of GLUT1. The methodology of this work will be applicable to studies of other carrier-mediated membrane transport processes, especially those with competition between simultaneously transported species. The GLUT1-specific results can be applied to the design of probes of glucose transport or inhibitors of glucose metabolism in cells, including those exhibiting the Warburg effect.

The S1 helix critically regulates the finely tuned gating of Kv11.1 channels.

Congenital mutations in the cardiac Kv11.1 channel can cause long QT syndrome type 2 (LQTS2), a heart rhythm disorder associated with sudden cardiac death. Mutations act either by reducing protein expression at the membrane and/or by perturbing the intricate gating properties of Kv11.1 channels. A number of clinical LQTS2-associated mutations have been reported in the first transmembrane segment (S1) of Kv11.1 channels, but the role of this region of the channel is largely unexplored. In part, this is due to problems defining the extent of the S1 helix, as a consequence of its low sequence homology with other Kv family members. Here, we used NMR spectroscopy and electrophysiological characterization to show that the S1 of Kv11.1 channels extends seven helical turns, from Pro-405 to Phe-431, and is flanked by unstructured loops. Functional analysis suggests that pre-S1 loop residues His-402 and Tyr-403 play an important role in regulating the kinetics and voltage dependence of channel activation and deactivation. Multiple residues within the S1 helix also play an important role in fine-tuning the voltage dependence of activation, regulating slow deactivation, and modulating C-type inactivation of Kv11.1 channels. Analyses of LQTS2-associated mutations in the pre-S1 loop or S1 helix of Kv11.1 channels demonstrate perturbations to both protein expression and most gating transitions. Thus, S1 region mutations would reduce both the action potential repolarizing current passed by Kv11.1 channels in cardiac myocytes, as well as the current passed in response to premature depolarizations that normally helps protect against the formation of ectopic beats.

Sub-minute kinetics of human red cell fumarase: 1 H spin-echo NMR spectroscopy and 13 C rapid-dissolution dynamic nuclear polarization.

Fumarate is an important probe of metabolism in hyperpolarized magnetic resonance imaging and spectroscopy. It is used to detect the release of fumarase in cancer tissues, which is associated with necrosis and drug treatment. Nevertheless, there are limited reports describing the detailed kinetic studies of this enzyme in various cells and tissues. Thus, we aimed to evaluate the sub-minute kinetics of human red blood cell fumarase using nuclear magnetic resonance (NMR) spectroscopy, and to provide a quantitative description of the enzyme that is relevant to the use of fumarate as a probe of cell rupture. The fumarase reaction was studied using time courses of 1 H spin-echo and 13 C-NMR spectra. 1 H-NMR experiments showed that the fumarase reaction in hemolysates is sufficiently rapid to make its kinetics amenable to study in a period of approximately 3 min, a timescale characteristic of hyperpolarized 13 C-NMR spectroscopy. The rapid-dissolution dynamic nuclear polarization (RD-DNP) technique was used to hyperpolarize [1,4-13 C]fumarate, which was injected into concentrated hemolysates. The kinetic data were analyzed using recently developed FmRα analysis and modeling of the enzymatic reaction using Michaelis-Menten equations. In RD-DNP experiments, the decline in the 13 C-NMR signal from fumarate, and the concurrent rise and fall of that from malate, were captured with high spectral resolution and signal-to-noise ratio, which allowed the robust quantification of fumarase kinetics. The kinetic parameters obtained indicate the potential contribution of hemolysis to the overall rate of the fumarase reaction when 13 C-NMR RD-DNP is used to detect necrosis in animal models of implanted tumors. The analytical procedures developed will be applicable to studies of other rapid enzymatic reactions using conventional and hyperpolarized substrate NMR spectroscopy.

Hypoxia-Responsive Cobalt Complexes in Tumor Spheroids: Laser Ablation Inductively Coupled Plasma Mass Spectrometry and Magnetic Resonance Imaging Studies.

Dense tumors are resistant to conventional chemotherapies due to the unique tumor microenvironment characterized by hypoxic regions that promote cellular dormancy. Bioreductive drugs that are activated in response to this hypoxic environment are an attractive strategy for therapy with anticipated lower harmful side effects in normoxic healthy tissue. Cobalt bioreductive pro-drugs that selectively release toxic payloads upon reduction in hypoxic cells have shown great promise as anticancer agents. However, the bioreductive response in the tumor microenvironment must be better understood, as current techniques for monitoring bioreduction to Co(II) such as X-ray absorption near-edge structure and extended X-ray absorption fine structure provide limited information on speciation and require synchrotron radiation sources. Here, we present magnetic resonance imaging (MRI) as an accessible and powerful technique to monitor bioreduction by treating the cobalt complex as an MRI contrast agent and monitoring the change in water signal induced by reduction from diamagnetic Co(III) to paramagnetic Co(II). Cobalt pro-drugs built upon the tris(2-pyridylmethyl)amine ligand scaffold with varying charge were investigated for distribution and activity in a 3D tumor spheroid model by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and MRI. In addition, paramagnetic 1H NMR spectroscopy of spheroids enabled determination of the speciation of activated Co(II)TPAx complexes. This study demonstrates the utility of MRI and associated spectroscopy techniques for understanding bioreductive cobalt pro-drugs in the tumor microenvironment and has broader implications for monitoring paramagnetic metal-based therapies.

Anisotropic diffusion in stretched hydrogels containing erythrocytes: evidence of cell-shape distortion recorded by PGSE NMR spectroscopy.

The remarkable flexibility of human red blood cells (RBCs) allows them to assume a range of shapes in normal and disease states. Biochemical mechanisms and energetic requirements associated with changes in RBC geometry are not well understood because of a lack of experimental procedures to fix and study cells in different morphological forms. By incorporating RBCs into stretchable gelatin hydrogels, we created conditions for adjustable elongation of their normal discocytic shape in all orientations. As the RBC-containing gels were stretched or compressed, the changes in the cell morphology were studied by using 1 H-PGSE-NMR spectroscopy. Measurements of the apparent diffusion coefficient of water along the three orthogonal directions revealed tuneable anisotropy in the environment of the hydrogel samples. Light microscopy was also used for recording the extent to which RBCs were distorted in a stretched gel that had been set around them. Having demonstrated the applicability of NMR diffusometry to detect morphological changes of immobilised cells, we have laid the groundwork for future investigations of controllably distorted RBCs. Specifically, we expect studies of metabolic and biophysical properties of the physically deformed cells, thus inferring the connection between intracellular physico-chemical processes and RBC morphology. Copyright © 2016 John Wiley & Sons, Ltd.

Na+ and solute diffusion in aqueous channels of Myverol bicontinuous cubic phase: PGSE NMR and computer modelling.

The apparent diffusion coefficients of 23 Na+ ions and the solute 2-fluoroethylamine present in the aqueous domain of a Myverol/water bulk bicontinuous cubic phase (BCP) were measured using pulsed field-gradient spin echo (PGSE) NMR spectroscopy. The measured values were dependent on the diffusion time interval, which is a characteristic of restricted diffusion. The translational motion of 23 Na+ and water in the aqueous channels of a cubic phase were simulated using a Monte-Carlo random walk algorithm, and the simulation results were compared with those from real PGSE NMR experiments. The simulations indicated that diffusion of 23 Na+ ions and water would appear to be restricted even on the shortest timescales available to PGSE NMR experiments. The micro-viscosity of the aqueous domain of the BCPs was estimated from the longitudinal relaxation times of 23 Na+ and 2-fluoroethylamine; this was three times higher than in free solution and suggests one of (but not the only) likely impediments to the release of hydrophilic drugs from stabilised aqueous dispersions of BCPs (cubosomes) when they are used therapeutically in vivo. Monte Carlo simulations of diffusive efflux from cubosomes suggest that the principal impediment to drug release is presented by a surfactant or lipid barrier at the cubosome surface, which separates the BCP aqueous channels from the bulk solution. The dynamics inferred from these studies informs quantitative predictions of drug delivery from cubosomes. Copyright © 2016 John Wiley & Sons, Ltd.

NMR Spectra of Glycine Isotopomers in Anisotropic Media: Subtle Chiral Interactions.

NMR spectra of deuterated glycine-2-(13)C revealed interactions between chiral anisotropic gelatin and κ-carrageenan gels and the prochiral and chiral isotopomers. The (1)H, (2)H and (13)C NMR spectra of mixtures of racemic mono- and prochiral bis-deuterated glycine-2-(13)C were resolved and well simulated using distinct dipolar coupling constants DCαH and DCαD for the enantiomers and also for the -(13)CαD2- group (DC,DA, and DC,DB). The orientation of the proton or deuteron on the (13)Cα-atom of glycine was assigned by analogy with alanine and lactate assuming that the molecular orientation of glycine isotopomers is the same. The assignment of the prochiral sites was derived from chiral analogues.

FmRα analysis: Rapid and direct estimation of relaxation and kinetic parameters from dynamic nuclear polarization time courses.

PURPOSE: To introduce a direct method for estimating relaxation and kinetic parameter values from rapid dissolution dynamic nuclear polarization (RD-DNP) NMR time courses. THEORY AND METHODS: The analysis relied on a kinetic model that is often used to analyze data in these studies-a unidirectional (bio)chemical reaction with rate constant k1 , coupled to longitudinal relaxation of the magnetization of substrate and product that is characterized by the time constant T1 . The latter value was estimated from the width of the product curve (peak) at the height α relative to the maximum height. We showed α ∼ 0.8 under most conditions, so we measured the interval between the falling and rising parts of the curve at the relative height 0.8. We called this the "fall-minus-rise time at height α," or FmRα , and found that FmR0.8 ∼ T1 . The ratio ß = (product signal/substrate signal) when the product is maximal was shown to be equal to k1 T1 . Therefore, k1 = ß/FmR0.8 . RESULTS: FmRα analysis was demonstrated with (13) C NMR RD-DNP data recorded from hemolysates and from previously published data. CONCLUSION: FmRα analysis enables immediate estimates of kinetic and relaxation parameters from (13) C NMR RD-DNP data. The values can be used as initial estimates in more extensive computer-based data-regression analysis.

Long-lived spin state of a tripeptide in stretched hydrogel.

The longitudinal (T 1), transverse (T 2), and singlet state (T s) relaxation times of the geminal backbone protons (CH2) of L-Leu-Gly-Gly were studied by NMR spectroscopy at 9.4 T in a bovine hide gelatin gel composed in D2O at 25 °C. Gelatin granules were dissolved in a hot solution of the tripeptide and then the solution was allowed to gel inside a flexible silicone tubing. With increases in gelatin content, the T 2 and T s of the CH2 protons correspondingly decreased (T s/T 2 ~ constant), while the change in T 1 was relatively small. The largest observed T s/T 1 value was 3.3 at 46% w/v gelatin that was the lowest gelatin content examined. Stretching the tubing, and hence the gel, brought about anisotropic alignment of the constituents resulting in residual quadrupolar splitting of the resonance from D2O in (2)H NMR spectra, and residual dipolar splitting of the CH2 resonance in (1)H NMR spectra. WALTZ-16 decoupling during the relaxation intervals extended the singlet state relaxation time, but the efficacy diminished as the gels were stretched. Theoretically predicted T 1, T 2, and T s values, assuming intramolecular dipolar coupling as the only source of relaxation, were within the same order of magnitude as the experimentally observed values. Overall we showed that it is possible to observe a long-lived spin state in an anisotropic medium when T 2 is shorter than T 1 in the presence of non-zero residual dipolar couplings.

Hyperpolarized [1,(13)C]pyruvate in lysed human erythrocytes: effects of co-substrate supply on reaction time courses.

Hyperpolarized [1,(13)C]pyruvate was injected rapidly into haemolysates in which hydrolysis of nicotinamide adenine dinucleotide (phosphate) (NAD(P))/NAD(P)H had been inhibited with nicotinamide. Haemolysates provide a stable glycolytic system in which membrane permeability is not a flux-controlling step, and they enable the concentration of NADH to be adjusted experimentally while keeping the rest of the sample with the same composition as that of the cytoplasm of the cell (albeit diluted twofold at the time of injection of the [1,(13)C]pyruvate). We showed that the maximum amplitude of the (13)C NMR signal from the [1,(13)C]L-lactate, produced from [1,(13)C]pyruvate, and the time at which it occurred was dependent on NADH concentration, as predicted by enzyme-kinetic analysis. The main feature of such curves was dictated by the immediacy of the supply of the co-substrate of lactate dehydrogenase (LDH, EC 1.1.1.27), and we posit that this also pertains in vivo in various tissues including neoplasms. By constructing an appropriate mathematical model and by using a Markov-chain Monte Carlo approach, we fitted experimental data to estimate LDH and NADH concentrations. Experiments carried out with only endogenous NADH present enabled the estimation of its effective concentration in human RBCs; the ability to make this estimate is a special feature of the rapid-dissolution dynamic nuclear polarization method. We found an endogenous NADH concentration in human RBCs two to four times higher than previously reported.

Membrane flickering of the human erythrocyte: constrained random walk used with Bayesian analysis.

The involvement of adenosine triphosphate (ATP) in erythrocyte (red blood cell; RBC) membrane flickering is of particular interest, because ATP turnover in the cell as a whole is not yet fully accounted for. We sought the origins of flickering by deriving a mathematical model of it, on the basis of the idea of thermally driven collisions of small molecules with the membrane, which responds like an over-damped spring. The model gave simulated responses that were similar to a constrained random walk and had the same frequency-spectral characteristics of membrane displacement as those recorded from RBCs by use of differential interference contrast light microscopy. Bayesian analysis was used as the basis for determination, from experimental results, of the values of the parameters in the model. The analysis was used in the accompanying article in which we investigated the response of membrane flickering to different effector molecules and physicochemical conditions. The results implied ATP was involved only indirectly in membrane flickering.

Membrane flickering of the human erythrocyte: physical and chemical effectors.

Recent studies suggest a link between adenosine triphosphate (ATP) concentration and the amplitude of cell membrane flickering (CMF) in the human erythrocyte (red blood cell; RBC). Potentially, the origin of this phenomenon and the unique discocyte shape could be active processes that account for some of the ATP turnover in the RBC. Active flickering could depend on several factors, including pH, osmolality, enzymatic rates and metabolic fluxes. In the present work, we applied the data analysis described in the previous article to study time courses of flickering RBCs acquired using differential interference contrast light microscopy in the presence of selected effectors. We also recorded images of air bubbles in aqueous detergent solutions and oil droplets in water, both of which showed rapid fluctuations in image intensity, the former showing the same type of spectral envelope (relative frequency composition) to RBCs. We conclude that CMF is not directly an active process, but that ATP affects the elastic properties of the membrane that flickers in response to molecular bombardment in a manner that is described mathematically by a constrained random walk.

Adeno-associated virus-mediated rescue of neonatal lethality in argininosuccinate synthetase-deficient mice.

Viral vectors based on adeno-associated virus (AAV) are showing exciting promise in gene therapy trials targeting the adult liver. A major challenge in extending this promise to the pediatric liver is the loss of episomal vector genomes that accompanies hepatocellular proliferation during liver growth. Hence maintenance of sufficient transgene expression will be critical for success in infants and children. We therefore set out to explore the therapeutic efficacy and durability of liver-targeted gene transfer in the challenging context of a neonatal lethal urea cycle defect, using the argininosuccinate synthetase deficient mouse. Lethal neonatal hyperammonemia was prevented by prenatal and early postnatal vector delivery; however, hyperammonemia subsequently recurred limiting survival to no more than 33 days despite vector readministration. Antivector antibodies acquired in milk from vector-exposed dams were subsequently shown to be blocking vector readministration, and were avoided by crossfostering vector-treated pups to vector-naive dams. In the absence of passively acquired antivector antibodies, vector redelivery proved efficacious with mice surviving to adulthood without recurrence of significant hyperammonemia. These data demonstrate the potential of AAV vectors in the developing liver, showing that vector readministration can be used to counter growth-associated loss of transgene expression provided the challenge of antivector humoral immunity is addressed.

Disrupting Na+ ion homeostasis and Na+/K+ ATPase activity in breast cancer cells directly modulates glycolysis in vitro and in vivo.

BACKGROUND: Glycolytic flux is regulated by the energy demands of the cell. Upregulated glycolysis in cancer cells may therefore result from increased demand for adenosine triphosphate (ATP), however it is unknown what this extra ATP turnover is used for. We hypothesise that an important contribution to the increased glycolytic flux in cancer cells results from the ATP demand of Na+/K+-ATPase (NKA) due to altered sodium ion homeostasis in cancer cells. METHODS: Live whole-cell measurements of intracellular sodium [Na+]i were performed in three human breast cancer cells (MDA-MB-231, HCC1954, MCF-7), in murine breast cancer cells (4T1), and control human epithelial cells MCF-10A using triple quantum filtered 23Na nuclear magnetic resonance (NMR) spectroscopy. Glycolytic flux was measured by 2H NMR to monitor conversion of [6,6-2H2]D-glucose to [2H]-labelled L-lactate at baseline and in response to NKA inhibition with ouabain. Intracellular [Na+]i was titrated using isotonic buffers with varying [Na+] and [K+] and introducing an artificial Na+ plasma membrane leak using the ionophore gramicidin-A. Experiments were carried out in parallel with cell viability assays, 1H NMR metabolomics of intracellular and extracellular metabolites, extracellular flux analyses and in vivo measurements in a MDA-MB-231 human-xenograft mouse model using 2-deoxy-2-[18F]fluoroglucose (18F-FDG) positron emission tomography (PET). RESULTS: Intracellular [Na+]i was elevated in human and murine breast cancer cells compared to control MCF-10A cells. Acute inhibition of NKA by ouabain resulted in elevated [Na+]i and inhibition of glycolytic flux in all three human cancer cells which are ouabain sensitive, but not in the murine cells which are ouabain resistant. Permeabilization of cell membranes with gramicidin-A led to a titratable increase of [Na+]i in MDA-MB-231 and 4T1 cells and a Na+-dependent increase in glycolytic flux. This was attenuated with ouabain in the human cells but not in the murine cells. 18FDG PET imaging in an MDA-MB-231 human-xenograft mouse model recorded lower 18FDG tumour uptake when treated with ouabain while murine tissue uptake was unaffected. CONCLUSIONS: Glycolytic flux correlates with Na+-driven NKA activity in breast cancer cells, providing evidence for the 'centrality of the [Na+]i-NKA nexus' in the mechanistic basis of the Warburg effect.

Transmembrane exchange of hyperpolarized 13C-urea in human erythrocytes: subminute timescale kinetic analysis.

The rate of exchange of urea across the membranes of human erythrocytes (red blood cells) was quantified on the 1-s to 2-min timescale. (13)C-urea was hyperpolarized and subjected to rapid dissolution and the previously reported (partial) resolution of (13)C NMR resonances from the molecules inside and outside red blood cells in suspensions was observed. This enabled a stopped-flow type of experiment to measure the (initially) zero-trans transport of urea with sequential single-pulse (13)C NMR spectra, every second for up to ~2 min. Data were analyzed using Bayesian reasoning and a Markov chain Monte Carlo method with a set of simultaneous nonlinear differential equations that described nuclear magnetic relaxation combined with transmembrane exchange. Our results contribute to quantitative understanding of urea-exchange kinetics in the whole body; and the methodological approach is likely to be applicable to other cellular systems and tissues in vivo.

Stoichiometric relationship between Na(+) ions transported and glucose consumed in human erythrocytes: Bayesian analysis of (23)Na and (13)C NMR time course data.

We examined the response of Na(+),K(+)-ATPase (NKA) to monensin, a Na(+) ionophore, with and without ouabain, an NKA inhibitor, in suspensions of human erythrocytes (red blood cells). A combination of (13)C and (23)Na NMR methods allowed the recording of intra- and extracellular Na(+), and (13)C-labeled glucose time courses. The net influx of Na(+) and the consumption of glucose were measured with and without NKA inhibited by ouabain. A Bayesian analysis was used to determine probability distributions of the parameter values of a minimalist mathematical model of the kinetics involved, and then used to infer the rates of Na(+) transported and glucose consumed. It was estimated that the numerical relationship between the number of Na(+) ions transported by NKA per molecule of glucose consumed by a red blood cell was close to the ratio 6.0:1.0, agreeing with theoretical prediction.

Cardiac function and lipid distribution in rats fed a high-fat diet: in vivo magnetic resonance imaging and spectroscopy.

Obesity is a major risk factor in the development of cardiovascular disease, type 2 diabetes, and its pathophysiological precondition insulin resistance. Very little is known about the metabolic changes that occur in the myocardium and consequent changes in cardiac function that are associated with high-fat accumulation. Therefore, cardiac function and metabolism were evaluated in control rats and those fed a high-fat diet, using magnetic resonance imaging, magnetic resonance spectroscopy, mRNA analysis, histology, and plasma biochemistry. Analysis of blood plasma from rats fed the high-fat diet showed that they were insulin resistant (P < 0.001). Our high-fat diet model had higher heart weight (P = 0.005) and also increasing trend in septal wall thickness (P = 0.07) compared with control diet rats. Our results from biochemistry, magnetic resonance imaging, and mRNA analysis confirmed that rats on the high-fat diet had moderate diabetes along with mild cardiac hypertrophy. The magnetic resonance spectroscopy results showed the extramyocellular lipid signal only in the spectra from high-fat diet rats, which was absent in the control diet rats. The intramyocellular lipids in high-fat diet rats was higher (8.7%) compared with rats on the control diet (6.1%). This was confirmed by electron microscope and light microscopy studies. Our results indicate that lipid accumulation in the myocardium might be an early indication of the cardiovascular pathophysiology associated with type 2 diabetes.