Charles Storey

Hyperpolarized 13C allows a direct measure of flux through a single enzyme-catalyzed step by NMR

13C NMR is a powerful tool for monitoring metabolic fluxes in vivo. The recent availability of automated dynamic nuclear polarization equipment for hyperpolarizing 13C nuclei now offers the potential to measure metabolic fluxes through select enzyme-catalyzed steps with substantially improved sensitivity. Here, we investigated the metabolism of hyperpolarized [1-13C1]pyruvate in a widely used model for physiology and pharmacology, the perfused rat heart. Dissolved 13CO2, the immediate product of the first step of the reaction catalyzed by pyruvate dehydrogenase, was observed with a temporal resolution of ≈1 s along with H13CO3−, the hydrated form of 13CO2 generated catalytically by carbonic anhydrase. In hearts presented with the medium-chain fatty acid octanoate in addition to hyperpolarized [1-13C1]pyruvate, production of 13CO2 and H13CO3− was suppressed by ≈90%, whereas the signal from [1-13C1]lactate was enhanced. In separate experiments, it was shown that O2 consumption and tricarboxylic acid (TCA) cycle flux were unchanged in the presence of added octanoate. Thus, the rate of appearance of 13CO2 and H13CO3− from [1-13C1]pyruvate does not reflect production of CO2 in the TCA cycle but rather reflects flux through pyruvate dehydrogenase exclusively.

Inhibition of carbohydrate oxidation during the first minute of reperfusion after brief ischemia: NMR detection of hyperpolarized 13CO2 and H13CO3-.

Isolated rat hearts were studied by 31P NMR and 13C NMR. Hyperpolarized [1‐13C]pyruvate was supplied to control normoxic hearts and production of [1‐13C]lactate, [1‐13C]alanine, 13CO2 and H13CO  −3 was monitored with 1‐s temporal resolution. Hearts were also subjected to 10 min of global ischemia followed by reperfusion. Developed pressure, heart rate, oxygen consumption, [ATP], [phosphocreatine], and pH recovered within 3 min after the ischemic period. During the first 90 s of reperfusion, [1‐13C]alanine and [1‐13C]lactate appeared rapidly, demonstrating metabolism of pyruvate through two enzymes largely confined to the cytosol, alanine aminotransferase, and lactate dehydrogenase. 13CO2 and H13CO  −3 were not detected. Late after ischemia and reperfusion, the products of pyruvate dehydrogenase, 13CO2 and H13CO  −3 were easily detected. Using this multinuclear NMR approach, we established that during the first 90 s of reperfusion PDH flux is essentially zero and recovers within 20 min in reversibly‐injured myocardium. Magn Reson Med 60:1029–1036, 2008.

Effects of insulin and cytosolic redox state on glucose production pathways in the isolated perfused mouse liver measured by integrated 2H and 13C NMR

A great deal is known about hepatic glucose production and its response to a variety of factors such as redox state, substrate supply and hormonal control, but the effects of these parameters on the flux through biochemical pathways which integrate to control glucose production are less clear. A combination of 13C and [2H]water tracers and NMR isotopomer analysis were used to investigate metabolic fluxes in response to altered cytosolic redox state and insulin. In livers isolated from fed mice and perfused with a mixture of substrates including lactate/pyruvate (10:1, w/w), hepatic glucose production had substantial contributions from glycogen, PEP (phosphoenolpyruvate) and glycerol. Inversion of the lactate/pyruvate ratio (1:10, w/w) resulted in a surprising decrease in the contribution from glycogen and an increase in that from PEP to glucose production. A change in the lactate/pyruvate ratio from 10:1 to 1:10 also stimulated flux through the tricarboxylic acid cycle (2-fold), while leaving oxygen consumption and overall glucose output unchanged. When lactate and pyruvate were eliminated from the perfusion medium, both gluconeogenesis and tricarboxylic-acid-cycle flux were dramatically lower. Insulin lowered glucose production by inhibiting glycogenolysis at both low and high doses, but only at high levels of insulin did gluconeogenesis or tricarboxylic-acid-cycle flux tend towards lower values (P<0.1). Our data demonstrate that, in the isolated mouse liver, substrate availability and cellular redox state have a dramatic impact on liver metabolism in both the tricarboxylic acid cycle and gluconeogenesis. The tight correlation of these two pathways under multiple conditions suggest that interventions which increase or decrease hepatic tricarboxylic-acid-cycle flux will have a concomitant effect on gluconeogenesis and vice versa.

Use of a single 13C NMR resonance of glutamate for measuring oxygen consumption in tissue

A kinetic model of the citric acid cycle for calculating oxygen consumption from13C nuclear magnetic resonance (NMR) multiplet data has been developed. Measured oxygen consumption (MV˙o 2) was compared with MV˙o 2 predicted by the model with 13C NMR data obtained from rat hearts perfused with glucose and either [2-13C]acetate or [3-13C]pyruvate. The accuracy of MV˙o 2 measured from three subsets of NMR data was compared: glutamate C-4 and C-3 resonance areas; the doublet C4D34 (expressed as a fraction of C-4 area); and C-4 and C-3 areas plus several multiplets of C-2, C-3, and C-4. MV˙o 2 determined by set 2(C4D34 only) gave the same degree of accuracy as set 3(complete data); both were superior to set 1(C-4 and C-3 areas). Analysis of the latter suffers from the correlation between citric acid cycle flux and exchange between α-ketoglutarate and glutamate, resulting in greater error in estimating MV˙o 2. Analysis of C4D34 is less influenced by correlation between parameters, and this single measurement provides the best opportunity for a noninvasive measurement of oxygen consumption.A kinetic model of the citric acid cycle for calculating oxygen consumption from (13)C nuclear magnetic resonance (NMR) multiplet data has been developed. Measured oxygen consumption (MVO(2)) was compared with MVO(2) predicted by the model with (13)C NMR data obtained from rat hearts perfused with glucose and either [2-(13)C]acetate or [3-(13)C]pyruvate. The accuracy of MVO(2) measured from three subsets of NMR data was compared: glutamate C-4 and C-3 resonance areas; the doublet C4D34 (expressed as a fraction of C-4 area); and C-4 and C-3 areas plus several multiplets of C-2, C-3, and C-4. MVO(2) determined by set 2 (C4D34 only) gave the same degree of accuracy as set 3 (complete data); both were superior to set 1 (C-4 and C-3 areas). Analysis of the latter suffers from the correlation between citric acid cycle flux and exchange between alpha-ketoglutarate and glutamate, resulting in greater error in estimating MVO(2). Analysis of C4D34 is less influenced by correlation between parameters, and this single measurement provides the best opportunity for a noninvasive measurement of oxygen consumption.

Predicting functional recovery from ischemia in the rat myocardium

SummaryDepletion of high-energy phosphates, accumulation of inorganic phosphate and intracellular acidosis have each been proposed as important events in the transition from reversible to irreversible ischemic injury. To assess whether each variable is predictive of functional recovery on reperfusion, these were measured in the isolated isovolumic rat heart using31P NMR. Perfused hearts were subjected to either 10, 12 or 40 min of normothermic ischemia followed by 40 min of reperfusion. Hearts were then freeze-clamped for further analysis of phosphate metabolites by NMR and ion chromatography. High-energy phosphates, Pi, phosphomonoesters and pH were measured by31P NMR spectroscopy at 2 minute intervals. Heart rate and developed pressure were monitored simultaneously. All hearts undergoing 10 min of ischemia and 40% of hearts subjected to 12 min of ischemia demonstrated good functional recovery. The remainder of hearts ischemic for 12 min went into contracture on reperfusion with little return of function. Hearts subject to 40 min of ischemia went into ischemic contracture and showed no recovery on reperfusion. Intracellular pH, [ATP], and [Pi] measured prior to reperfusion did not predict the extent of recovery. However, phosphomonoesters were detected prior to reperfusion in all hearts that did not recover well, but were not observed in hearts that showed good mechanical recovery. Analysis of tissue extracts by31P NMR and ion chromatography indicated that the most prominent components of the phosphomonoesters were glucose 6-phosphate, α-glycerol phosphate and AMP. In conclusion, of the various phosphorus metabolites that can be measured by31P NMR, only one group, the phosphomonoesters, was predictive of functional recovery.

Measurement of glycine in human prefrontal brain by point-resolved spectroscopy at 7.0 tesla in vivo

Measurement of glycine in human frontal brain by an optimized point‐resolved spectroscopy sequence at 7 T is reported. Echo time dependencies of the overlapping coupled resonances of myo‐inositol, free choline, and threonine were investigated with density matrix simulations, incorporating the slice‐selective radiofrequency and gradient pulses. The numerical simulations indicated that the selectivity of the 3.55‐ppm glycine singlet is maximized at (TE1, TE2) = (101, 51) ms. Phantom experiments indicated that the myo‐inositol peak amplitude between 3.5 and 3.6 ppm is reduced by a factor of 30 following the optimized point‐resolved spectroscopy, as predicted by the simulation. From LCModel analyses, the glycine concentration in the medial prefrontal cortex in healthy adults was estimated, with a mean Cramér‐Rao lower bound of 7 ± 1% (mean ± standard deviation; n = 7), to be 0.8 ± 0.1 mM, with reference to total creatine at 8 mM. Magn Reson Med, 2009.

Effects of different oxidative insults on intermediary metabolism in isolated perfused rat hearts

13C and 31P NMR were used to evaluate exogenous substrate utilization and endogenous phosphate metabolites in perfused rat hearts exposed to tert-butylhydroperoxide (tert-BOOH) and hydrogen peroxide (H2O2). Both reagents caused a reduction in developed pressure compared to controls and, in agreement with previous 31P NMR data, had different effects on intracellular high-energy phosphates and glycolysis. 13C Isotopomer analysis of tissue extracts showed that H2O2 and tert-BOOH also had significantly different effects on substrate utilization by the citric acid cycle. The contribution of exogenous lactate and glucose to acetyl-CoA was 43% in controls and increased to over 80% in the presence of either oxidant. With tert-BOOH, exogenous glucose and lactate were both significant contributors to acetyl-CoA (44 +/- 2 and 41 +/- 3%). However, with H2O2, exogenous lactate supplied a much higher fraction of acetyl-CoA (72 +/- 2%) than glucose (9 +/- 1%). Also, when [2-(13)C] glucose was supplied, accumulation of [2-(13)C] and [5-(13)C] fructose 1,6-bisphosphate was observed in the presence of H2O2, indicating inhibition of glyceraldehyde-3-phosphate dehydrogenase. These results indicate that despite this glycolytic inhibition, H2O2 increased the utilization of pyruvate precursors when lactate was present as an alternative carbohydrate substrate.

Right-shifting the oxyhemoglobin dissociation curve with RSR13: Effects on high-energy phosphates and myocardial recovery after low-flow ischemia

RSR13[2-(4[[(3,5-Dimethylanilino)carbonyl] methyl] phenoxy)-2-methyl propionic acid], a synthetic allosteric modifier of hemoglobin, increases O2 release from hemoglobin at low oxygen tension. The isolated blood-perfused rat heart was examined during potassium-arrest to determine the effects of RSR13 on the concentration of phosphocreatine (PCr) and adenosine triphosphate (ATP) by using 31P nuclear magnetic resonance (NMR) spectroscopy throughout an episode of low-flow ischemia. All hearts were perfused at constant flow during control (2.0 ml/min) and low-flow (0.2 ml/min) conditions. In normoxic hearts, RSR13 had no effect on either the 31P NMR spectrum or the rate-pressure product. In hearts subjected to 30 min of reduced flow, treatment with RSR13 improved mechanical function on reperfusion (p = 0.026 after 20 min; p = 0.032 after 25 min; and p = 0.045 after 30 min) at 2.0 ml/min with normokalemic blood perfusate. In potassium-arrested hearts, the rate of decrease of [ATP] was reduced in hearts exposed to RSR13 (p < or = 0.05 between 10 and 35.8 min of ischemia except at 28.4 min) during low flow. These results indicate a protective effect of RSR13 on high-energy phosphates during low-flow ischemia and mechanical recovery after reperfusion.