Oxidation of hyperpolarized [1-13 C]pyruvate in isolated rat kidneys.

Kidneys play a central role in numerous disorders but current imaging methods have limited utility to probe renal metabolism. Hyperpolarized (HP) 13 C magnetic resonance imaging is uniquely suited to provide metabolite-specific information about key biochemical pathways and it offers the further advantage that renal imaging is practical in humans. This study evaluated the feasibility of hyperpolarization examinations in a widely used model for analysis of renal physiology, the isolated kidney, which enables isolation of renal metabolism from the effects of other organs and validation of HP results by independent measurements. Isolated rat kidneys were supplied with either HP [1-13 C]pyruvate only or HP [1-13 C]pyruvate plus octanoate. Metabolic activity in both groups was confirmed by stable renal oxygen consumption. HP [1-13 C]pyruvate was readily metabolized to [13 C]bicarbonate, [1-13 C]lactate, and [1-13 C]alanine, detectable seconds after HP [1-13 C]pyruvate was injected. Octanoate suppressed but did not eliminate the production of HP [13 C]bicarbonate from [1-13 C]pyruvate. Steady-state flux analyses using non-HP 13 C substrates validated the utilization of HP [1-13 C]pyruvate, as observed by HP 13 C NMR. In the presence of octanoate, lactate is generated from a tricarboxylic acid cycle intermediate, oxaloacetate. The isolated rat kidney may serve as an excellent model for investigating and establishing new HP 13 C metabolic probes for future kidney imaging applications.

Co-Polarized [1-13C]Pyruvate and [1,3-13C2]Acetoacetate Provide a Simultaneous View of Cytosolic and Mitochondrial Redox in a Single Experiment.

Cellular redox is intricately linked to energy production and normal cell function. Although the redox states of mitochondria and cytosol are connected by shuttle mechanisms, the redox state of mitochondria may differ from redox in the cytosol in response to stress. However, detecting these differences in functioning tissues is difficult. Here, we employed 13C magnetic resonance spectroscopy (MRS) and co-polarized [1-13C]pyruvate and [1,3-13C2]acetoacetate ([1,3-13C2]AcAc) to monitor production of hyperpolarized (HP) lactate and ß-hydroxybutyrate as indicators of cytosolic and mitochondrial redox, respectively. Isolated rat hearts were examined under normoxic conditions, during low-flow ischemia, and after pretreatment with either aminooxyacetate (AOA) or rotenone. All interventions were associated with an increase in [Pi]/[ATP] measured by 31P NMR. In well-oxygenated untreated hearts, rapid conversion of HP [1-13C]pyruvate to [1-13C]lactate and [1,3-13C2]AcAc to [1,3-13C2]ß-hydroxybutyrate ([1,3-13C2]ß-HB) was readily detected. A significant increase in HP [1,3-13C2]ß-HB but not [1-13C]lactate was observed in rotenone-treated and ischemic hearts, consistent with an increase in mitochondrial NADH but not cytosolic NADH. AOA treatments did not alter the productions of HP [1-13C]lactate or [1,3-13C2]ß-HB. This study demonstrates that biomarkers of mitochondrial and cytosolic redox may be detected simultaneously in functioning tissues using co-polarized [1-13C]pyruvate and [1,3-13C2]AcAc and 13C MRS and that changes in mitochondrial redox may precede changes in cytosolic redox.

13C-Labeled Diethyl Ketoglutarate Derivatives as Hyperpolarized Probes of 2-Ketoglutarate Dehydrogenase Activity.

The TCA cycle is a central metabolic pathway for energy production and biosynthesis. A major control point of metabolic flux through the cycle is the decarboxylation of 2-ketoglutarate by the TCA cycle enzyme 2-ketoglutarate dehydrogenase (2-KGDH). In this project, we developed 13C labeled 2-ketoglutarate derivatives to monitor 2-KGDH activity in vivo. 13C NMR analysis of liver extracts revealed that uniformly 13C labeled 2-ketogutarate, in its cell permeable ester form, was rapidly taken up and hydrolyzed in liver and underwent extensive metabolism to produce labeled glutamate, succinate, lactate and other metabolites. Diethyl [1,2-13C2]-2-ketoglutarate was successfully polarized by dynamic nuclear polarization and within seconds after injection into rats, the probe produced hyperpolarized [13C]bicarbonate in the liver reflecting flux through the TCA cycle. These experiments demonstrate that this tracer offers the possibility of directly monitoring flux through 2-KGDH in vivo.

Lactate Dehydrogenase A Governs Cardiac Hypertrophic Growth in Response to Hemodynamic Stress.

The heart manifests hypertrophic growth in response to high blood pressure, which may decompensate and progress to heart failure under persistent stress. Metabolic remodeling is an early event in this process. However, its role remains to be fully characterized. Here, we show that lactate dehydrogenase A (LDHA), a critical glycolytic enzyme, is elevated in the heart in response to hemodynamic stress. Cardiomyocyte-restricted deletion of LDHA leads to defective cardiac hypertrophic growth and heart failure by pressure overload. Silencing of LDHA in cultured cardiomyocytes suppresses cell growth from pro-hypertrophic stimulation in vitro, while overexpression of LDHA is sufficient to drive cardiomyocyte growth. Furthermore, we find that lactate is capable of rescuing the growth defect from LDHA knockdown. Mechanistically, lactate stabilizes NDRG3 (N-myc downregulated gene family 3) and stimulates ERK (extracellular signal-regulated kinase). Our results together suggest that the LDHA/NDRG3 axis may play a critical role in adaptive cardiomyocyte growth in response to hemodynamic stress.

Mitochondrial Substrate Utilization Regulates Cardiomyocyte Cell Cycle Progression.

The neonatal mammalian heart is capable of regeneration for a brief window of time after birth. However, this regenerative capacity is lost within the first week of life, which coincides with a postnatal shift from anaerobic glycolysis to mitochondrial oxidative phosphorylation, particularly towards fatty-acid utilization. Despite the energy advantage of fatty-acid beta-oxidation, cardiac mitochondria produce elevated rates of reactive oxygen species when utilizing fatty acids, which is thought to play a role in cardiomyocyte cell-cycle arrest through induction of DNA damage and activation of DNA-damage response (DDR) pathway. Here we show that inhibiting fatty-acid utilization promotes cardiomyocyte proliferation in the postnatatal heart. First, neonatal mice fed fatty-acid deficient milk showed prolongation of the postnatal cardiomyocyte proliferative window, however cell cycle arrest eventually ensued. Next, we generated a tamoxifen-inducible cardiomyocyte-specific, pyruvate dehydrogenase kinase 4 (PDK4) knockout mouse model to selectively enhance oxidation of glycolytically derived pyruvate in cardiomyocytes. Conditional PDK4 deletion resulted in an increase in pyruvate dehydrogenase activity and consequently an increase in glucose relative to fatty-acid oxidation. Loss of PDK4 also resulted in decreased cardiomyocyte size, decreased DNA damage and expression of DDR markers and an increase in cardiomyocyte proliferation. Following myocardial infarction, inducible deletion of PDK4 improved left ventricular function and decreased remodelling. Collectively, inhibition of fatty-acid utilization in cardiomyocytes promotes proliferation, and may be a viable target for cardiac regenerative therapies.

Real-time hyperpolarized 13C magnetic resonance detects increased pyruvate oxidation in pyruvate dehydrogenase kinase 2/4-double knockout mouse livers.

The pyruvate dehydrogenase complex (PDH) critically regulates carbohydrate metabolism. Phosphorylation of PDH by one of the pyruvate dehydrogenase kinases 1-4 (PDK1-4) decreases the flux of carbohydrates into the TCA cycle. Inhibition of PDKs increases oxidative metabolism of carbohydrates, so targeting PDKs has emerged as an important therapeutic approach to manage various metabolic diseases. Therefore, it is highly desirable to begin to establish imaging tools for noninvasive measurements of PDH flux in rodent models. In this study, we used hyperpolarized (HP) 13C-magnetic resonance spectroscopy to study the impact of a PDK2/PDK4 double knockout (DKO) on pyruvate metabolism in perfused livers from lean and diet-induced obese (DIO) mice and validated the HP observations with high-resolution 13C-nuclear magnetic resonance (NMR) spectroscopy of tissue extracts and steady-state isotopomer analyses. We observed that PDK-deficient livers produce more HP-bicarbonate from HP-[1-13C]pyruvate than age-matched control livers. A steady-state 13C-NMR isotopomer analysis of tissue extracts confirmed that flux rates through PDH, as well as pyruvate carboxylase and pyruvate cycling activities, are significantly higher in PDK-deficient livers. Immunoblotting experiments confirmed that HP-bicarbonate production from HP-[1-13C]pyruvate parallels decreased phosphorylation of the PDH E1α subunit (pE1α) in liver tissue. Our findings indicate that combining real-time hyperpolarized 13C NMR spectroscopy and 13C isotopomer analysis provides quantitative insights into intermediary metabolism in PDK-knockout mice. We propose that this method will be useful in assessing metabolic disease states and developing therapies to improve PDH flux.

Metabolism of hyperpolarized 13 C-acetoacetate to ß-hydroxybutyrate detects real-time mitochondrial redox state and dysfunction in heart tissue.

Mitochondrial dysfunction is considered to be an important component of many metabolic diseases yet there is no reliable imaging biomarker for monitoring mitochondrial damage in vivo. A large prior literature on inter-conversion of ß-hydroxybutyrate and acetoacetate indicates that the process is mitochondrial and that the ratio reflects a specifically mitochondrial redox state. Therefore, the conversion of [1,3-13 C]acetoacetate to [1,3-13 C]ß-hydroxybutyrate is expected to be sensitive to the abnormal redox state present in dysfunctional mitochondria. In this study, we examined the conversion of hyperpolarized (HP) 13 C-acetoacetate (AcAc) to 13 C-ß-hydroxybutyrate (ß-HB) as a potential imaging biomarker for mitochondrial redox and dysfunction in perfused rat hearts. Conversion of HP-AcAc to ß-HB was investigated using 13 C magnetic resonance spectroscopy in Langendorff-perfused rat hearts in four groups: control, global ischemic reperfusion, low-flow ischemic, and rotenone (mitochondrial complex-I inhibitor)-treated hearts. We observed that more ß-HB was produced from AcAc in ischemic hearts and the hearts exposed to complex I inhibitor rotenone compared with controls, consistent with the accumulation of excess mitochondrial NADH. The increase in ß-HB, as detected by 13 C MRS, was validated by a direct measure of tissue ß-HB by 1 H nuclear magnetic resonance in tissue extracts. The redox ratio, NAD+ /NADH, measured by enzyme assays of homogenized tissue, also paralleled production of ß-HB from AcAc. Transmission electron microscopy of tissues provided direct evidence for abnormal mitochondrial structure in each ischemic tissue model. The results suggest that conversion of HP-AcAc to HP-ß-HB detected by 13 C-MRS may serve as a useful diagnostic marker of mitochondrial redox and dysfunction in heart tissue in vivo.

Effects of deuteration on transamination and oxidation of hyperpolarized 13C-Pyruvate in the isolated heart.

This study was designed to determine the effects of deuteration in pyruvate on exchange reactions in alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and flux through pyruvate dehydrogenase (PDH). Although deuteration of a 13C enriched substrate is commonly used to increase the lifetime of a probe for hyperpolarization experiments, the potential impact of kinetic isotope effects on such substitutions has not been studied in detail. Metabolism of deuterated pyruvate was investigated in isolated rat hearts. Hearts were perfused with a 1:1 mixture of [U-13C3]pyruvate and [2-13C1]pyruvate or a 1:1 mixture of [U-13C3]pyruvate plus [2-13C1, U-2H3]pyruvate for 30 min before being freeze clamped. Another set of hearts received [2-13C1, U-2H3]pyruvate and was freeze-clamped at 3 min or 6 min. Tissue extracts were analyzed by 1H and 13C{1H} NMR spectroscopy. The chemical shift isotope effect of 2H was monitored in the 13C NMR spectra of the C2 resonance of lactate and alanine plus the C5 of glutamate. There was little kinetic isotope effect of 2H in pyruvate on flux through PDH, LDH or ALT as detected by the distribution of 13C, but the distribution of 2H differed markedly between alanine and lactate. At steady-state, alanine was a mixture of deuterated species, while lactate was largely perdeuterated. Consistent with results at steady-state, hearts freeze-clamped at 3 min or 6 min showed rapid removal of deuterium in alanine but not in lactate. Metabolism of hyperpolarized [1-13C1]pyruvate was compared to [1-13C1,U-2H3]pyruvate in isolated hearts. Consistent with the results from tissue extracts, there was little effect of deuteration on the kinetics of appearance of lactate, alanine or bicarbonate, but there was a small, time-dependent upfield chemical shift in the HP[1-13C1]alanine signal reflecting exchange of methyl deuterons with water protons. Together, these results demonstrate that (1) the kinetics of pyruvate metabolism in hearts detected by 13C NMR are not affected by replacement of the pyruvate methyl protons with deuterons and (2) that the loss of deuterium from the methyl position occurs rapidly during the conversion of pyruvate to alanine. The majority of the deuterium atoms are lost on the time-scale of a hyperpolarization experiment.

Brain metabolism modulates neuronal excitability in a mouse model of pyruvate dehydrogenase deficiency.

Glucose is the ultimate substrate for most brain activities that use carbon, including synthesis of the neurotransmitters glutamate and γ-aminobutyric acid via mitochondrial tricarboxylic acid (TCA) cycle. Brain metabolism and neuronal excitability are thus interdependent. However, the principles that govern their relationship are not always intuitive because heritable defects of brain glucose metabolism are associated with the paradoxical coexistence, in the same individual, of episodic neuronal hyperexcitation (seizures) with reduced basal cerebral electrical activity. One such prototypic disorder is pyruvate dehydrogenase (PDH) deficiency (PDHD). PDH is central to metabolism because it steers most of the glucose-derived flux into the TCA cycle. To better understand the pathophysiology of PDHD, we generated mice with brain-specific reduced PDH activity that paralleled salient human disease features, including cerebral hypotrophy, decreased amplitude electroencephalogram (EEG), and epilepsy. The mice exhibited reductions in cerebral TCA cycle flux, glutamate content, spontaneous, and electrically evoked in vivo cortical field potentials and gamma EEG oscillation amplitude. Episodic decreases in gamma oscillations preceded most epileptiform discharges, facilitating their prediction. Fast-spiking neuron excitability was decreased in brain slices, contributing to in vivo action potential burst prolongation after whisker pad stimulation. These features were partially reversed after systemic administration of acetate, which augmented cerebral TCA cycle flux, glutamate-dependent synaptic transmission, inhibition and gamma oscillations, and reduced epileptiform discharge duration. Thus, our results suggest that dysfunctional excitability in PDHD is consequent to reduced oxidative flux, which leads to decreased neuronal activation and impaired inhibition, and can be mitigated by an alternative metabolic substrate.

Probing carbohydrate metabolism using hyperpolarized 13 C-labeled molecules.

Glycolysis is a fundamental metabolic process in all organisms. Anomalies in glucose metabolism are linked to various pathological conditions. In particular, elevated aerobic glycolysis is a characteristic feature of rapidly growing cells. Glycolysis and the closely related pentose phosphate pathway can be monitored in real time by hyperpolarized 13 C-labeled metabolic substrates such as 13 C-enriched, deuterated D-glucose derivatives, [2-13 C]-D-fructose, [2-13 C] dihydroxyacetone, [1-13 C]-D-glycerate, [1-13 C]-D-glucono-δ-lactone and [1-13 C] pyruvate in healthy and diseased tissues. Elevated glycolysis in tumors (the Warburg effect) was also successfully imaged using hyperpolarized [U-13 C6 , U-2 H7 ]-D-glucose, while the size of the preexisting lactate pool can be measured by 13 C MRS and/or MRI with hyperpolarized [1-13 C]pyruvate. This review summarizes the application of various hyperpolarized 13 C-labeled metabolites to the real-time monitoring of glycolysis and related metabolic processes in normal and diseased tissues.

Esterase-Catalyzed Production of Hyperpolarized 13C-Enriched Carbon Dioxide in Tissues for Measuring pH.

13C Magnetic resonance imaging of hyperpolarized (HP) 13C-enriched bicarbonate (H13CO3-) and carbon dioxide (13CO2) is a novel and sensitive technique for tissue pH mapping in vivo. Administration of the HP physiological buffer pair is attractive, but poor polarization and the short T1 of 13C-enriched inorganic bicarbonate salts are major drawbacks for this approach. Here, we report a new class of mixed anhydrides for esterase-catalyzed production of highly polarized 13CO2 and H13CO3- in tissue. A series of precursors with different alkoxy and acyl groups were synthesized and tested for chemical stability and T1. 13C-enriched ethyl acetyl carbonate (13C-EAC) was found to be the most suitable candidate due to the relatively long T1 and good chemical stability. Our results showed that 13C-EAC can be efficiently and rapidly polarized using BDPA. HP 13C-EAC was rapidly hydrolyzed by esterase to 13C-enriched monoacetyl carbonate (13C-MAC), which then decomposed to HP 13CO2. Equilibrium between the newly produced 13CO2 and H13CO3- was quickly established by carbonic anhydrase, producing a physiological buffer pair with 13C NMR signals that can be quantified for pH measurements. Finally, in vivo tissue pH measurements using HP 13C-EAC was successfully demonstrated in the liver of healthy rats. These results suggest that HP 13C-EAC is a novel imaging probe for in vivo pH measurements.

MOXI Is a Mitochondrial Micropeptide That Enhances Fatty Acid ß-Oxidation.

Micropeptide regulator of ß-oxidation (MOXI) is a conserved muscle-enriched protein encoded by an RNA transcript misannotated as non-coding. MOXI localizes to the inner mitochondrial membrane where it associates with the mitochondrial trifunctional protein, an enzyme complex that plays a critical role in fatty acid ß-oxidation. Isolated heart and skeletal muscle mitochondria from MOXI knockout mice exhibit a diminished ability to metabolize fatty acids, while transgenic MOXI overexpression leads to enhanced ß-oxidation. Additionally, hearts from MOXI knockout mice preferentially oxidize carbohydrates over fatty acids in an isolated perfused heart system compared to wild-type (WT) animals. MOXI knockout mice also exhibit a profound reduction in exercise capacity, highlighting the role of MOXI in metabolic control. The functional characterization of MOXI provides insight into the regulation of mitochondrial metabolism and energy homeostasis and underscores the regulatory potential of additional micropeptides that have yet to be identified.

A novel inhibitor of pyruvate dehydrogenase kinase stimulates myocardial carbohydrate oxidation in diet-induced obesity.

The pyruvate dehydrogenase complex (PDC) is a key control point of energy metabolism and is subject to regulation by multiple mechanisms, including posttranslational phosphorylation by pyruvate dehydrogenase kinase (PDK). Pharmacological modulation of PDC activity could provide a new treatment for diabetic cardiomyopathy, as dysregulated substrate selection is concomitant with decreased heart function. Dichloroacetate (DCA), a classic PDK inhibitor, has been used to treat diabetic cardiomyopathy, but the lack of specificity and side effects of DCA indicate a more specific inhibitor of PDK is needed. This study was designed to determine the effects of a novel and highly selective PDK inhibitor, 2((2,4-dihydroxyphenyl)sulfonyl) isoindoline-4,6-diol (designated PS10), on pyruvate oxidation in diet-induced obese (DIO) mouse hearts compared with DCA-treated hearts. Four groups of mice were studied: lean control, DIO, DIO + DCA, and DIO + PS10. Both DCA and PS10 improved glucose tolerance in the intact animal. Pyruvate metabolism was studied in perfused hearts supplied with physiological mixtures of long chain fatty acids, lactate, and pyruvate. Analysis was performed using conventional 1H and 13C isotopomer methods in combination with hyperpolarized [1-13C]pyruvate in the same hearts. PS10 and DCA both stimulated flux through PDC as measured by the appearance of hyperpolarized [13C]bicarbonate. DCA but not PS10 increased hyperpolarized [1-13C]lactate production. Total carbohydrate oxidation was reduced in DIO mouse hearts but increased by DCA and PS10, the latter doing so without increasing lactate production. The present results suggest that PS10 is a more suitable PDK inhibitor for treatment of diabetic cardiomyopathy.

Targeting hepatic pyruvate dehydrogenase kinases restores insulin signaling and mitigates ChREBP-mediated lipogenesis in diet-induced obese mice.

OBJECTIVE: Mitochondrial pyruvate dehydrogenase kinases 1-4 (PDKs1-4) negatively regulate activity of the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation. PDKs play a pivotal role in maintaining energy homeostasis and contribute to metabolic flexibility by attenuating PDC activity in various mammalian tissues. Cumulative evidence has shown that the up-regulation of PDK4 expression is tightly associated with obesity and diabetes. In this investigation, we test the central hypothesis that PDKs1-4 are a pharmacological target for lowering glucose levels and restoring insulin sensitivity in obesity and type 2 diabetes (T2D). METHODS: Diet-induced obese (DIO) mice were treated with a liver-specific pan-PDK inhibitor 2-[(2,4-dihydroxyphenyl) sulfonyl]isoindoline-4,6-diol (PS10) for four weeks, and results compared with PDK2/PDK4 double knockout (DKO) mice on the same high fat diet (HFD). RESULTS: Both PS10-treated DIO mice and HFD-fed DKO mice showed significantly improved glucose, insulin and pyruvate tolerance, compared to DIO controls, with lower plasma insulin levels and increased insulin signaling in liver. In response to lower glucose levels, phosphorylated AMPK in PS10-treated DIO and HFD-fed DKO mice is upregulated, accompanied by decreased nuclear carbohydrate-responsive element binding protein (ChREBP). The reduced ChREBP signaling correlates with down-regulation of hepatic lipogenic enzymes (ACC1, FAS, and SCD1), leading to markedly diminished hepatic steatosis in both study groups, with lower circulating cholesterol and triacylglyceride levels as well as reduced fat mass. PS10-treated DIO as well as DKO mice showed predominant fatty acid over glucose oxidation. However, unlike systemic DKO mice, increased hepatic PDC activity alone in PS10-treated DIO mice does not raise the plasma total ketone body level. CONCLUSION: Our findings establish that specific targeting of hepatic PDKs with the PDK inhibitor PS10 is an effective therapeutic approach to maintaining glucose and lipid homeostasis in obesity and T2D, without the harmful ketoacidosis associated with systemic inhibition of PDKs.

Rational Design of [13 C,D14 ]Tert-butylbenzene as a Scaffold Structure for Designing Long-lived Hyperpolarized 13 C Probes.

Dynamic nuclear polarization (DNP) is a technique to polarize the nuclear spin population. As a result of the hyperpolarization, the NMR sensitivity of the nuclei in molecules can be dramatically enhanced. Recent application of the hyperpolarization technique has led to advances in biochemical and molecular studies. A major problem is the short lifetime of the polarized nuclear spin state. Generally, in solution, the polarized nuclear spin state decays to a thermal spin equilibrium, resulting in loss of the enhanced NMR signal. This decay is correlated directly with the spin-lattice relaxation time T1 . Here we report [13 C,D14 ]tert-butylbenzene as a new scaffold structure for designing hyperpolarized 13 C probes. Thanks to the minimized spin-lattice relaxation (T1 ) pathways, its water-soluble derivative showed a remarkably long 13 C T1 value and long retention of the hyperpolarized spin state.

The rate of lactate production from glucose in hearts is not altered by per-deuteration of glucose.

This study was designed to determine whether perdeuterated glucose experiences a kinetic isotope effect (KIE) as glucose passes through glycolysis and is further oxidized in the tricarboxylic acid (TCA) cycle. Metabolism of deuterated glucose was investigated in two groups of perfused rat hearts. The control group was supplied with a 1:1 mixture of [U-13C6]glucose and [1,6-13C2]glucose, while the experimental group received [U-13C6,U-2H7]glucose and [1,6-13C2]glucose. Tissue extracts were analyzed by 1H, 2H and proton-decoupled 13C NMR spectroscopy. Extensive 2H-13C scalar coupling plus chemical shift isotope effects were observed in the proton-decoupled 13C NMR spectra of lactate, alanine and glutamate. A small but measureable (∼8%) difference in the rate of conversion of [U-13C6]glucose vs. [1,6-13C2]glucose to lactate, likely reflecting rates of CC bond breakage in the aldolase reaction, but conversion of [U-13C6]glucose versus [U-13C6,U-2H7]glucose to lactate did not differ. This shows that the presence of deuterium in glucose does not alter glycolytic flux. However, there were two distinct effects of deuteration on metabolism of glucose to alanine and oxidation of glucose in the TCA. First, alanine undergoes extensive exchange of methyl deuterons with solvent protons in the alanine amino transferase reaction. Second, there is a substantial kinetic isotope effect in metabolism of [U-13C6,U-2H7]glucose to alanine and glutamate. In the presence of [U-13C6,U-2H7]glucose, alanine and lactate are not in rapid exchange with the same pool of pyruvate. These studies indicate that the appearance of hyperpolarized 13C-lactate from hyperpolarized [U-13C6,U-2H7]glucose is not substantially influenced by a deuterium kinetic isotope effect.

In vivo assessment of intracellular redox state in rat liver using hyperpolarized [1-13 C]Alanine.

PURPOSE: The intracellular lactate to pyruvate concentration ratio is a commonly used tissue assay biomarker of redox, being proportional to free cytosolic [NADH]/[NAD+ ]. In this study, we assessed the use of hyperpolarized [1-13 C]alanine and the subsequent detection of the intracellular products of [1-13 C]pyruvate and [1-13 C]lactate as a useful substrate for assessing redox levels in the liver in vivo. METHODS: Animal experiments were conducted to measure in vivo metabolism at baseline and after ethanol infusion. A solution of 80-mM hyperpolarized [1-13 C]alanine was injected intravenously at baseline (n = 8) and 45 min after ethanol infusion (n = 4), immediately followed by the dynamic acquisition of 13 C MRS spectra. RESULTS: In vivo rat liver spectra showed peaks from [1-13 C] alanine and the products of [1-13 C]lactate, [1-13 C]pyruvate, and 13 C-bicarbonate. A significantly increased 13 C-lactate/13 C-pyruvate ratio was observed after ethanol infusion (8.46 ± 0.58 at baseline versus 13.58 ± 0.69 after ethanol infusion; P < 0.001) consistent with the increased NADH produced by liver metabolism of ethanol to acetaldehyde and then acetate. A decrease in 13 C-bicarbonate production was also noted, potentially reflecting ethanol-induced mitochondrial redox changes. CONCLUSION: A method to measure in vivo tissue redox using hyperpolarized [1-13 C]alanine is presented, with the validity of the proposed 13 C-pyruvate/13 C-lactate metric tested using an ethanol challenge to alter liver redox state. Magn Reson Med 77:1741-1748, 2017. © 2017 International Society for Magnetic Resonance in Medicine.

Influence of Dy3+ and Tb3+ doping on 13C dynamic nuclear polarization.

Dynamic nuclear polarization (DNP) is a technique that uses a microwave-driven transfer of high spin alignment from electrons to nuclear spins. This is most effective at low temperature and high magnetic field, and with the invention of the dissolution method, the amplified nuclear magnetic resonance (NMR) signals in the frozen state in DNP can be harnessed in the liquid-state at physiologically acceptable temperature for in vitro and in vivo metabolic studies. A current optimization practice in dissolution DNP is to dope the sample with trace amounts of lanthanides such as Gd3+ or Ho3+, which further improves the polarization. While Gd3+ and Ho3+ have been optimized for use in dissolution DNP, other lanthanides have not been exhaustively studied for use in C13 DNP applications. In this work, two additional lanthanides with relatively high magnetic moments, Dy3+ and Tb3+, were extensively optimized and tested as doping additives for C13 DNP at 3.35 T and 1.2 K. We have found that both of these lanthanides are also beneficial additives, to a varying degree, for C13 DNP. The optimal concentrations of Dy3+ (1.5 mM) and Tb3+ (0.25 mM) for C13 DNP were found to be less than that of Gd3+ (2 mM). W-band electron paramagnetic resonance shows that these enhancements due to Dy3+ and Tb3+ doping are accompanied by shortening of electron T1 of trityl OX063 free radical. Furthermore, when dissolution was employed, Tb3+-doped samples were found to have similar liquid-state C13 NMR signal enhancements compared to samples doped with Gd3+, and both Tb3+ and Dy3+ had a negligible liquid-state nuclear T1 shortening effect which contrasts with the significant reduction in T1 when using Gd3+. Our results show that Dy3+ doping and Tb3+ doping have a beneficial impact on C13 DNP both in the solid and liquid states, and that Tb3+ in particular could be used as a potential alternative to Gd3+ in C13 dissolution DNP experiments.

Impact of Ho(3+)-doping on (13)C dynamic nuclear polarization using trityl OX063 free radical.

We have investigated the effects of Ho-DOTA doping on the dynamic nuclear polarization (DNP) of [1-(13)C] sodium acetate using trityl OX063 free radical at 3.35 T and 1.2 K. Our results indicate that addition of 2 mM Ho-DOTA on 3 M [1-(13)C] sodium acetate sample in 1 : 1 v/v glycerol : water with 15 mM trityl OX063 improves the DNP-enhanced (13)C solid-state nuclear polarization by a factor of around 2.7-fold. Similar to the Gd(3+) doping effect on (13)C DNP, the locations of the positive and negative (13)C maximum polarization peaks in the (13)C microwave DNP sweep are shifted towards each other with the addition of Ho-DOTA on the DNP sample. W-band electron spin resonance (ESR) studies have revealed that while the shape and linewidth of the trityl OX063 ESR spectrum was not affected by Ho(3+)-doping, the electron spin-lattice relaxation time T1 of trityl OX063 was prominently reduced at cryogenic temperatures. The reduction of trityl OX063 electron T1 by Ho-doping is linked to the (13)C DNP improvement in light of the thermodynamic picture of DNP. Moreover, the presence of Ho-DOTA in the dissolution liquid at room temperature has negligible reduction effect on liquid-state (13)C T1, in contrast to Gd(3+)-doping which drastically reduces the (13)C T1. The results here suggest that Ho(3+)-doping is advantageous over Gd(3+) in terms of preservation of hyperpolarized state-an important aspect to consider for in vitro and in vivo NMR or imaging (MRI) experiments where a considerable preparation time is needed to administer the hyperpolarized (13)C liquid.

Hyperpolarized 15N-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 (15)N-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 (15)N chemical shifts were found to be highly sensitive to pH. These probes displayed sharp (15)N resonances and large differences in chemical shifts (Δδ > 90 ppm) 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.