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.

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.

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.

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.

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.

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.