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 13 C 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-13 C 3 ]pyruvate and [2-13 C 1 ]pyruvate or a 1:1 mixture of [U-13 C 3 ]pyruvate plus [2-13 C 1 , U-2 H 3 ]pyruvate for 30 min before being freeze clamped. Another set of hearts received [2-13 C 1 , U-2 H 3 ]pyruvate and was freeze-clamped at 3 min or 6 min. Tissue extracts were analyzed by 1 H and 13 C{ 1 H} NMR spectroscopy. The chemical shift isotope effect of 2 H was monitored in the 13 C NMR spectra of the C2 resonance of lactate and alanine plus the C5 of glutamate. There was little kinetic isotope effect of 2 H in pyruvate on flux through PDH, LDH or ALT as detected by the distribution of 13 C, but the distribution of 2 H 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 [l-13 C 1 ]pyruvate was compared to [l-13 C 1 ,U-2 H 3 ]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[l-13 C 1 ]alanine signal reflecting exchange of methyl deuterons with water protons. Together, these results

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 C–C 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.

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 β-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]­β-hydroxybutyrate ([1,3-13C2]­β-HB) was readily detected. A significant increase in HP [1,3-13C2]­β-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]­β-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.