Abstract
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 [1-(13)C(1)]pyruvate was compared to [1-(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[1-(13)C(1)]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 (13)C 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.