Dataset Information

Evaluation of enzymatic and magnetic properties of γ-glutamyl-[1-¹³C]glycine and its deuteration toward longer retention of the hyperpolarized state.

ABSTRACT: Dynamic nuclear polarization (DNP) is an emerging cutting-edge method of acquiring metabolic and physiological information in vivo. We recently developed γ-glutamyl-[1-¹³C]glycine (γ-Glu-[1-¹³C]Gly) as a DNP nuclear magnetic resonance (NMR) molecular probe to detect γ-glutamyl transpeptidase (GGT) activity in vivo. However, the detailed enzymatic and magnetic properties of this probe remain unknown. Here, we evaluate a γ-Glu-Gly scaffold and develop a deuterated probe, γ-Glu-[1-¹³C]Gly-d ₂, that can realize a longer lifetime of the hyperpolarized signal. We initially evaluated the GGT-mediated enzymatic conversion of γ-Glu-Gly and the magnetic properties of ¹³C-enriched γ-Glu-Gly (γ-Glu-[1-¹³C]Gly and γ-[5-¹³C]Glu-Gly) to support the validity of γ-Glu-[1-¹³C]Gly as a DNP NMR molecular probe for GGT. We then examined the spin-lattice relaxation time (T ₁) of γ-Glu-[1-¹³C]Gly and γ-Glu-[1-¹³C]Gly-d ₂ under various conditions (D₂O, PBS, and serum) and confirmed that the T ₁ of γ-Glu-[1-¹³C]Gly and γ-Glu-[1-¹³C]Gly-d ₂ was maintained for 30 s (9.4 T) and 41 s (9.4 T), respectively, even in serum. Relaxation analysis of γ-Glu-[1-¹³C]Gly revealed a significant contribution of the dipole-dipole interaction and the chemical shift anisotropy relaxation pathway (71% of the total relaxation rate at 9.4 T), indicating the potential of deuteration and the use of a lower magnetic field for realizing a longer T ₁. In fact, by using γ-Glu-[1-¹³C]Gly-d ₂ as a DNP probe, we achieved longer retention of the hyperpolarized signal at 1.4 T.

SUBMITTER: Kondo Y

PROVIDER: S-EPMC9043577 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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Evaluation of enzymatic and magnetic properties of γ-glutamyl-[1-13C]glycine and its deuteration toward longer retention of the hyperpolarized state.

Kondo Yohei Y Saito Yutaro Y Elhelaly Abdelazim Elsayed AE Hyodo Fuminori F Nishihara Tatsuya T Itoda Marino M Nonaka Hiroshi H Matsuo Masayuki M Sando Shinsuke S

RSC advances 20211117 59

Dynamic nuclear polarization (DNP) is an emerging cutting-edge method of acquiring metabolic and physiological information in vivo. We recently developed γ-glutamyl-[1-13C]glycine (γ-Glu-[1-13C]Gly) as a DNP nuclear magnetic resonance (NMR) molecular probe to detect γ-glutamyl transpeptidase (GGT) activity in vivo. However, the detailed enzymatic and magnetic properties of this probe remain unknown. Here, we evaluate a γ-Glu-Gly scaffold and develop a deuterat ...[more]

PMID: 35496407

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Project description: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.

Dataset Information

Evaluation of enzymatic and magnetic properties of γ-glutamyl-[1-¹³C]glycine and its deuteration toward longer retention of the hyperpolarized state.

Publications

Evaluation of enzymatic and magnetic properties of γ-glutamyl-[1-<sup>13</sup>C]glycine and its deuteration toward longer retention of the hyperpolarized state.

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