Hyperpolarized 13C allows a direct measure of flux through a single enzyme-catalyzed step by NMR

Abstract

(13)C NMR is a powerful tool for monitoring metabolic fluxes in vivo. The recent availability of automated dynamic nuclear polarization equipment for hyperpolarizing (13)C nuclei now offers the potential to measure metabolic fluxes through select enzyme-catalyzed steps with substantially improved sensitivity. Here, we investigated the metabolism of hyperpolarized [1-(13)C(1)]pyruvate in a widely used model for physiology and pharmacology, the perfused rat heart. Dissolved (13)CO(2), the immediate product of the first step of the reaction catalyzed by pyruvate dehydrogenase, was observed with a temporal resolution of approximately 1 s along with H(13)CO(3)(-), the hydrated form of (13)CO(2) generated catalytically by carbonic anhydrase. In hearts presented with the medium-chain fatty acid octanoate in addition to hyperpolarized [1-(13)C(1)]pyruvate, production of (13)CO(2) and H(13)CO(3)(-) was suppressed by approximately 90%, whereas the signal from [1-(13)C(1)]lactate was enhanced. In separate experiments, it was shown that O(2) consumption and tricarboxylic acid (TCA) cycle flux were unchanged in the presence of added octanoate. Thus, the rate of appearance of (13)CO(2) and H(13)CO(3)(-) from [1-(13)C(1)]pyruvate does not reflect production of CO(2) in the TCA cycle but rather reflects flux through pyruvate dehydrogenase exclusively.

Fig. 1.

¹³C NMR spectrum of an isolated rat heart. This spectrum is the sum of 100 scans. Resonances assigned to dioxane (external standard, 67.4 ppm), CO₂, HCO₃⁻, pyruvate C1, lactate C1, alanine C1, and pyruvate hydrate C1. The resonance labeled with the question mark at 169.2 ppm is an impurity in the pyruvate.

Fig. 2.

Stacked plot of ¹³C NMR spectra from hearts supplied with [1-¹³C₁]pyruvate. Each resonance is a single scan from the chemical shift ± ≈10 Hz from the region assigned to pyruvate C1, H¹³CO₃⁻, or lactate C1. The Left column is from a heart supplied with [1-¹³C₁]pyruvate. The Right column is from a heart supplied with [1-¹³C₁]pyruvate plus unlabeled octanoate.

Fig. 3.

Proton-decoupled ¹³C NMR spectrum of the C4 resonance of glutamate in heart tissue extracts. (A) The spectrum taken from a heart supplied with [U-¹³C₃]pyruvate, shows multiplets in the glutamate C4 due to TCA cycle turnover. From the chemical shift and J coupling pattern, all of the ¹³C signal is due to oxidation of [1-¹³C]pyruvate. (B) Spectrum taken from a heart supplied with [U-¹³C₃]pyruvate and [2,4,6,8-¹³C₄]octanoate. The C4 resonance of glutamate was changed from a six-line multiplet due to oxidation of [U-¹³C₃]pyruvate, to a multiplet dominated by a singlet and a doublet, due to oxidation of [2,4,6,8-¹³C₄]octanoate. P, resonance due to oxidation of pyruvate; D_xx, doublets or doublet of doublets, derived from J-coupling between the appropriate ¹³C-labeled positions of glutamate.

Fig. 4.

Effect of octanoate on appearance of ¹³CO₂ and H¹³CO₃⁻. The average ¹³C NMR signals from dissolved ¹³CO₂ and H¹³CO₃⁻ are shown relative to the maximum pyruvate signal from hearts supplied with [1-¹³C₁]pyruvate (A) or [1-¹³C₁]pyruvate plus octanoate and propionate (B). Data are the mean signal from three to four hearts acquired each second. Standard deviation was ≈10%; the error bars are omitted for clarity. Flux through PDH and the citric acid cycle are shown to the right. Under conditions in which ¹³CO₂ and H¹³CO₃⁻ do not appear in the intact heart, independent measurements made by using isotopomer analysis of freeze-clamped hearts showed near-complete suppression of PDH flux, but no significant effect on TCA cycle flux.