Hyperpolarized 13 C Magnetic Resonance Imaging of Fumarate Metabolism by Parahydrogen-induced Polarization: A Proof-of-Concept in vivo Study

Abstract

Hyperpolarized [1-¹³ C]fumarate is a promising magnetic resonance imaging (MRI) biomarker for cellular necrosis, which plays an important role in various disease and cancerous pathological processes. To demonstrate the feasibility of MRI of [1-¹³ C]fumarate metabolism using parahydrogen-induced polarization (PHIP), a low-cost alternative to dissolution dynamic nuclear polarization (dDNP), a cost-effective and high-yield synthetic pathway of hydrogenation precursor [1-¹³ C]acetylenedicarboxylate (ADC) was developed. The trans-selectivity of the hydrogenation reaction of ADC using a ruthenium-based catalyst was elucidated employing density functional theory (DFT) simulations. A simple PHIP set-up was used to generate hyperpolarized [1-¹³ C]fumarate at sufficient ¹³ C polarization for ex vivo detection of hyperpolarized ¹³ C malate metabolized from fumarate in murine liver tissue homogenates, and in vivo ¹³ C MR spectroscopy and imaging in a murine model of acetaminophen-induced hepatitis.

Keywords: density functional calculations; fumarate; hyperpolarized 13C MRS/I; matabolism; parahydrogen-induced polarization.

Scheme 1

Four‐step synthesis pathway for the [1‐¹³C]fumarate precursor, [1‐¹³C] acetylenedicarboxylic acid, using [1‐¹³C]sodium acetate as a starting material. Full details are provided in the Experimental Section and the Supporting Information.

Figure 1

Free energy diagram for the trans‐selective hydrogenation reaction of acetylenedicarboxylate using [Cp*Ru(MeCN)₃]PF₆ at SMD/B3PW91/6‐311++G(2df,2p)(C,H,N,O),DGDZVP(Ru) //SMD/B3PW91/DGDZVP calculated by DFT simulation. Values in parentheses are the differences of the energies from the previous steps.

Figure 2

An optimized structure of stable reactant complex of A1.

Figure 3

Comparative free energy diagram for the hydrogenation of acetylenedicarboxylic acid using [Cp*Ru(MeCN)₃]PF₆ in basic and neutral conditions from C2. Values in parentheses are the differences of the energies from the previous steps.

Figure 4

a) Schematic of the conversion of [1‐¹³C]fumarate into [1‐¹³C] and [4‐¹³C]malate by fumarase. b) Dynamic ¹³C NMR spectra of hyperpolarized [1‐¹³C]fumarate at 176 ppm produced by MFC‐based PHIP (left) and the fitted signal decay (right). c) Generation of hyperpolarized [1‐¹³C] and [4‐¹³C]malate at around 181 ppm by mixing hyperpolarized [1‐¹³C]fumarate with mouse liver homogenates in vitro (left) and signal kinetics of the [1‐¹³C]fumarate and [1‐¹³C] and [4‐¹³C]malate peaks fitted with a two‐compartment exchange model (yielding T₁ ∼80 s for fumarate) (right). Note: the “DC offset” at the beginning of the purple curve suggests that considerable metabolic exchange occurred prior to the acquisition; this prevented us from obtaining a realistic fitted estimate for the fumarate‐malate conversion rate.

Figure 5

In vivo CSI of hyperpolarized [1‐¹³C]fumarate metabolism in an acetaminophen‐induced hepatitis mouse. a) Representative ¹³C NMR spectrum of hyperpolarized [1‐¹³C]fumarate and its metabolite for a CSI pixel at the liver. b) Map of hyperpolarized [1‐¹³C]fumarate CSI signal intensity overlaid on an anatomical ¹H MRI image. c) Map of hyperpolarized [1‐¹³C] and [4‐¹³C]malate. d) parametric map of the malate/fumarate ratio; a biomarker of cellular necrosis.