Introduction

Magnetic resonance spectroscopy of hyperpolarized [2-¹³C] pyruvate (pyr) and its metabolic products is a powerful tool to study real-time in vivo glycolysis (GLY) as [2-¹³C] pyruvate is converted to [2-¹³C] lactate (lac), as well as oxidative phosphorylation (OXPHOS), as the ¹³C label is retained in tricarboxylic acid (TCA) cycle compounds citrate, glutamate and acetylcarnitine, with applications in studying cellular metabolism, tumor metabolism, response to therapy and cardiac energetics^1-3. Despite this advantage of using [2-¹³C] pyr to obtain a direct measure of OXPHOS and GLY simultaneously, the large chemical shift dispersion of [2-¹³C] pyr and its metabolites, the resulting chemical shift displacement artifact during slice excitation, the dipolar relaxation & J-coupling effects caused by the proton attached to the C₂ carbon limit its practical use. Previously we presented a hybrid technique that utilizes fully resolved spectroscopic imaging to assess mitochondrial metabolites and a two-shot quadrature imaging method to image [2-¹³C] lac⁴. In this work, we propose a faster single-shot narrrowband radiofrequency (RF) excitation method for resolution of J-modulated artifacts arising from the [2-¹³C] lac doublet. Using product operator formalism (POF) we show that the combination of an in-phase and an anti-phase doublet due to the long duration of the narrowband excitation pulse resolves the J-modulated ghosting artifact. Results from simulations, phantoms and rat brain are presented that highlight the utility of this method for potential real-time simultaneous assessment of GLY and OXPHOS in vivo.

Theory

The starting state of a weakly coupled I-S spin system $$$\hat{\rho} = \hat{I}_{z}+\hat{S}_{z}$$$ with C₂ carbon as the I-spin and the attached proton as the S-spin, in the presence of a narrow bandwidth off-resonance radiofrequency field evolves under the Hamiltonian (rotating frame of reference): $$\hat{H}=-{\Omega_{I}}\hat{I}_{z}-{\Omega_{S}}\hat{S}_{z}-{\omega_{1}}\hat{I}_{x}+2{\pi}J\widehat{I_{z}{S}_{z}} \qquad \qquad \qquad[1]$$ where $$$\Omega_{I}$$$, $$$\Omega_{S}$$$ are the off-resonance frequencies (rad/s) of the I & S spins respectively, J (Hz) is the J-coupling constant between the I and S spins and $$$\omega_{1}$$$ is the frequency (rad/s) of the applied radio frequency pulse. After a 90⁰ RF pulse, the spin density operator evolves to: $$\hat{\rho}=\frac{1}{2}\hat{I}_{y}+\frac{1}{2}\hat{I}_{z}+\frac{1}{2}2\widehat{I_{y}{S}_{z}}-\frac{1}{2}2\widehat{I_{z}{S}_{z}}+\hat{S}_{z}\qquad \qquad \qquad [2]$$ The sum of the in-phase doublet arising from the $$$\hat{I}_{y}$$$ term combines with anti-phase doublet from $$$2\widehat{I_{y}{S}_{z}}$$$ to produce a singlet (Fig 1), eliminating all J-coupling associated artifacts during signal acquisition. Therefore, a narrowband spectrally and spatially selective pulse with a spectral bandwidth (BW) of 75Hz centered 70Hz (J/2) away from the [2-¹³C] lac resonance, followed by a fast echoplanar imaging (EPI) readout, can be used to image [2-¹³C] lac artifact free (Fig 2).

Methods

Simulation & in vitro experiments:A full-density matrix simulator for a coupled 2-spin I-S system in MATLAB (Mathworks Inc., MA, USA) developed in-house was used to simulate the evolution of coherences. A syringe filled with 5ml of 2.5M ¹³C formate(dia. 15mm), doped with 8μl/ml of Gadolinium agent to shorten the in-vitro T₁, was placed in a ¹³C transmit-receive surface coil (dia ~35mm) and imaged using a clinical 3T PET-MR scanner(GE Medical systems). A narrowband spectrally selective radiofrequency excitation pulse(BW=100Hz,T_RF=40ms,slice thickness=20mm,100Hz off-resonance), followed by free-induction decay signal acquisition(BW=5000Hz,N=2048,TR=2s), was used to test the narrowband technique for eliminating the splitting from J-coupling. Another syringe filled with 0.5M [2-¹³C] lac was used in the EPI imaging experiment.
Hyperpolarization and in vivo imaging of [2-¹³C] lac: 54mL of 15.5M [2-¹³C]Pyruvic acid (Sigma Aldrich) mixed with 15mM trityl radical AH11141(GE Medical systems) was polarized between 3-4 hours in SPINLab system(GE Medical systems) and dissolved using 16g solution of 40mM tris(hydroxymethyl aminomethane),100mg/L ethylenediaminetetraacetic acid(EDTA), and 50mM NaCl. The resulting 100mM pyruvate solution was neutralized with 640mL of 125mM NaOH buffer. 2.5ml of this solution was injected at a rate of 0.25mL/s into a male Wistar rat through a tail-vein catheter and imaged in a clinical 3T scanner(Signa MR750,GE Systems) 20s post injection to maximize the signal from lactate. A slice and frequency selective narrowband RF pulse centered on one of the lac peaks(slice-thickness=10mm, BW=125Hz,pulse-width=25.6ms,flip-angle=90⁰) followed by an EPI readout (FOV=128mm,matrix=32x32, TR=200ms,TE=24ms) was used to acquire the [2-¹³C] lac image from an axial slice placed in the rat-brain, and this was overlaid on a T₂-weighted proton image acquired using a custom-built ¹H T/R bird-cage coil for anatomical reference (FOV=128mm,256x256 matrix,2mm slice-thickness,TR/TE=6000/125ms).

Results

Spectrum from the numerical simulation as well as the in-vitro ¹³C formate using full bandwidth RF pulse(2kHz, 1.8ms) demonstrates the splitting caused due to the J-coupling between the ¹³C labeled carbon and its attached proton (left panel, Fig 3a & 3b respectively). Use of a narrow bandwidth RF pulse (100Hz, 40ms) results in a singlet as seen in the simulation (Fig 3a, right panel) and the in-vitro spectrum (Fig 3b, right panel). This singlet can then be imaged using a single-shot fast imaging EPI readout as seen in Fig 3c ([2-¹³C] lac phantom). An artifact-free in vivo [2-¹³C] lac map acquired from a rat brain after an injection of hyperpolarized [2-¹³C] pyr, and overlaid on a T₂-weighted image is shown in Fig 4.

Conclusion

We propose a fast single-shot narrowband RF excitation based imaging method to resolve the artifacts due to the J-coupling evolution during signal collection of [2-¹³C] lac and show that it can facilitate simultaneous in vivo measurement of glycolysis and mitochondrial metabolism during a hyperpolarized [2-¹³C] pyr experiment.