Direct assessment of renal mitochondrial redox state using hyperpolarized 13 C-acetoacetate

Abstract

Purpose: The purpose of this study was to investigate the hyperpolarized ketone body ¹³ C-acetoacetate (AcAc) and its conversion to ¹³ C-β-hydroxybutyrate (βOHB) in vivo, catalyzed by β-hydroxybutyrate dehydrogenase (BDH), as a novel direct marker of mitochondrial redox state.

Methods: [1,3-¹³ C₂ ]AcAc was synthesized by hydrolysis of the ethyl ester, and hyperpolarized via dissolution DNP. Cold storage under basic conditions resulted in sufficient chemical stability for use in hyperpolarized (HP) MRI studies. Polarizations and relaxation times of HP [1,3-¹³ C₂ ]AcAc were measured in a clinical 3T MRI scanner, and 8 rats were scanned by dynamic HP ¹³ C MR spectroscopy of a slab through the kidneys. Four rats were scanned after acute treatment with high dose metformin (125 mg/kg, intravenous), which is known to modulate mitochondrial redox via inhibition of mitochondrial complex I. An additional metformin-treated rat was scanned by abdominal 2D CSI (8 mm × 8 mm).

Results: Polarizations of 7 ± 1% and 7 ± 3%, and T₁ relaxation times of 58 ± 5 s and 52 ± 3 s, were attained at the C₁ and C₃ positions, respectively. Rapid conversion of HP AcAc to βOHB was detected in rat kidney in vivo, via the C₁ label. The product HP βOHB was resolved from closely resonating acetate. Conversion to βOHB was also detected via 2D CSI, in both kidney as well as liver regions. Metformin treatment resulted in a significant increase (40%, P = 0.01) of conversion of HP AcAc to βOHB.

Conclusion: Rapid conversion of HP AcAc to βOHB was observed in rat kidney in vivo and is a promising new non-invasive marker of mitochondrial redox state. Magn Reson Med 79:1862-1869, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Keywords: beta-hydroxybutyrate; dynamic nuclear polarization; ketone bodies.

Figure 1

Simplified depiction of the metabolic pathways for ketone bodies acetoacetate (AcAc) and β-hydroxybutyrate (βOHB) as compared with pyruvate and lactate (A), showing the compartmentalization of their respective NAD-dependent biochemical equilibria to the mitochondria and cytoplasm, respectively. Mitochondrial equilibrium between [1,3-¹³C₂]AcAc and [1,3-¹³C₂]βOHB, catalyzed by β-hydroxybutyrate dehydrogenase (BDH), with ¹³C chemical shifts (B). PEP= phosphoenolpyruvate, OAA= oxaloacetate, AA’s= amino acids, NAD= nicotinamide adenine dinucleotide (hydride).

Figure 2

¹³C NMR spectrum of product sodium acetoacetate material (unlabeled precursor) acquired at 11.7T (no ¹H decoupling), with peak assignments. A small quantity of [¹³C]urea was added to the sample for concentration reference. The peak at 170ppm could not be definitively assigned but is most likely due to carbonate formed via decarboxylation of AcAc.

Figure 3

Initial 5° HP solution spectrum (A) of 20mM [1,3-¹³C₂]AcAc taken from dynamic series acquired for estimation of percent polarization and T₁’s, alongside averaged 90° spectrum (B) from a series of thermally polarized scans collected after complete decay of hyperpolarization.

Figure 4

Conversion of HP [1,3-¹³C₂]AcAc to [1,3-¹³C₂]βOHB in rat kidney in vivo, detected via the C₁ label. Magnitude spectra are scaled to approximate SNR units. Conversion was found to be increased in a rat pre-treated with high-dose metformin (C), as compared to a normal rat (B). Position of a 10mm axial ¹³C kidney slice from one of the scans is shown on a coronal ¹H anatomic image (A).

Figure 5

Coronal 2D projection CSI of HP [1,3-¹³C₂]AcAc in a normal rat pre-treated with high-dose metformin. Spectra from individual voxels corresponding to liver and left kidney are shown, with peaks labeled.

Figure 6

Effect of acute high-dose metformin treatment on the extent of renal conversion of HP AcAc to βOHB in eight rats. A statistically significant (p=0.01) increase in conversion of 40% was detected in the observed HP βOHB/AcAc ratio.

Figure 7

Effect of HP [1,3-¹³C₂]AcAc injection on blood βOHB levels in seven rats, as measured using a blood ketone meter. βOHB levels were measured from a small sample of tail vein blood obtained just before, and one minute after, the start of HP injection. Lines connect the individual data pairs that correspond to each rat. Only six (as opposed to seven) lines are seen because one data pair result (0.3mM–>0.4mM) was observed twice.