The branched-chain α-keto acid dehydrogenase (BCKDH) complex has been established as a major regulator of branched chain amino acid (BCAA) catabolism. In several diseases including liver cirrhosis, decreased BCKDH activation results in an increase in BCAA breakdown through the branched-chain amino transferase (BCAT) and subsequent protein and energy deficiency [1]. Hyperpolarized α-keto[1-¹³C]isocaproate (KIC) magnetic resonance spectroscopy has been proposed as a non-invasive measure of BCAT activity in vivo in preclinical models of cancer and in brain BCAA metabolism studies [2-4]. In addition to transamination to leucine (Leu), KIC is decarboxylated to form isovaleryl-CoA. This process releases CO2, which is in fast exchange with bicarbonate (HCO₃^-)(figure 1). In this work, we demonstrate the feasibility of using hyperpolarized HCO₃^- generation as a readout for BCKDH activity in liver. We took advantage of the known differences in BCKDH and BCAT activity in rodent liver and kidney [5-7] to show the sensitivity of our approach.

Chemical preparation of ¹³C-KIC: A 40ul sample containing 8.1 M of α-keto[1-¹³C]isocaproic acid (Sigma Aldrich, Miamisburg, OH), 15 mM of trityl radical OX063 (Oxford Intruments, Abingdon, UK) and 1.5 mM of gadolinium-DOTA chelate (Guerbet, Roissy, France) was polarized in an Oxford Hypersense (Abingdon, UK) operating at 3.35 T and 1.3 K. The frozen sample was then dissolved in 4 ml of a superheated buffer that neutralized the acid.

Animal studies: Data were acquired in 7 month-old CD1 naive mice (n=3) under anesthesia in a vertical 14.1T Agilent scanner using a volume ¹H-¹³C birdcage coil. Acquisition started after a 10 s injection of 350 ul of the KIC solution through a tail vein catheter. Fifteen spectra were acquired dynamically from a 8 mm axial slice placed on the liver or the kidneys (receiver BW=20kHz, 4096 points, TR=3 s, flip angle=30º).

Data processing: Data were processed in MestReNova software with 5 Hz line broadening, phase and baseline correction, and correction for progressive loss of signal due to flip angle. Peak integration ranges were [175-178.5 ppm] for Leu, [175-169 ppm] for KIC and [160-163 ppm] for HCO₃^-. Results are reported as metabolite ratios: Leu/KIC and HCO₃^-/KIC calculated from the summed spectra.

The T₁ and polarization level of [1-¹³C]KIC in solution were measured to be 42.5 s at 500 MHz and 17%, respectively. In kidney, only Leu (176.8 ppm) and KIC (172.6 ppm) were detected. As expected from the high BCKDH activity in liver, a HCO₃^- (161 ppm) peak was observed in addition to Leu. Dynamic acquisitions showed that KIC signal was at its maximum at the end of the injection in our acquisitions, whereas HCO₃^- and Leu signal maximum was delayed by about 3 s. Analysis of the metabolites ratios showed excellent agreement with rodent liver and kidney BCKDH and BCAT activities reported in the literature for both organs [5-7], with significantly lower Leu/Kic in liver as compared to kidney (t-test, p=0.049) (Figure 2).We have shown that both hyperpolarized Leu and HCO₃^- signals can be detected in liver in vivo at 14.1 T as byproducts of [1-¹³C]KIC metabolism. This provided information on BCAT and BCKDH activities in mouse liver and kidney, consistent with literature values. Future studies will explore the relationship between the hyperpolarized metabolite ratios measured in vivo and BCAT/BCKDH enzyme activities assayed ex vivo in preclinical models of liver injury and treatment response. The assessment of the effect of BCAA supplementation on liver cirrhosis, as now recommended by practical clinical guidelines [8], would have a large impact on patient monitoring and treatment titration.