In Vivo Hyperpolarized 13C-MRS of Ethanol-Modulated Pyruvate Metabolism in the Rat

Introduction: This work demonstrates the application of in vivo MRS of hyperpolarized [1-C]pyruvate to interrogate a metabolic pathway involved in neither glycolysis nor the Krebs cycle. In particular, ethanol is metabolized in the liver via the breakdown of ethanol to acetaldehyde and acetaldehyde to acetate. These reactions are catalyzed by the enzymes alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), with both requiring the reduction of the coenzyme nicotinamide adenine dinucleotide (NAD) to NADH [1]. Hence, ethanol consumption leads to an accumulation of excess NADH in the liver and when extreme is associated with pathologies including fatty liver disease, hepatitis, cirrhosis, and hepatocellular carcinoma [2]. Here we present a method for noninvasively monitoring this important process in vivo. Using a bolus injection of hyperpolarized [1-C]pyruvate, we demonstrate significantly increased rat liver lactate production with the coadministration of ethanol, an effect attributable to increased liver NADH in combination with NADH’s role as a coenzyme in pyruvate-to-lactate conversion [3,4]. Methods: Ten male Wistar rats (370 ± 21 g) were anesthetized with 1-3% isoflurane and fitted with a tail vein catheter for administration of ethanol (or saline) and pyruvate. Using a DNP polarizer (HyperSense, Oxford Instruments), we polarized a 32μl formulation of 14 M [1-C]pyruvate for each injection. The frozen sample was dissolved in 4.6 cc of 185 C buffered NaOH, yielding a 100 mM pyruvate solution (pH = 7.4 ± 0.34) hyperpolarized to approximately 20%. Each rat was placed in an 80-mm inner-diameter dual-tuned C-H volume RF coil centered in the bore of a 3T clinical MR scanner (GE Healthcare, Waukesha, WI). After the acquisition of conventional MRI for anatomical reference, 2.5 cc of hyperpolarized pyruvate solution was injected into the tail vein catheter at a rate of 0.25 ml/s. C spectra (FID acquisition, flip angle = 5, 5kHz spectral width, 2048 points) were then acquired every 3 s over a 4-minute period from a single 15-mm axial slice through the liver (N=3) or one slice through the liver and a second through kidneys (N=3). Following the acquisition of the baseline pyruvate measurements, 1.0 gm/kg of a 20% ethanol solution was injected into the tail vein in order to achieve a targeted steady-state blood alcohol level of 100 mg/dl at the time of the second C MRS acquisition. A second bolus of hyperpolarized pyruvate was injected into the animal 45 min after the administration of the ethanol, and C spectra were again acquired every 3 s over the next 4 minutes. Control animals (N = 4) were scanned using the same procedures with the exception that the ethanol injection was replaced by an injection of normal saline (same volume and temperature as the ethanol injection). The time-resolved metabolic signals following each bolus injection were fit using a three-site exchange model in order to estimate the apparent rate constants. Statistical analysis of the data was performed using two-factor repeated-measures ANOVA.