The aim of this study was to explore hyperpolarized MRI as a method of assessing the metabolism of [1-¹³C]-pyruvate in the chinchilla placenta and fetus.

The study was performed in chinchillas (n=4) because this rodent usually only gives birth to 1-2 babies and has a gestation period for about 111 days. Two animals were scanned one time and two animals were scanned two times between day 69 and 98 of pregnancy (6 scans in total). The animal was anaesthetized with 2,5% sevoflurane in atmospheric air as breathing gas. Tail vein catheterization was performed for administration of hyperpolarized [1-¹³C]-pyruvate and an isotonic glucose infusion (6 mL/h) one hour prior to the experiment. [1-¹³C]-pyruvate was polarized in a SpinLab (GE Healthcare). MRI measurements were performed on a 3T GE HDx scanner (GE Healthcare) equipped with a 1H 8-Channel MR Cardiac Array Coil for anatomical scans (GE Healthcare) and a 13C Helmholtz loop coil (PulseTeq Limited, Surrey, UK) (ø=20cm) for hyperpolarized MRS. A dynamic (50 sec, 1 image/5 sec) sequence was initiated 30 sec after start of [1-¹³C]-pyruvate injection. Regions-of-interests (fig. 1) of placenta, fetus and muscle tissue in the mother were manually drawn on the 1H anatomic images in OsiriX, and its signals from the 13C images were translated into a signal-to-noise ratio.

After the MRI experiments placental, maternal renal and cardiac tissue as well as fetal renal and cardiac tissue were taken out to validate the MRI results, with a sample size of 4 for all tissue types. In order to validate the mRNA expressions of lactate dehydrogenase (LDH), pyruvate dehydrogenase (PDH) and alanine aminotransferase (ALT), RNA extraction was carried out using a NucleoSpin RNA II kit (Stratagene, AH diagnostics, Aarhus, Denmark), cDNA was synthesized using a RevertAid First strand cDNA synthesis kit (MBI Fermentas, Burlington, Canada), and qPCR was performed using the SYBR Green qPCR Master Mix (Stratagene, AH diagnostics, Aarhus, Denmark). LDH activity was assessed using LDH activity assay kits (Sigma-Aldrich, Brøndby, Danmark) and NAD+/NADH-ratios were validated using NAD+/NADH quantification kits (Sigma-Aldrich, Brøndby, Danmark).

Hyperpolarized [1-¹³C]-pyruvate significantly accumulated in the placenta compared to the fetus and muscle tissue in the mother (fig. 1+2). Furthermore the MRI measurements showed a significant high [1-¹³C]-lactate signal produced in the placenta (fig. 1). This is in agreement with our initial hypothesis. This states that a relative large fraction of glucose in the placenta is metabolized anaerobically to lactate, which is supported by previous in vivo studies¹. We found no change in the lactate/pyruvate ratio from day 69 to day 98 in the 111 days gestation period. Also we did not observe any signal from alanine or bicarbonate in the placenta as well as any signal in the fetus. It is therefore doubtful whether the method is able to examine fetoplacental transport and fetal metabolism.

Recently, a similar study by Friesen-Waldner et al.² showed significant amounts of [1-¹³C]-pyruvate and [1-¹³C]-lactate in the guinea pig placenta, which relates to our results. However, they did observe signals from [1-¹³C]-pyruvate and [1-¹³C]-lactate in the fetus, which differs from our findings.

The LDH activity in the maternal organs was high compared to the fetal organs (fig. 3), and suggests that the mother in general has a higher anaerobic metabolism than the fetus. Additionally, the low LDH activity in the fetal kidney could be due to the fetus receiving most of the lactate needed for energy metabolism from the placenta. NAD+/NADH ratios were the same for all tissue types except for the maternal and fetal kidney (fig. 4). The placental mRNA expression of LDHA1 was significantly higher than the placental expression of PDH and ALT (fig. 5). This indicates a higher anaerobic than aerobic metabolism in the placenta, which is consistent with our MRI results.

This study highlights the potential of hyperpolarized MRI as a novel method for assessing placental and fetal metabolism of [1-¹³C]-pyruvate. The chinchilla model shows great promise for quantitative validation of placental metabolism.