Metabolite time curves averaged over three animals after injections with hyperpolarized [1-13C]lactate and [1-13C]pyruvate are compared in . Also shown are metabolic images, superimposed onto SPGR images, from slices through the center of the heart and through the liver acquired after injection of lactate. The metabolic images, averaged over data acquired between 5 s and 35 s relative to the start of injection, indicate that the slice-selective metabolite signals were predominantly from the heart. The images from the heart slice demonstrate that there were only small contributions from surrounding tissue such as skeletal muscle. Due to the limited spatial resolution, contributions from blood, in particular for the substrate, could not be resolved from signal in the myocardium. However, comparing signals from ROIs in the heart and the aorta (in the liver slice) suggests that the bicarbonate was predominantly from the heart tissue as no bicarbonate was detected in the aorta. Although the sensitivity from the liver slice was reduced because of the slab profile, given the SNR of 22 for bicarbonate in the heart and the 1:2 ratio of lactate signal from aorta and heart, bicarbonate should have been detected in the aorta with an estimated SNR of 11 if all of the bicarbonate in the heart slice would have been from blood.
Figure 1 (a) Metabolite time courses (mean ± standard error, N = 3) from a 15-mm slice through the heart after the first injection of hyperpolarized [1-13C]lactate (group 1). Conversion to pyruvate as well as the secondary products bicarbonate and alanine (more ...)
For a better comparison of absolute signal levels, the plotted time courses for both substrates have been normalized to a polarization of 25%. Additionally, the small differences in the delay between dissolution and time of injection were corrected using a T1
in solution of 39 s for lactate and 60 s for pyruvate. Following the injection of lactate, both the primary oxidation of lactate to pyruvate and the subsequent conversion of pyruvate to alanine and bicarbonate were observed. The signal from pyruvate hydrate (Pyh), which is in exchange with pyruvate, was below the detection threshold. As expected from the different substrate concentrations and their longitudinal relaxation rates, the maximum in the time course of the injected lactate was approximately 35%–40% of the maximum in the pyruvate curve in . The time course for alanine after lactate injection was clearly delayed by approximately 6 s compared to the alanine curve after pyruvate injection. The corresponding delay for bicarbonate appeared to be less than 3 s. However, considering the 3-s sampling interval used here, experiments at higher temporal resolution are necessary to assess the different metabolite dynamics more accurately. Whereas the maximum bicarbonate signal after lactate injection was approximately 30% of the bicarbonate level after pyruvate injection, the corresponding value for alanine was only 20%. This difference can be explained by the fact that bicarbonate is only generated through the non-reversible conversion of pyruvate to acetyl-CoA, whereas alanine is produced by the reversible reductive amination of pyruvate. Therefore, hyperpolarized labeled alanine is also generated through fast isotopic exchange. It has been shown that a 1-mL dose of 80 mM pyruvate does not saturate the conversion of pyruvate to alanine in the rat heart whereas the conversion to bicarbonate is saturated at a concentration of 40 mM (7
). The low levels of pyruvate measured here suggest that even the PDH flux was not saturated at the given lactate dose. Therefore, even the SNR of bicarbonate, which was 9 after lactate injection in comparison to 25 in the time course acquired after the pyruvate injection, could be increased by using a higher concentration of the injected lactate. This can be achieved by preparing the sample with lactic acid instead of the sodium salt of lactate as it was done in this study. A higher concentration would also benefit from the high Km
of approximately 2.5 mM for the monocarboxylate transporter (MCT)-mediated uptake of lactate into the myocytes as proposed by Schroeder et al. (7
Representative sum spectra from single animals in groups 1 and 3 shown in demonstrate the effect of DCA on cardiac metabolism. Note that for each spectrum a first-order phase correction was performed and the baseline was subtracted by fitting a spline to the signal-free regions of the smoothed spectrum. As the administration of DCA led to an increase in PDH flux via the inhibition of PDK, a more than 2.5-fold increase in bicarbonate signal was detected with both substrates. The ratios of the metabolic products to the respective substrate for all animals are plotted in . For group 1, the average Bic/Lac increased from 1.53% ± 0.22% at baseline to 4.15% ± 0.28% (mean ± sd, p = 0.00056) after infusion of DCA whereas the other two products both decreased (Ala/Lac: from 4.31% ± 0.28% to 2.81% ± 0.08%, p = 0.0084; Pyr/Lac: from 1.86% ± 0.12% to 0.80% ± 0.14%, p = 0.0013). In contrast, none of the metabolite-to-Lac ratios were different at injection 1 and 2 in the control animals (group 2) that received saline without DCA, (Bic/Lac: from 1.51% ± 0.09% to 1.27% ± 0.10%, p = 0.1632; Ala/Lac: from 3.95% ± 0.50% to 4.74% ± 0.15%, p = 0.0675; Pyr/Lac: from 2.19% ± 0.11% to 2.21% ± 0.29%, p = 0.9032). Furthermore, the percentage change between the first and second injection differed significantly between group 1 and 2 for all three metabolite-to-lactate (%change Bic/Lac: 173 ± 22 for group 1, −15 ± 12 for group 2, p = 0.0002; %change Ala/Lac: −35 ± 3 for group 1, 21 ± 11 for group 2, p = 0.0012; %change Pyr/Lac: −57 ± 6 for group 1, 1 ± 13 for group 2, p = 0.0022). For comparison, in the animals that were injected with hyperpolarized pyruvate, only the change in Bic/Pyr was significant (Bic/Pyr: from 2.45% ± 0.54% to 7.87% ± 0.10%, p = 0.0030; Ala/Pyr: from 10.22% ± 1.34% to 10.99% ± 1.66%, p = 0.0593; Lac/Pyr: from 17.29% ± 2.73% to 16.49% ± 2.19%, p = 0.5828). In contrast to the results from group 1 when lactate first has to be converted to pyruvate, the concentration of labeled pyruvate in group 3 is high enough to leave the conversion to alanine or lactate unchanged despite the increase in PDH flux. The increase of Bic-to-substrate ratio due to DCA administration, 2.7 ± 0.2 with lactate and 3.3 ± 0.8 with pyruvate as the substrate, agrees well with the 2.6-fold difference in Pyr-to-Bic conversion rate constant measured between animals that received DCA prior to the injection of 1 mL of hyperpolarized [1-13
C]pyruvate and control animals reported in (10
). The slightly higher increase in Bic/Pyr found in our study could be due to differences in the acquisition protocols or the different types of metrics, i.e., metabolite ratios vs. estimated rate constants. Additionally, the pyruvate dose in (10
) was only about a third of the amount injected in this study. Therefore, a smaller increase could be expected if the higher PDH flux with DCA was not completely saturated at this lower dose.
Figure 2 (a) Representative slice-selective spectra from the heart (averaging data from time points 2 to 30) with lactate as the substrate from a single animal at baseline (blue) and after administration of DCA (red). DCA inhibits PDK that leads to an increase (more ...)
Figure 3 (a) Metabolite-to-lactate ratios for individual animals at baseline and after administration of DCA (blue) and saline (red) with lactate as the substrate. The difference between DCA and saline groups for percentage change of metabolite ratios between (more ...)
One of the limitations of the presented study was the large variation in the polarization level for the lactate samples. The variations could be due to the more complicated glassing required for sodium lactate. However, this needs further investigation. A potential remedy is using a substrate formulation based on lactic acid that should have similar properties as the pyruvic acid substrate, which showed more consistent polarization levels. Another source of uncertainty is the scaling procedure correcting for the small differences in the delay between dissolution and injection that uses, for each substrate, only a single T1
measured at 3 T. Recently, Miéville et al. (25
) showed that the paramagnetic polarizing agent can lead to accelerated relaxation during the transport in low-field regions between polarizer and NMR magnet. However, the concentration of the TEMPO radical in that study was about an order of magnitude higher than the OX063 concentration in the final solution here. Furthermore, it is most likely that the majority of the ±2-s difference in the delay between dissolution and injection were due to differences in filling the syringe at the polarizer and degassing and connecting it to the tail vein catheter at the MR scanner instead of differences in the transfer time. All these uncertainties with respect to the absolute level of polarization, however, did not affect the findings for the effect of DCA on cardiac metabolism as the results were evaluated using metabolite-to-substrate ratios. Another limitation of the study is the fact that the resonance of 13
could not reliably be detected in all the groups due to its low SNR. Using the bicarbonate-to-CO2
ratio and the Henderson-Hasselbalch equation would have permitted to determine any contributions to the change in bicarbonate due to a change in pH (5
). However, the concentration of CO2
is approximately 10–20 times lower than bicarbonate at physiological pH levels (13
). Therefore, even the worst-case scenario, i.e., all CO2
being converted to bicarbonate after the DCA injection, would only lead to an increase in bicarbonate on the order of 5–10% compared to the baseline level.