shows a comparison of hyperpolarized spectra obtained from injecting [1-13C]pyruvate and [1-13C]alanine. As illustrated by , in the hyperpolarized [1-13C]pyruvate injection experiment, hyperpolarized [1-13C]lactate and hyperpolarized [1-13C]alanine are produced by LDH and ALT catalyzed reactions respectively. Note that for clarity, the MCT step and the transport of pyruvate into the mitochondria/decarboxylation to acetyl-CoA and 13CO2 are not shown. In contrast, when hyperpolarized [1-13C]alanine is injected, hyperpolarized [1-13C]lactate only appears after passage through hyperpolarized [1-13C]pyruvate as an intermediate. Therefore, detection of lactate also indicates initial conversion to pyruvate. shows some of the anatomical regions (experiments were non-localized), including liver, kidneys, stomach, intestines, and muscle, from which spectral data ( and 1d) were obtained. shows a typical spectrum (sum of first 15 FIDs) after injection of 3 mL of 100 mM hyperpolarized [1-13C]pyruvate. Pyruvate (and some pyruvate-hydrate), alanine, and lactate were the primary resonances detected. Resonances from 13C-bicarbonate, from the carbonic anhydrase mediated equilibrium with 13CO2 after the pyruvate decarboxylation step, were also detected. The pyruvate, alanine, and lactate SNRs were 8310, 3140, and 3070 respectively (total SNR = 14520), and the lactate to pyruvate ratio of SNRs was 0.37. The lactate to pyruvate ratio calculated using peak areas was 0.39. shows a corresponding typical spectrum after injection of 3 mL of 100 mM hyperpolarized [1-13C]alanine. The inset of , which has the same vertical scale as , shows that most of signal is from the injected alanine. With a 92-fold zoom, the lactate and pyruvate peaks become apparent (lipid subtracted). The pyruvate, alanine, and lactate SNRs in this spectrum were 16.2, 7150, and 70.0 respectively (total SNR = 7240). The lactate to pyruvate ratios using SNR and peak areas were 4.32 and 4.14 respectively.
Figure 1 A) Schematic of the interconversion between pyruvate, alanine, and lactate and the redistribution of the 13C label. (Note: for clarity, the entry of pyruvate into the mitochondria and its subsequent metabolism is not shown.) Of particular relevance in (more ...)
shows the identification of pyruvate in the alanine injection experiments and highlights the benefit of fat subtraction. shows FIDs 16 through 64 of a spectrum following a hyperpolarized [1-13
C]pyruvate injection. The earlier FIDs, which contain overwhelming pyruvate signal, were omitted to make the lipid resonance visible. As shown in , the distance from lactate to pyruvate was 391 Hz, and the distance between the fat peak and pyruvate was 46 Hz. The lipid resonance, due to its proximity in the spectrum to pyruvate, most likely originates from a fatty acyl chain in pure adipose tissue (6
). As expected, the pyruvate peak increased only when earlier dynamic time points were included, and the fat peak increased even when later time points after the hyperpolarized signal had decayed away were averaged. When hyperpolarized [1-13
C]alanine was injected (, first 15 FIDs), the same resonances with the same peak separations were detected. The distance between the outermost peaks, lactate and pyruvate, was 393 Hz, and the distance from pyruvate to fat was 44 Hz, which was in close agreement with considering the spectral resolution of the acquisitions was 2.44 Hz. shows the pyruvate peak after lipid subtraction. The top part of (same data as ) shows the original unsubtracted spectrum with pyruvate as a shoulder on the lipid resonance. The middle of shows a non-hyperpolarized baseline scan immediately following the hyperpolarized alanine injection scan in which there was no shoulder on the lipid resonance. The bottom of is the resulting difference spectrum, showing the lipid completely removed and only the pyruvate present. These data show that pyruvate, albeit very little, was detected after injection of hyperpolarized alanine. Lactate, on the other hand was abundantly detected compared with pyruvate.
Figure 2 A) Hyperpolarized 13C non-localized data after injection of [1-13C]pyruvate with the earlier time points in the summed spectrum omitted (shown are time points 16 through 64) to allow for visualization of a lipid resonance ~1.4 ppm to the left of pyruvate. (more ...)
and summarize all the hyperpolarized SNR and peak area data. As shown in , the total SNR when injecting alanine was only a little more than a factor of 2 lower than when injecting pyruvate, which is consistent with the polarization values measured (reported in the methods section). However, following hyperpolarized 13Calanine injections, the majority of the SNR came from the pre-polarized alanine, and the SNRs of pyruvate and lactate were much lower. The same trend can be seen in , which reports integrated peak areas (normalized to total carbon area) instead of SNR. Both and show the measured lactate to pyruvate ratio was an order of magnitude higher when alanine was injected instead of pyruvate. The lactate to pyruvate ratios were comparable between the two tables, thus corroborating each other and showing that the ratios were insensitive to the quantification method.
Table 1 Summary of pyruvate injection (n = 4) and alanine injection (n = 4) hyperpolarized SNR data. SNR was taken to be maximal metabolite peak height in a phased spectrum divided by noise standard deviation measured from a non-peak region. Quantification was (more ...)
Table 2 Summary pyruvate injection (n = 4) and alanine injection (n = 4) hyperpolarized peak area data. The peak areas were calculated by integrating over the full width of metabolite peaks in a phased spectrum, with the noise contribution subtracted out. Quantification (more ...)
As demonstrates, non-localized SNRs of pyruvate and lactate were relatively low for the alanine injection experiments. Sensitivity enhancements would be needed to enable finely localized studies. However, as shows, slice-localized data with adequate lactate SNR may be feasible with current techniques. The data in were acquired with the same parameters described in the methods section except RF excitation was limited to a slice localized to the rat liver (). The lactate SNR in the alanine injection spectrum in was 11.2, and the pyruvate SNR was too low to measure. If the lowest detectable SNR is ~3, then the lactate to pyruvate ratio would be greater than at least 3.5. The lactate to pyruvate SNR ratio for this same slice when pyruvate was injected () was 0.55. Thus, the difference in 13C-lactate to 13C-pyruvate ratio between the alanine injection and pyruvate injection acquisitions in this initial liver-localized experiment is in agreement with what was detected in the non-localized studies.
Figure 3 A) T2-weighted coronal image with a box depicting the 20 mm slice localized to the rat liver from which the hyperpolarized data were acquired. B) A hyperpolarized 13C liver-localized summed spectrum after injection of [1-13C]alanine. A clear lactate signal (more ...)