We report a technique to measure localized T2
relaxation times of 13
C-labeled metabolites in vivo
, and the comparison between an HCC rat model and normal rat liver. To the best of our knowledge, this is the first report on in vivo
measurements of 13
C metabolites. The large signal enhancement afforded by the hyperpolarized 13
C MRS technique made such measurements possible in vivo
. Atomic motion influences the rate of transverse relaxation, and such motion varies in different microscopic environment depending on viscosity, temperature, local magnetic fields, and surrounding molecules. Therefore, the T2
measured in vivo
may be particularly relevant to the design of imaging sequences and contrast generation strategies for in vivo
C applications. The 13
C-alanine and 13
relaxation times were found to be about half a second in the normal liver. For comparisons, the relaxation time for 13
C-lactate due to spin-spin coupling (JCH
) was about 25 ms (41
) and a similar JCH
coupling for 13
C-alanine. Imaging sequences acquiring T2
signal may have advantages in SNR and/or large matrix size. The large difference in T2
relaxation time between HCC tumor and normal liver could provide an opportunity to develop novel strategies for enhancing image contrast and improving cancer detection.
One of the limitations with the in vivo
measurement presented here was that metabolic conversions continued during data acquisition. Typical conversion rates from pyruvate to lactate or pyruvate to alanine range from 0.004 to 0.07 s−1
), corresponding to time constants longer than 14 s. Hence, during the 8-s T2
acquisition time, a very small number of 13
C labels may have exchanged from one metabolite to another.
Another limitation of this technique is the possible variations in voxel placement due to respiratory motion. Although no appreciable liver displacement relative to the voxel was observed in repeated T2W proton images, contribution of extrahepatic tissues such as fat during respiration was almost unavoidable when measuring T2 in vivo. Extend of this variability contributed to the intra and inter subject variations reported in this work. The partial volume effect due to respiratory motion may also affect tumor T2 measurements, resulting an underestimate of T2 if non-cancerous liver tissue contributed to the measured signal, or overestimate of T2 if vasculature contributed to the measured signal. Given the highly heterogeneous nature of this tumor model as examined by necropsies and the limited spatial resolution of this approach, further uncertainty was added to this circumstance. Gd enhanced images (or SPIO images) of the same hepatic tumors, after 13C MRS, would have provided a more clear picture of tumor heterogeneity and partial volume effects, helping a better interpretation of the results. For future studies, respiratory gating can be used to improve the consistency of voxel placements between proton scout scans and 13C acquisitions.
We found that T2
C-alanine and 13
C-lactate in rat HCC tumors were longer than those in the normal liver tissue. This may be related to tumor cell morphology and leaky vessels in the fast growing tumors. The improved localization of T2
in this voxel-based study eliminated some of the complexity in the previous whole-slice study (13
). Most notably, a very long T2
component (2–3 s) previously observed in the whole-slice study was not observed in this study even when we tried bi- or tri-exponential fittings (results not shown here). This may not be surprising since significant signal from the intravascular space was included in the whole-slice data, and the proper noise correction (39
) was not applied in the data analysis of the whole-slice study. The sensitivity of this technique to very short T2
components (100 to 250 ms as previously reported (13
)) was somewhat limited by the delayed acquisition (78 ms) due to the voxel selection pulses.
Besides variations in T2
relaxation, injection dose and T1
relaxation from dissolution to injection, which were corrected in the data analysis, the total carbon signal could also be affected by the DNP solid-state build-up of the 13
C-pyruvate sample, variations in the liquid state polarization due to the dissolution process, and/or the voxel position and heterogeneity of the liver. Despite these variations, there is a trend towards higher total carbon signal in HCC tumors than that in normal livers. Since all detectable 13
C signal came from the injected 13
C-pyruvate, the high total carbon signal found in tumors may be due to the high metabolism expected in tumor cells or the high perfusion in these highly vascular tumors, or both. Techniques capable of differentiating signals from within cells versus signals in blood (42
) will be very important for more detailed characterization of cancerous tumors using hyperpolarized 13
The ratio of individual metabolite signals to the total carbon signal is, however, independent of many of the variations in experimental conditions mentioned above; and therefore, the ratio is a good ‘first-order’ indicator of enzymatic activity. We found the alanine to total carbon ratio is significantly higher in HCC tumors than in normal livers. This may be related to the reported high ALT level in the Morris HCC rat model (35
). Even though the same amount of hyperpolarized 13
C-pyruvate solution was injected in each animal, the up-take of 13
C-pyruvate by each HCC tumor varies, as indicated by the wide range of total carbon signal found in HCC tumors (). The up-take of 13
C-pyruvate may depend on the tumor vasculature, monocarboxylate transporters, and metabolic activity. But the almost linear relationship between the total carbon and the labeled alanine signals observed in HCC tumors (red stars in ) is very interesting. This may imply that the enzymatic activity in the HCC tumor cell is so high that it stays ‘unsaturated’ even with the high doses of 13
C-pyruvate injected in this study. An alternative interpretation as proposed by Day et al.
) is that the observed differences in alanine to total carbon ratios between HCC tumor and normal liver result from passive exchange with a pre-existing alanine pool. Based on this interpretation, our data showing higher alanine than lactate production in the Morris 7777 tumors may imply there is more alanine than lactate in this tumor model. Unfortunately, we are not able to find alanine and lactate concentrations measured in Morris 7777 tumors in literature. This may be of future interest but is outside of the scope of this work.
This study showed similar lactate to total carbon ratios between HCC tumors and normal livers (); very different from the elevated lactate to total carbon ratios previously reported by other tumor studies (2
). This may also be due to the particular animal model used here. Studies done by Weber et al.
) and Shonk et al.
) on metabolism regulations in Morris HCC models found that in spite of correlation between the increasing growth rate and increased lactate production, the LDH level in the fast growing HCC model [approximately 135 μmole/min/g w-w in average (30
)] is not higher than the LDH level in the normal liver [162 μmole/min/g w-w (30
)]. In another study comparing a fast-growing HCC model to a slow-growing one, Fields et al.
) found that the fast-growing model (Morris hepatoma 7777, in particular) is not able to oxidize fat. Therefore, it must derive all of its calories from protein or carbohydrate sources. We postulate that the Morris hepatoma 7777 model may have elevated lactate production due to high consumption of pyruvate as a fuel but the pyruvate to lactate conversion rate may be similar to normal liver, as indicated to the first-order by the similar lactate to total carbon ratios found here () in the presence of a pyruvate concentration higher than the normal physiological level. A better experiment to evaluate the pyruvate to lactate conversion in vivo
using hyperpolarized 13
C technique would be to perform a localized dynamic acquisition from the start of hyperpolarized 13
C-pyruvate injection and to extract the apparent Vmax
(the maximum reaction rate) from the Michaelis-Menten kinetics (43
). Such an experiment is beyond the scope of this study but will be of future interest.
It is important to note that in addition to the LDH activity, the labeled lactate signal to the total carbon ratio also depends on in flow and transport. The monocarboxylate transporters will equilibrate pyruvate and lactate, as determined by the intra- and extracellular pH gradient, exchanging these metabolites much faster between the intracellular and extracellular spaces than LDH activity. This exchange between different vascular, extracellular and intracellular environments in control and HCC may be an additional source of the wide range of total carbon and lactate signals found in HCC tumors.
The similar ratio of labeled lactate to total carbon discovered here was after injections of unphysiologically high concentration of pyruvate. Under physiological conditions, LDH is not a rate-limiting enzyme of glycolysis. The glycolytic flux and lactate production are determined upstream in the glycolytic pathway at the hexokinase, phosphofructokinase and pyruvate kinase steps. Thus the finding of similar ratio of 13C labeled lactate to the total 13C in normal liver and HCC tumor does not imply that the HCC tumor will not produce more lactate from glucose than the normal liver under physiological conditions since all the rate limiting steps prior to LDH in tumor may be different or altered.