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The development of dynamic nuclear polarization (DNP) in solution has enabled in vivo 13C MR studies at high signal to noise ratio (SNR) following injection of pre-polarized 13C substrates. While prior studies have demonstrated the ability to observe metabolism following injection of hyperpolarized 13C-pyruvate, the goal of this study was to develop and test a new hyperpolarized agent for investigating in vivo metabolism, [1-13C] lactate. A preparation for pre-polarized 13C-lactate and the requisite dissolution media were developed to investigate the feasibility for in vivo 13C MRS/MRSI studies following injection of this hyperpolarized agent. This study demonstrated, for the first time, not only the ability to detect hyperpolarized [1-13C] lactate in vivo, but also the metabolic products 13C pyruvate, 13C alanine and 13C bicarbonate following injection in normal rats. Using 13C-lactate as a substrate provided the ability to study the conversion of lactate to pyruvate in vivo and to detect the secondary conversions to alanine and bicarbonate through pyruvate. This study also demonstrated the potential value of this hyperpolarized agent to investigate in vivo lactate uptake and metabolism in pre-clinical animal models.
Recent studies have demonstrated that signal enhancements from dynamic nuclear polarization (DNP) can be retained in solution (1). This development has enabled in vivo 13C MR studies at high signal to noise ratio (SNR) following injection of pre-polarized 13C substrates (2). It has been shown by using [l-13C]pyruvate as the substrate for hyperpolarized 13C MRS/MRSI experiment, real time cellular metabolism can be studied in normal and diseased tissues in vivo (3–5). These studies have shown the ability to detect the metabolic conversion of the hyperpolarized [l-13C]pyruvate into [l-13C]lactate, [l-13C]alanine and 13C-bicarbonate after in vivo injection in animal models. In these studies using pre-polarized 13C pyruvate as the substrate, the concentration of pyruvate in the injected solution was much higher than the endogenous concentration (3–5). Thus one of the processes that can be probed in these conditions is the cellular conversion of the 13C pyruvate bolus to 13C lactate (6).
All these prior studies (2–6) have used 13C-pyruvic acid, however, if 13C-lactate could be used as the substrate in pre-polarized 13C MRS/MRSI study, it then may be possible to probe the reverse enzymatic conversion of hyperpolarized 13C lactate into the unlabeled pyruvate pool. This could allow better understanding of the lactate dehydrogenase (LDH) activity in vivo in different tissue types or diseases at different stages. The development of 13C-lactate as a new hyperpolarized agent for in vivo studies requires overcoming several obstacles. [l-13C]Pyruvic-acid is an especially good compound for hyperpolarized studies since as a neat solution it naturally forms an amorphous solid at 1°K, polarizes rapidly to a high percentage in under 1 hour, and retains polarization well due to its long T1 in solution. For [l-13C]lactate, a different preparation is required with a high substrate concentration, a glassing agent/solvent to insure an amorphous solid at 1°K, a short DNP enhancement rate constant and a high a degree of polarization. The goal of this study was to develop a hyperpolarized MR probe based on [l-13C]lactate as the substrate for in vivo 13C MRS/MRSI studies and to determine if metabolic products could be detected following injection in animal models.
A HyperSense DNP polarizer (Oxford Instruments, Abingdon, UK) was used in this study. The preparation for pre-polarization developed in this project was a mixture of [l-13C]lactate (Isotec, Miamisburg, OH), water, DMSO and OX63 trityl radical (Oxford Instruments, Abingdon, UK). The mixture contained 38.5 % 13C1-lactate and 30% DMSO. The trityl radical concentration was 15 mM. The [l-13C]lactate mixture was polarized in a field of 3.35T at approximately 1.4° K by irradiation of 94.116 GHz microwaves similarly to what was described previously for [l-13C]pyruvate (1). Each sample used in the animal experiments was polarized for ~ 100 minutes prior to dissolution.
A normal saline and 100mg/L sodium EDTA solution was used as the dissolution medium. The 13C-lactate concentration in the hyperpolarized lactate solution was 44 mM. Immediately after the dissolution, an aliquot of the hyperpolarized lactate solution was used to measure the percent polarization (ranging from 6.9 – 12.4%, average 10.0 % for these experiments). The duration for the dissolution process and transferring of the solution was approximately 10 – 15s. In each rat studied, ~2.5 ml (ranging from 2.2 – 3 ml, average 2.6 ml) of the final lactate solution was injected into the rat over a 12 s period followed by a normal saline flush.
All animal studies were carried out under a protocol approved by the UCSF Institutional Animal Care and Use Committee. Male Sprague-Dawley rats ranging in weight from 250 to 350 grams were placed on a heated pad and anesthetized with isoflurane (2–3%). A catheter was introduced into the tail vein for the eventual intra-venous administration of hyperpolarized lactate solution, and the rat was transferred to a heated pad in the RF coil in the MR scanner. While in the scanner, anesthesia was maintained by a continual delivery of isoflurane (1–2%) via a long tube to a cone placed over the rat’s nose and mouth. The rat’s vital signs (heart rate and oxygen saturation) were continually monitored. Care was taken to ensure that body temperature was maintained at 37° C throughout the imaging procedures by maintaining a flow of heated water through the pad underneath the rat. At the end of the study, the rat was euthanized by a combination of over-anesthesia with isoflurane and bilateral thoracotomy following an approved protocol.
All studies were performed using a 3T GE Signa™ scanner (GE Healthcare, Waukesha, WI) equipped with the MNS (multinuclear spectroscopy) hardware package. The RF coil used in these experiments was a dual-tuned 1H-13C coil with a quadrature 13C channel and linear 1H channel constructed based on an earlier design (7) and used in prior hyperpolarized 13C pyruvate studies (3–5). The inner coil diameter was 8 cm and the length of the coil was 9 cm to accommodate rats of varying size.
T2-weighted anatomical images were obtained in all three planes using a fast spin-echo (FSE) sequence. Axial and sagittal images were each acquired in ~10 minutes with a 10 cm FOV, 192 × 192 matrix, 2 mm thick slices and NEX = 6. Coronal images were acquired with a 12 cm FOV, 192 × 192 matrix, 1.5 mm thick slices and NEX = 6 with a scan time of 10 minutes. The total imaging time required to obtain images in all three planes was thus approximately 35 minutes.
One polarized/dissolved sample was used to estimate the T1 relaxation time of the hyperpolarized [1-13C]lactate in solution. The polarized [1-13C]lactate solution was transfer into a syringe and placed inside the dual tuned coil used in this study. A small tip angle, non-localized MRS pulse sequence (identical to what is used in the 13C in vivo dynamic MRS study described in the next paragraph) was used to measured the signal decay due to T1 relaxation. A mono-exponential fit was used to estimate the T1 and the reported T1 value was corrected for RF tipping (8).
Five 13C dynamic MRS studies were performed in five different rats using a double spin-echo pulse sequence with a non-selective 5 degree flip angle RF excitation pulse and a pair of 180 degree hyperbolic secant refocusing pulses (also without localization) (4). A TE of 35 ms, a repetition time (TR) of 3 s and a readout filter of 5000 Hz / 2048 pts were used for these studies. The acquisition started at the beginning of a 12-second manual injection of the 13C-lactate into the rat tail vein.
3D 13C MRSI data were acquired in 10 seconds in two normal rats using a double spin-echo pulse sequence with a slice selective small tip angle excitation pulse and a flyback echo-planar readout trajectory (4). A 8 × 6 × 1 phase encoding matrix with flyback echo-planar trajectory on the Z-axis (8 × 6 × 16 effective matrix) was used with 10 mm × 10 mm × 10 mm spatial resolution (1.0 cc voxel resolution) that resulted with a 80 mm × 60 mm × 160 mm FOV to cover the rat torso and abdomen. The flyback echo-planar trajectory was designed for a 581 Hz spectral bandwidth to include 13C lactate, 13C alanine and 13C pyruvate without spectral aliasing. A total of 59 readout/rewind lobes were included during each readout for a spectral resolution of 9.83 Hz. With a readout filter of 25,000 Hz / 2,538 points, 16 k-space points were acquired during each TR and the SNR efficiency of this waveform was 61% (9). The TE for the MRSI acquisition was 140 ms (readout was centered on the center of the second spin-echo), and the TR was 215 ms.
Dynamic data were apodized in the time domain with a 10 Hz Gaussian filter prior to a one-dimensional Fourier transform. 3D 13C MRSI data acquired using the flyback echo-planar trajectory were processed using the same procedures described previously (4,9). The k-space points corresponding to the constant portion of the flyback trajectory were selected out and the data was reordered into a 4D dataset with the time decay in the first dimension. The reordered dataset was then processed in the same manner as a conventional 4D MRSI dataset with the exception that the k-space points in the flyback dimension were each acquired at a slightly different time point (9). This was corrected during the 4D Fourier reconstruction using the fact that an origin shift in k-space is equivalent to a phase shift in the transformed domain. The time domain signal was apodized by 16 Hz Gaussian filter and zero-filled from 59 pts to 128 pts. No apodization in the spatial domains was applied.
The T1 of hyperpolarized [1-13C] lactate estimated from the MRS study performed on the syringe of hyperpolarized lactate solution was 45s. In this experiment, polarization in solution was measured to be ~7%, which corresponds to a 28,500 fold increase in polarization compared to the thermal equilibrium polarization of 2.47ppm at 3T and 300°K.
From the dynamic MRS studies performed starting at the same time as the pre-polarized 13C lactate was injected, the time course of hyperpolarized 13C lactate uptake in the animal, as well as the time course of the formation of its metabolic products were determined. Representative data in figure 1 shows a stacked plot of dynamic 13C data (first 25 time points, each 3 seconds apart) and a graph of the signal amplitude vs time from one of the in vivo dynamic studies. Resonances for 13C lactate, 13C alanine, and 13C pyruvate were observed in the spectra (figure 1). In the time course graph in figure 1, the peak amplitudes of 13C alanine and 13C pyruvate were scaled up by 10 fold for easier viewing. Also observed in this graph was that the 13C pyruvate resonance appeared in the dynamic MRS study prior to the appearance of the 13C alanine resonance, but the 13C alanine signal amplitude becomes higher than that of the 13C pyruvate at approximately 15s after the start of the injection/experiment in this particular case. No discernable 13C bicarbonate resonance was observed in this dynamic study. Table 1 summarized the lactate arrival time as well as the lactate, pyruvate and alanine time to peak (maximum amplitude in the time course) relative to the start of the injection time. The alanine time to peak was on average 14.4s after the lactate time to peak and and 7.4s after the pyruvate time to peak.
In two out of the five dynamic studies, 13C bicarbonate resonance was observed, the representative data is shown in figure 2. Due to limited SNR of the 13C bicarbonate signal, the 13C bicarbonate peak was more clearly observed in the summed spectrum (figure 2b) as compared the individual spectra in the stacked plot (figure 2a).
From the 3D 13C MRSI studies, high amplitudes of 13C lactate were observed (figure 3), 13C alanine and pyruvate were also observed in voxels centered on rat kidney, muscle and vasculature. Consistent with the data from the 13C dynamic MRS studies, higher 13C alanine signal amplitude compared to that of 13C pyruvate were observed in the MRSI voxels where both resonances were present.
Hyperpolarized 13C MRS/MRSI provides an exciting new opportunity to study dynamic metabolic processes in vivo since small organic molecules including many metabolic intermediates have the potential to be compatible with the DNP process (3). Oxidation, transamination, and oxidative decarboxylation of pre-polarized [l-13C]pyruvate to produce 13C lactate, 13C alanine and 13C bicarbonate have been reported in vivo (3). Since high, non-physiological concentrations of [l-13C]pyruvate were used in these studies, this substrate allowed investigation of the differences in the enzymatic conversion of pyruvate to lactate for different tissue types. However, the lactate to pyruvate process could not be studied with 13C-pyruvate as the substrate. Using pre-polarized 13C-lactate as the substrate, provides a new opportunity to probe the LDH activity that converts lactate to pyruvate and to determine if secondary conversions from lactate through pyruvate to alanine and bicarbonate would be observable in vivo by hyperpolarized 13C MRSI.
In this study, after injection of the hyperpolarized [l-13C]lactate into rats, 13C pyruvate was indeed observed (figure 1–3). In addition, 13C alanine resonance was also observed in these experiments. The spectral pattern observed in these studies were very different from that observed in the studies in which [1-13C]pyruvate was injected. The alanine signal intensity was higher than the signal intensity for pyruvate in both the dynamic MRS studies as well as the MRSI studies. It can be surmised that the 13C pyruvate produced by the 13C lactate substrate was rapidly consumed to produce lactate, alanine and bicarbonate, resulting in the relatively low level of 13C pyruvate. The slight temporal offset between 13C pyruvate curve and 13C alanine curve in figure 1 (pyruvate signal was higher initially compared to that of alanine) and the delayed time to peak of the lactate seen in the dynamic MRS studies (figure 1, table 1) further suggest that 13C pyruvate was produced first after the injection of the 13C lactate, and the 13C pyruvate was then metabolized to 13C alanine, 13C bicarbonate, and perhaps back to 13C lactate. It is worth noting that while the pre-polarized magnetization may have been decreased through T1 loses in various enzymatic and membrane transport processes, 13C bicarbonate was still observable in some cases through three enzyme catalyzed reactions (lactate dehydrogenase, pyruvate dehydrogenase and carbonic anhydrase). The fact that multiple down stream products from 13C lactate was observed in this study demonstrates the added potential of using pre-polarized 13C substrates for in vivo metabolic studies in which multiple enzyme steps are required.
In the MRSI experiments performed in this study, high intensity 13C lactate signals were observed in most voxels within the body of the rat. 13C pyruvate and 13C alanine were observed mostly in the kidney and the muscle/central vasculature regions in the rat body. It appeared that the uptake of lactate did not differ significantly between different tissues in a normal healthy rat. The low amplitudes of the 13C pyruvate and 13C alanine signals observed in these studies relative to the 13C lactate signal indicate that the conversion of the 13C lactate to pyruvate is fairly low in normal tissues. More 13C pyruvate and alanine were detected in voxels containing rat kidney, but with the low signal level of these metabolic products and the large voxel sizes used, it was not possible to see clear differences in metabolic activities between different tissue types in this study. Further more, the flyback echo-planar readout trajectory didn’t have adequate spectral bandwidth to include the 13C bicarbonate resonance without spectral aliasing. And base on the relative SNR of the 13C bicarbonate compare to the other metabolites, the 13C bicarbonate signal was most likely below the detection limit in these 3D MRSI experiments. To allow observation of 13C bicarbonate, perhaps a 2D MRSI experiment (5) without using the flyback readout trajectory (to give larger spectral bandwidth), and larger voxel size (to give more SNR) are required.
In all the in vivo experiments reported in this study, the boluses given corresponded to an approximate 5–6 mM concentration of 13C lactate in blood of the animals. This concentration is 4–5 times the endogenous blood lactate concentration in a resting animal (1 – 2 mM) but within physiological range for an exercising mammal. Thus the results of this study demonstrate the feasibility for studying hyperpolarized lactate metabolism at concentrations within the physiological range for blood lactate levels.
A prior study using hyperpolarized pyruvate reported that higher levels of 13C lactate were observed in regions of prostate cancer in a mouse model of prostate cancer after injection of pre-polarized 13C pyruvate (4). But it was not clear that if the high level of lactate observed in the tumor reflected solely the higher level of LDH activity within the tumor, or higher uptake of the 13C lactate (produced in the blood and other organs) as well. Although beyond the scope of this initial feasibility study, using pre-polarized 13C lactate as a substrate for in vivo MRSI studies may allow improved elucidation of the mechanism behind the high lactate observed in region of tumor in pre-clinical cancer models.
The feasibility of using [l-13C]lactate as a substrate for pre-polarized MRS and MRSI in vivo was demonstrated in this study. The metabolic products 13C pyruvate, 13C alanine and 13C bicarbonate were observed in normal rats following injection of hyperpolarized 13C lactate. Using [l-13C]lactate as a substrate provides the opportunity to study the conversion of lactate to pyruvate in vivo and to detect the secondary conversions to alanine and bicarbonate through pyruvate.
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