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Spin echo measurements of the extravasation and tumor cell uptake of hyperpolarized [1-13C]lactate and [1-13C]pyruvate†
Article first published online: 27 DEC 2012
Copyright © 2012 Wiley Periodicals, Inc.
Magnetic Resonance in Medicine
Volume 70, Issue 5, pages 1200–1209, November 2013
How to Cite
Kettunen, M. I., Kennedy, B. W. C., Hu, D.-e. and Brindle, K. M. (2013), Spin echo measurements of the extravasation and tumor cell uptake of hyperpolarized [1-13C]lactate and [1-13C]pyruvate. Magn Reson Med, 70: 1200–1209. doi: 10.1002/mrm.24591
- Issue published online: 25 OCT 2013
- Article first published online: 27 DEC 2012
- Manuscript Accepted: 16 NOV 2012
- Manuscript Revised: 14 NOV 2012
- Manuscript Received: 9 JUL 2012
- Cancer Research UK Program Grant. Grant Number: C197/A3514
- Translational Research Program Award from The Leukemia and Lymphoma Society
- monocarboxylate transporter
To assess the blood-tissue distribution of hyperpolarized 13C-labeled molecules in vivo.
Spin-echo experiments with simultaneous acquisition of the free induction decay (FID) signal following the excitation pulse and the spin-echo signal, were used to monitor hyperpolarized [1-13C]lactate, [1-13C]pyruvate, and the perfusion marker, [13C]HP001, following their intravenous injection into tumor-bearing mice. Apparent T2 relaxation times and diffusion coefficients were also measured.
An increasing tumor echo/FID ratio was observed for all three molecules, which could be explained by their extravasation into the tumor interstitial space, where T2 relaxation times were longer and diffusion coefficients smaller. Inhibition of the monocarboxylate transporter, which decreased by 40% the label exchange between pyruvate and lactate, reduced the increase in the echo/FID ratio for pyruvate and lactate, but not for HP001, demonstrating that some of the increase in the echo/FID ratio was due to cell uptake of lactate and pyruvate. The different relaxation and diffusion behavior of the intravascular and extravascular signals affected measurements of the apparent label exchange rate constants.
Simultaneous collection of both FID and echo signals can provide information on cell uptake thus giving further insight into the kinetics of hyperpolarized 13C label exchange. Care is needed when comparing exchange rate constants determined in spin-echo-based studies. Magn Reson Med 70:1200–1209, 2013. © 2012 Wiley Periodicals, Inc.
The development of dynamic nuclear polarization in combination with a rapid dissolution process that brings the polarized material to physiological temperatures  has allowed real-time MR spectroscopic imaging of metabolism using hyperpolarized 13C-labeled cell metabolites [2-4]. The most widely used 13C-labeled substrate to date has been pyruvate, which has found applications in imaging metabolism, particularly in tumors [4, 5] and heart . In tumors, following injection of hyperpolarized [1-13C]pyruvate, a high level of lactate labeling was observed , which was subsequently shown to be due to exchange of the hyperpolarized 13C label between pyruvate and the large endogenous lactate pool that results from the high glycolytic rate that characterizes tumors [8, 9]. This lactate labeling was decreased in tumors that showed a positive response to treatment (4,5,8).
The short lifetime of the hyperpolarized 13C-label in [1-13C]pyruvate (the T1 of the 13C label is ∼30 s in vivo), means that the labeled compound can only be imaged for 2–3 min postinjection. Therefore, during the measurement of hyperpolarized 13C label exchange between pyruvate and endogenous lactate there is redistribution of pyruvate between the vascular, interstitial, and intracellular spaces, which necessarily complicates analysis of the isotope exchange kinetics. In tumors, the injected [1-13C]pyruvate is assumed to be predominantly in the intravascular space whereas the lactate, which is labeled in the reaction catalyzed by lactate dehydrogenase, is presumed to be largely intracellular, or at least in the extravascular space. Red blood cells also catalyze label exchange between pyruvate and lactate [10, 11], although there is no evidence for significant wash-in into the tumor of labeled lactate from the blood or other tissues . Depending on the pulse sequence used for signal acquisition these differences in the distribution of labeled lactate and pyruvate will affect their relative tissue intensities and thus influence the estimated rate constants for label exchange. Previous studies have shown that it is possible to attenuate intravascular flowing spins with saturation or tagging pulses [12, 13], double-spin echoes , stimulated echoes , or via injection of exogenous contrast agents . However, only some of these approaches allow repetitive sampling for measurements of the kinetics of metabolite labeling. In this study, we used a spin-echo pulse sequence in which we also acquired the signal immediately following the initial pulse in the sequence [termed here the free induction decay (FID) signal] as well as the echo signal . This allowed us to monitor simultaneously the total 13C signal (FID) as well as the signal attenuated by vascular flow (echo signal). Previously this has only been achieved by measuring the FID and echo signals separately after two sequential injections of pyruvate [14, 15]. In a recent study, in which we measured exchange of deuterium label between the C2 position of hyperpolarized L-[U-2H,1-13C]lactate and the C2 protons in endogenous lactate, where the exchange was monitored indirectly by phase modulation of the spin-coupled hyperpolarized 13C label in a heteronuclear spin echo experiment (TE = 310 ms) , we observed an increase in the echo signal relative to the FID signal. The increase in this echo/FID ratio can be explained by suppression of the lactate signal in the vascular space due to flow, with the increase in the ratio reflecting redistribution of lactate between the vascular and extravascular spaces.
The main aim of this study was to investigate the increase in echo/FID ratio when hyperpolarized [1-13C]lactate and [1-13C]pyruvate were injected into tumor-bearing mice. We show, by measuring the apparent T2 relaxation times and diffusion coefficients (ADC) for [1-13C]lactate and [1-13C]pyruvate as well as a perfusion marker, bis-1,1-(hydroxymethyl)-[1-13C]cyclopropane-d8 ([13C]HP001) [18, 19], that the increase in these ratios can be explained by their different relaxation and diffusion properties in the intravascular and extravascular spaces. We also investigated the extent to which the increases in the echo/FID ratios reflect lactate and pyruvate transport into the cell via the monocarboxylate transporters (MCT) [20-22] by inhibiting this transport. The transport of both pyruvate and lactate were inhibited by using a noncompetitive inhibitor of the transporter, α-cyano-4-hydroxycinnamate (4-CIN)  and lactate transport was also inhibited by coinjecting unlabeled lactate. The influence of the different relaxation times and ADCs for hyperpolarized 13C-labeled pyruvate and lactate in the intravascular and extravascular spaces on the apparent isotope exchange kinetics measured using echo-based pulse sequences was examined.
Tumors were implanted into female C57BL/6 mice (n = 60, 6–8 weeks of age; Charles River Ltd., Margate, UK) by subcutaneous injection, in the lower flank, of 5 × 106 EL-4 murine lymphoma cells. MRS was performed when the tumors had grown to a size of ∼2 cm3 (which was reached typically at 10 days following implantation) . At this location, there was no detectable respiratory motion in MR images of the tumors.
For 13C MRS experiments, animals were anesthetized with intraperitoneal injections of Hypnorm (VetaPharma, Leeds, UK)/Hypnovel (Roche, Welwyn Garden City, UK)/dextrose-saline (4:0.18%) in a 5:4:31 ratio (10 mL kg−1 body weight) and a catheter was inserted into the tail vein for injection of hyperpolarized [1-13C]lactate, [1-13C]pyruvate, or [13C]HP001. Cell uptake of [1-13C]lactate or [1-13C]pyruvate was inhibited either by injecting the MCT inhibitor, 4-CIN (150 mg kg−1, 200 μL i.v.), or, in the case of lactate, by injecting unlabeled L-lactate (200 μL of 2 M L-lactate i.p. 30 min before the MR experiment or 200 μL of 0.18 M L-lactate i.v., which was 3× the concentration of the 13C-labeled lactate) prior to injection of hyperpolarized [1-13C]lactate. We have previously measured lactate levels of up to 11 mM in this tumor model  and injection of 200 μL of 2 M L-lactate i.p. led to an ∼60% increase in the tumor lactate signal, measured using multiple-quantum coherence edited 1H lactate spectroscopy , and more than doubled the lactate 1H signal in blood samples by 30 min (data not shown). These data confirmed that there were increased levels of unlabeled lactate in the blood and tumor at the time of injection of the hyperpolarized L-[1-13C]lactate. The body temperature of the animals was maintained by blowing warm air through the magnet bore. All experiments were conducted in compliance with project and personal licenses issued under the Animals (Scientific Procedures) Act of 1986 and were designed with reference to the U.K. Co-ordinating Committee on Cancer Research guidelines for the welfare of animals in experimental neoplasia. The work was approved by a local ethical review committee.
Hyperpolarization of L-[1-13C1]lactate, [1-13C]pyruvate, and [13C]HP001
Trityl radical (15 mM, OX063; GE Healthcare, Amersham, UK) and gadolinium chelate (1.2 mM, Dotarem; Guerbet, Roissy, France) were dissolved in an aqueous solution of L-[1-13C] sodium lactate (∼50% w/v) and then 30% w/v dimethyl sulfoxide was added to ensure glass formation in the solid state. The [1-13C] pyruvic acid samples (44 mg) contained 15 mM trityl radical and 1.4 mM of a gadolinium chelate (Dotarem). Samples of the perfusion marker, [13C]HP001 [18, 19], were prepared by adding 1.2 mM gadolinium chelate to a solution containing 44 mg of HP001 and 15 mM OX063. Samples were hyperpolarized in an alpha-prototype hyperpolarizer (GE Healthcare, Amersham, UK), as described previously [17, 26]. The polarized samples were dissolved in pressurized buffer at ∼180°C and ∼1000 kPa. Lactate and HP001 samples were dissolved in 2–4 mL of PBS, to give a final concentration of 45–90 mM for lactate and 45 mM for HP001. Pyruvate samples were dissolved in 6 mL of HEPES buffer (40 mM HEPES, 94 mM NaOH, 30 mM NaCl, and 100 mg L−1 EDTA) to give a final concentration of 75 mM. For all substrates, the levels of polarization were typically >20%. The samples were cooled to ∼37°C before i.v. injection.
MR Spectroscopy In Vivo
Experiments were performed using a 9.4 T vertical bore magnet (Oxford Instruments, Oxford, UK) interfaced to a Varian UnityInova console (Varian Inc, Palo Alto, CA). Surface coil experiments were performed using a 13C-surface coil (diameter 24 mm) placed directly over the tumor and the animal holder was then placed inside a 1H volume coil (Millipede, Varian Inc, length 6 cm, diameter 4 cm). Volume-coil experiments were performed using a 13C/1H volume coil (Rapid Biomedical, Rimpar Germany, length 5 cm, diameter 3 cm) for transmission and a 20 mm diameter 13C surface coil for receive, with the volume coil actively detuned during data acquisition. Animals received a 200 μL i.v. injection of the hyperpolarized sample outside the magnet and data acquisition was started as soon as the animal was placed inside the magnet bore, typically 8 s after the beginning of injection. Either a single (S180) or dual-refocused (D180) spin-echo pulse sequences were used with a nonselective 13C excitation pulse (10° nominal flip angle) and the FID (TE = 1 ms, spectral width 8000 Hz, 512 complex points) and spin-echo signals (spectral width 8000 Hz, 512 or 1024 complex points centered around the echo time) were acquired. In the S180 sequence, a single 4 ms adiabatic 13C BIR4 (peak B1 = 2.5 kHz)  180° refocusing pulse, surrounded by crusher gradients, was placed at TE/2 (Fig. 1a). In the D180 pulse sequence, a pair of 4 ms hyperbolic secant (HS) refocusing pulses (R = 20, peak B1 = 2.5 kHz) , surrounded by crusher gradients, were placed at TE/4 and 3TE/4 (Fig. 1b). The S180 pulse sequence was used with a surface coil for transmit/receive while the D180 pulse sequence was used with both the surface coil transmit/receive arrangement and the volume coil transmit/surface coil receive arrangement. The amplitudes of the crusher gradients were adjusted to remove any transverse magnetization originating from the refocusing pulses and were used for diffusion-weighting (Table 1). Imperfections in these refocusing pulses are known to lead to slightly faster loss of longitudinal magnetization in hyperpolarized experiments .
|Sequence||Refocusing pulse||δ (ms)||Δ (ms)||G (G cm−1)a||b-Range (s mm−2)||TE (ms)|
|S180||4 ms BIR4||12||38||1–12||7–946||132|
|D180||2 × 4 ms HS||12||24||1–12||8–1133||170|
|D180||2 × 4 ms HS||11||23–73||11–6||730b||240|
Apparent T2s and ADCs were measured using both the S180 pulse sequence, with a second refocusing pulse applied immediately following acquisition of the echo signal, to return residual longitudinal magnetization to +z, and the D180 sequence. For T2 measurements, echo times between 100 and 600 ms were collected in mixed echo time order to minimize the effects of variation in T2s during the measurements. There was a constant repetition delay of 0.15 s between the end of data acquisition and the next excitation pulse. For diffusion experiments, the repetition delay was 0.25 s. The other acquisition parameters are summarized in Table 1. The data were collected in mixed gradient amplitude order. The b-value (s mm−2) was calculated using the equation:
where γ, G, δ, Δ, and nG are the gyromagnetic ratio (for 13C, 6.728 × 103 rad Gs−1), diffusion gradient amplitude (G mm−1), gradient length (s), the delay between the gradients (s) and the number of gradient pairs, respectively . For the number of measurements performed using each pulse sequence, see Table 2.
|Surface coil, S180, 4ms BIR4 180°|
|T2 (s)||–||–||0.18 ± 0.04 (n = 13)||0.27 ± 0.01 (n = 2)a|
|S0||–||–||0.78 ± 0.18||0.81 ± 0.05|
|ADC (×10−3 mm2 s−1)||–||–||2.3 ± 0.5 (n = 7)||2.8 (n = 1)|
|S0||–||–||0.64 ± 0.10||0.61|
|Surface coil, D180, 2 × 4 ms HS 180°|
|T2 (s)||0.17 ± 0.04 (n = 5)||0.27 ± 0.02 (n = 9)||0.21 ± 0.07 (n = 4)*b||0.40 ± 0.04 (n = 4)|
|S0||0.68 ± 0.08||1.00 ± 0.09||0.92 ± 0.10||0.99 ± 0.10|
|ADC (×10−3 mm2 s−1)||1.7 ± 0.6 (n = 6)||0.8 ± 0.2 (n = 17)||1.4 ± 0.5 (n = 6)**||1.4 ± 0.5 (n = 3)|
|S0||0.19 ± 0.06||0.53 ± 0.08||0.46 ± 0.16||0.61 ± 0.21|
|Volume coil, D180, 2 × 5 ms HS 180°|
|T2 (s)||0.11 (n = 1)||0.24 ± 0.02 (n = 2)||–||–|
|S0||0.74||1.00 ± 0.02||–||–|
|ADC (×10−3 mm2 s−1)||1.20 ± 0.30 (n = 2)||0.5 ± 0.1 (n = 2)||–||–|
|S0||0.14 ± 0.02||0.42 ± 0.05||–||–|
For studies of the effect of MCT inhibition on the echo/FID ratio following injection of [1-13C]lactate (n = 25), data were acquired using a surface coil and the S180 pulse sequence with an echo time of 310 ms. These acquisition conditions match those used in the heteronuclear spin-echo experiment described in our previous study with L-[U-2H,1-13C]lactate . Pairs of spectra were collected every 4 s with a repetition delay of 0.5 s between the first and the second spectrum. The phase of the excitation pulse was changed by 180° for the second member of each echo pair to account for inversion of the remaining longitudinal magnetization by the preceding 13C refocusing pulse. This pulse sequence and coil arrangement were also used for measurements of the echo/FID ratio for HP001 (n = 4).
As the effective bandwidth of the BIR4 pulse is dependent on B1 amplitude  it was not wide enough to give perfect refocusing of both the pyruvate and lactate magnetization. Previous studies on the kinetics of lactate labeling with [1-13C]pyruvate have used pulse sequences with two HS refocusing pulses (14,29,30). Therefore for all measurements of the echo/FID ratio for lactate and pyruvate, following injection of [1-13C]pyruvate, signal was acquired using the D180 pulse sequence.
Experiments studying the effects of MCT inhibition on the kinetics of label exchange between [1-13C]pyruvate and endogenous lactate (n = 8) were performed using a surface coil. Pairs of spectra were collected every 3 s with a 0.5 s delay between the first and second spectrum. The first, T2-weighted, spectrum in the pair was acquired with TE = 310 ms and b = 7 s mm−2 and the second, diffusion-weighted, spectrum with TE = 155 ms and b = 303 s mm−2 (δ = 8 ms, Δ = 20.1 ms, G = 10 G cm−1). This pulse sequence and coil arrangement were also used for measurements of the echo/FID ratio for HP001 (n = 2).
The effect of T2- or diffusion-weighting on estimation of the apparent first order rate constant describing label exchange between injected [1-13C]pyruvate and endogenous lactate, kP, was tested in another set of experiments using the volume coil transmit/surface coil receive arrangement. The D180 pulse sequence was used to collect pairs of signals every 2.5 s with varying TE and b-values (n = 10). Two echoes with TEs of 86 ms and 172 ms were collected after each excitation pulse and the amplitudes of the diffusion gradients were varied to give b-values of 98 and 196 s mm−2 (δ = 6 ms, Δ = 13.1 ms, G = 9.5 G cm−1) or 27 and 54 s mm−2 (G = 5 G cm−1). For these experiments, an 8 mm slice-selective excitation pulse was used while refocusing was performed using nonslice selective 5 ms HS pulses.
Spectra were phase- and baseline-corrected and the signals fitted in the time-domain using MATLAB (Mathworks, Natick). The signals were also integrated in the frequency-domain and similar results were obtained, although only results for time-domain fits are shown here. It should be noted that when the whole echo signal is Fourier transformed, the peak integral of the echo signal is twice the FID peak integral if relaxation is corrected for. Exchange of hyperpolarized 13C label between [1-13C]pyruvate and endogenous lactate was analyzed using a simple two-site exchange model , to give the first order rate constant describing flux of label between pyruvate and lactate (kP) and the relaxation rate constant for both species (R1). In experiments with [1-13C]lactate, there is minimal exchange of label with the small endogenous pyruvate pool  and therefore R1 for lactate was estimated directly from its apparently monoexponential signal decay.
For apparent T2 and diffusion coefficient calculations, each echo signal was normalized to the corresponding FID signal and the data were then fitted to a monoexponential function (Eq. (2)) using two parameters (T2 or the apparent diffusion coefficient, D, and the STE,b/SFID ratio when TE or b were zero, S0).
In most cases, either a constant b-value (in T2-weighted experiments) or TE (in diffusion-weighted experiments) was used and this exponential term is therefore incorporated into the S0 term, thus reducing the equation to normal monoexponential decay. Furthermore, this analysis assumes that the S0 term does not change significantly during the measurement, which is not strictly true. Therefore, to minimize the effect of systematic changes in S0, the data points were collected in mixed echo time or b-value order. Data sets where the FID signal was close to zero for any data point were not included and only data sets where the quality of fit, as assessed by the coefficient of determination (R2), was higher than 0.8 were included. All values are given as mean ± standard deviation. Statistical significance was assessed using Student's t-test.
Apparent Diffusion Coefficients and T2 Relaxation Times
Relaxation and diffusion measurements could be fitted using a monoexponential equation (Eq. (2), Fig. 2). Apparent T2 relaxation times and ADCs measured using the different pulse sequences (Fig. 1, Table 1) are summarized in Table 2. Estimates of S0 for the injected molecules tended to be <1, suggesting that there was a component with shorter T2 that could not be measured adequately with the echo times used. Measurements using the D180 pulse sequence gave longer T2 values and lower diffusion coefficients than the S180 pulse sequence. Similarly, diffusion coefficients measured using the volume coil for transmit were smaller than those observed when the surface coil was used for transmit. This likely reflects the improved refocusing of signal in the double-echo pulse sequence, especially when a volume coil was used. A significantly smaller diffusion coefficient and longer T2 relaxation time were measured for lactate labeled by exchange with hyperpolarized [1-13C] pyruvate than when hyperpolarized [1-13C]lactate was injected, consistent with the former being predominantly intracellular and the latter intravascular. Moreover, measurements using b = 730 s mm−2 and different diffusion times (23–73 ms, TE = 240 ms, Fig. 2e) suggest that lactate produced from labeled pyruvate displays more restricted diffusion. The pyruvate T2 measured in mouse blood in vitro using the D180 pulse sequence was 240 ms (n = 1). There was no significant production of labeled lactate in this experiment with blood.
L-[1-13C]Lactate Transport into Tumors and Tumor Cell Uptake
In a 13C spin echo experiment (S180, TE = 310 ms, b = 10 s mm−2, Fig. 1a) there was an increase in the echo/FID ratio following i.v. injection of hyperpolarized [1-13C]lactate (Fig. 3a,b), which was similar to what we have observed previously in this tumor model . Because of the delay between injection and signal acquisition, it was not possible to measure the echo/FID ratio immediately following injection, when all the lactate is in the blood. The initial ratio was 0.12 ± 0.04, at 8 s post-injection, and this increased to 0.20 ± 0.04 by 40 s, which corresponds to an increase in the apparent T2 relaxation time of the hyperpolarized 13C label, estimated using Eq. (1), from 150 ± 10 ms to 210 ± 70 ms (P < 0.01, n = 14). This is similar to T2 difference of 60 ms between injected lactate and lactate labeled by injected pyruvate measured using the D180 pulse sequence (Table 2). Thus, the increase in the echo/FID ratio with time following lactate injection could be explained by extravasation of lactate into extravascular space with longer T2, the increase in ratio reflecting differences in the rates of signal loss between vascular lactate and lactate that has left the vasculature. Apparent R1 measured for the echo signal (0.054 ± 0.010 s−1) was lower than that for the FID signal (0.063 ± 0.009 s−1, P < 0.01, and n = 14). A similar increase in the ratio was observed for [13C]HP001 (Fig. 3b), with the echo/FID ratio increasing by 0.1 between 8 and 40 s (n = 2, Fig. 3b).
The increase in the lactate echo/FID ratio might also reflect, at least in part, tumor cell uptake. We investigated this by examining the effects on the echo/FID ratio of inhibiting [1-13C]lactate transport, using either an MCT inhibitor (4-CIN) or by competing the labeled lactate with unlabeled lactate. Increasing the concentration of injected [1-13C]lactate from 45 to 90 mM had only a minor effect on the behavior of the ratio (Fig 3c). However, when injection of hyperpolarized [1-13C]lactate was preceded immediately by injection of either the MCT inhibitor, 4-CIN (150 mg kg−1), or 200 μL 0.18 M unlabeled lactate or by i.p. injection of 200 μL 2 M unlabeled lactate, both of which led to a blood lactate concentration that was 3× the labeled lactate concentration (see Methods section), the difference in the ratio between 8 and 40 s after injection was decreased by 30% (P < 0.05; Fig. 3c). Injecting 4-CIN 30 min before injection of the hyperpolarized [1-13C]lactate, however, had minimal effect (Fig. 3c). A trend towards lower R1 values for both the echo (0.049 ± 0.008 s−1) and the FID (0.057 ± 0.012 s−1) were observed following inhibition of lactate transport, although this was not significant. No significant change in [13C]HP001 kinetics was observed as a result of 4-CIN inhibition of the MCT (n = 2, data not shown).
Spin Echo Measurements of 13C Label Exchange Between Pyruvate and Lactate
The FID signal in the spin echo experiment showed substantial labeling of the endogenous lactate pool following i.v. injection of hyperpolarized [1-13C]pyruvate, as has been observed previously in this tumor model (Fig. 4a) . In accordance with a previous report , the spin-echo signal from pyruvate was reduced more than corresponding lactate signal, consistent with the observed pyruvate being predominantly in the extracellular space. MCT inhibition did not have an effect on the apparent T2s and ADCs estimated for both lactate and pyruvate from the echo/FID ratios during the first 15 s of the isotope exchange time courses and these values were similar to those measured using multiple echo times or b-values although a slightly lower T2 was estimated for pyruvate (compare Tables 2 and 3). Correction of the hyperpolarized [1-13C]pyruvate and lactate echo amplitudes for signal loss due to T2 relaxation and diffusion using the T2, ADC, and S0 values given in Table 2 gave a good match with the FID signal amplitudes when the diffusion-weighted spectra were corrected (compare Fig. 4a,b) but tended to underestimate slightly the echo amplitudes when the T2-weighted spectra were corrected. This showed that signal loss in the echoes could largely be accounted for by diffusion and T2 relaxation. The echo/FID ratio was found to be nearly constant for lactate between 8 and 40 s following injection of hyperpolarized [1-13C]pyruvate, regardless of the T2- or diffusion-weighting (Fig. 4c,d), and was much higher than the echo/FID ratio for pyruvate, consistent with the labeled lactate being largely intracellular and the pyruvate intravascular. This is in contrast to the increase in the ratio observed following injection of hyperpolarized [1-13C]lactate and suggests that the distribution of lactate labeled intracellularly, following injection of hyperpolarized [1-13C]pyruvate, between the intravascular and extravascular spaces does not change appreciably during this time period. In contrast to the stable echo/FID ratio observed for lactate, there was an approximately linear increase (2.5 ± 1.4) × 10−3 units s−1 over the first 30 s (n = 4) in the ratio observed for pyruvate, using either a long echo time (TE = 310 ms; b = 7 s mm−2; Fig. 4c) or moderate diffusion weighting (TE = 155 ms; b = 303 s mm−2; Fig. 4d). The MCT inhibitor, 4-CIN, resulted in a decrease in this slope to (0.6 ± 1.0) × 10−3 units s−1 over the first 30 s (P < 0.05, n = 4). There was no change in the lactate echo/FID ratio. The MCT inhibitor also resulted in and ∼40% decrease in the measured rate constant (kP) for hyperpolarized 13C label flux between pyruvate and lactate (summarized in Table 3), which is similar to that reported previously for this inhibitor . The differential loss of the pyruvate and lactate echo signals, with greater loss of the pyruvate signal at longer echo times and with increased diffusion-weighting, leads to overestimation of the apparent exchange rates (Table 3). Correction of the echo amplitudes, using the measured apparent T2s and diffusion coefficients for lactate and pyruvate (Table 2), decreased the fitted exchange rate constants to values that were close to those determined using the FID signal (Table 3), particularly when diffusion-weighted data were used.
|kP (s−1)||R1 (s−1)|
|Exchange modela||Control (n = 4)||MCT (n = 4)||Control (n = 4)||MCT (n = 4)|
|FID||0.13 ± 0.03||0.08 ± 0.04*b||0.043 ± 0.011||0.041 ± 0.004|
|Echo310–7||0.46 ± 0.14||0.25 ± 0.15*||0.035 ± 0.004||0.036 ± 0.001|
|Echo155–303||0.31 ± 0.07||0.19 ± 0.09*||0.036 ± 0.003||0.036 ± 0.002|
|Adj Echosc||0.15 ± 0.04||0.08 ± 0.04*||0.041 ± 0.008||0.044 ± 0.007|
|T2 (s)d||ADC (×10−3 mm2 s−1)|
|Lac||0.28 ± 0.05||0.26 ± 0.3||0.6 ± 0.2||0.5 ± 0.2|
|Pyr||0.11 ± 0.01||0.12 ± 0.03||2.0 ± 0.1||1.7 ± 0.5|
Similar results were obtained using a volume coil and a multiecho experiment (the echo/FID ratios are shown in Fig. 4e). The larger decrease in pyruvate signal observed at longer TE times and with increased diffusion weighting resulted in higher apparent exchange rates (Fig. 4f). The kP for the TE and b value combination of 172 ms and 196 s mm−2 was 180 ± 20% of that determined using the FID signal. The echo/FID ratios were higher when a volume coil was used; possibly reflecting improved signal refocusing (compare Fig. 4c–e) although direct comparison was not possible due to the different refocusing scheme used.
In previous studies with the EL4 lymphoma model used here we showed that multiexponential T2 relaxation behavior, measured using a multiecho pulse sequence with echo spacing of 10 ms, was observed for pyruvate and lactate following injection of hyperpolarized [1-13C]pyruvate  and that in a spin echo experiment (TE = 310 ms) the lactate echo/FID ratio showed a progressive increase following the injection of hyperpolarized [1-13C]lactate . Measurements made in this study of the T2 relaxation times and ADC values for injected pyruvate and lactate show that the extravascular species have longer T2 relaxation times and lower ADC values and therefore that the progressive increase in the lactate echo/FID ratio observed previously  can be explained by extravasation of the injected lactate. The T2 relaxation times measured here (Table 2), using the S180 and D180 pulse sequences (TEs 100–600 ms), after injection of hyperpolarized [1-13C]pyruvate or [1-13C]lactate showed only single components which, with the exception of lactate that was labeled by exchange with injected pyruvate, had T2s comparable with the shorter T2 components measured previously . This is consistent with the proposal that these species are predominantly intravascular at this time point. The lactate labeled by exchange with the injected hyperpolarized [1-13C]pyruvate, however, had a significantly longer T2, consistent with this species being predominantly intracellular. These assignments were also supported by the fitted S0 values (Eq. (2)). Injected lactate, and particularly pyruvate, had S0 values that were <1, reflecting the presence of a pool with very short T2. The amplitude of the missing signal for pyruvate (∼25%; Table 2) is similar to that reported previously to be removed using vascular saturation pulses  and could therefore reflect very fast flowing spins. The assignment of these species to the intravascular and extravascular spaces was also supported by measurements of their ADCs. The injected pyruvate and lactate had significantly higher ADC values than the lactate labeled by exchange, consistent with the former being predominantly intravascular and the latter intracellular. The measured diffusion coefficients for “intracellular” lactate are similar to those reported for water in tissue , but slightly higher than those measured previously for intracellular lactate using proton spectroscopy, ∼0.2 × 10−3 mm2 s−1 (28,32,33), which may be due to a contribution to the observed signal from extracellular lactate. A lower diffusion coefficient, ∼0.3 × 10−3 mm2 s−1, was observed for lactate when the echo time was increased to 240 ms and longer diffusion times were used with b = 730 s mm−2 (Fig. 4e). The values for injected pyruvate and lactate were an order of magnitude lower than those expected for water in flowing blood, ∼20 × 10−3 mm2 s−1 . This may be explained by the long echo time used, which will suppress signal from the fast flowing spins and thus bias the signal toward more slowly moving and extravascular molecules. The T2 and ADC measurements for “extracellular” [13C]HP001 are broadly in agreement with the results for injected pyruvate and lactate.
While the increase in the lactate echo/FID ratio following injection of hyperpolarized [1-13C]lactate  can be explained by extravasation of the molecule, it was not clear how much of this increase was due to cell uptake. We show here that inhibition of lactate transport into the cell, either by using an MCT inhibitor (4-CIN) or by competing the injected hyperpolarized 13C-labeled lactate with unlabeled lactate, resulted in a significant change in the echo/FID ratio curve, confirming that a part of the increase in the ratio is due to lactate transport into the cell. Confirmation that the injected 4-CIN was effective in inhibiting the MCT was obtained by showing that it also inhibited exchange of hyperpolarized 13C label between injected [1-13C]pyruvate and endogenous lactate (Table 3); the exchange rate constant, kP, being decreased by ∼40%. MCT activity is known to play a significant role in the observed pyruvate-lactate exchange in vivo [35, 36] and we have shown previously in EL4 cells in vitro that the rate of label exchange between injected pyruvate and endogenous lactate is affected by both MCT and lactate dehydrogenase activity . In a spin echo experiment with injected hyperpolarized [1-13C]pyruvate, the increase in the pyruvate echo/FID ratio was also decreased by 4-CIN, again suggesting that at least part of the increase in the ratio is due to cell uptake. Therefore, the simultaneous acquisition of both the FID and echo signals may help to determine the relative contribution that the activities of the MCTs and LDH make to the observed exchange kinetics. The echo/FID ratio for lactate in this experiment with injected hyperpolarized [1-13C]pyruvate was almost constant, suggesting that there was little washout of labeled lactate during the measured isotope exchange time course (8–40 s), which can be explained by competition of the lactate labeled by exchange with the large endogenous unlabeled lactate pool [8, 24]. This is consistent with previous PET studies using 11C-pyruvate, with tumors showing trapping of label in the endogenous lactate pool [37, 38], although unlike in 13C MRS the separation between the injected molecule and the products was not directly possible in PET studies.
The MCT inhibition data strongly suggest that some of the increase in the lactate and pyruvate echo/FID ratios is due to cell uptake of these metabolites. However a slower loss of blood signal in the 4-CIN treated animals, for example due to reduced pyruvate and lactate uptake by other tissues, could also lead to a reduction in the rate of increase in the echo/FID ratio for these molecules. Moreover, the overall shape of the echo/FID time course observed with HP001 showed that most of the increase in the lactate and pyruvate echo/FID ratios is due principally to their extravasation rather than cell uptake. Increasing the diffusion-weighting or lengthening the echo time might increase the contribution of cell uptake to the increase in the echo/FID ratio, but this would come at the cost of a decrease in signal-to-noise. Assuming that most of the pyruvate 13C label entering the cell is rapidly exchanged with endogenous lactate and that the relaxation time and diffusion coefficient measured for pyruvate are therefore representative of extracellular metabolites, a reduction of the pyruvate echo signal to <1% (echo/FID < 0.01) would require an echo time of ∼720 ms or b = 1700 s mm−2, at which point the remaining lactate echo signal would be ∼10% of the FID signal.
The measured apparent isotope exchange rate between injected hyperpolarized [1-13C]pyruvate and endogenous lactate is strongly dependent on the pulse sequence used for signal acquisition (Table 3) due to differences in apparent T2 and ADC values between intravascular, extravascular, and intracellular pyruvate and lactate and therefore care is needed when comparing results between studies. While the differences could be accounted for by correction of the signal losses due to relaxation and diffusion, nonecho based sequences with minimal diffusion and T2 weighting, may be preferable. However, simultaneous acquisition of both FID and echo signals can provide information on extravasation and cell uptake and therefore can give further insight into the kinetics of hyperpolarized 13C label exchange. This may be particularly important in a clinical setting, where the contribution that systemic delivery and the activities of MCT and LDH make to the observed kinetics of hyperpolarized 13C label exchange between pyruvate and lactate may vary between patients and within an individual patient's tumor before and after treatment.
B.W.C.K. was in receipt of a Cancer Research UK studentship. Hyperpolarizer was provided by GE.
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