Dynamic 1 H imaging of hyperpolarized [1-13 C]lactate in vivo using a reverse INEPT experiment.

PURPOSE: Dynamic magnetic resonance spectroscopic imaging of hyperpolarized 13 C-labeled cell substrates has enabled the investigation of tissue metabolism in vivo. Currently observation of these hyperpolarized substrates is limited mainly to 13 C detection. We describe here an imaging pulse sequence that enables proton observation by using polarization transfer from the hyperpolarized 13 C nucleus to spin-coupled protons.
METHODS: The pulse sequence transfers 13 C hyperpolarization to 1 H using a modified reverse insensitive nuclei enhanced by polarization transfer (INEPT) sequence that acquires a fully refocused echo. The resulting hyperpolarized 1 H signal is acquired using a 2D echo-planar trajectory. The efficiency of polarization transfer was investigated using simulations with and without T1 and T2 relaxation of both the 1 H and 13 C nuclei.
RESULTS: Simulations showed that 1 H detection of the hyperpolarized 13 C nucleus in lactate should increase significantly the signal-to-noise ratio when compared with direct 13 C detection at 3T. However the advantage of 1 H detection is expected to disappear at higher fields. Dynamic 1 H images of hyperpolarized [1-13 C]lactate, with a spatial resolution of 1.25 × 1.25 mm2 , were acquired from a phantom injected with hyperpolarized [1-13 C]lactate and from tumors in vivo following injection of hyperpolarized [1-13 C]pyruvate.
CONCLUSIONS: The sequence allows 1 H imaging of hyperpolarized 13 C-labeled substrates in vivo. Magn Reson Med 79:741-747, 2018.

INTRODUCTION

The development of hyperpolarized 13C MRI using dynamic nuclear polarization of 13C‐labeled substrates has enabled imaging of metabolic fluxes in vivo 1, 2. 13C‐labeled pyruvate has been the most widely used substrate because it plays a central role in carbohydrate metabolism, it is relatively easy to polarize, and the long C1 carbon T1 (∼30 s in vivo), which makes the polarization relatively long lived, means that there can be substantial delivery and metabolism of the labeled pyruvate within the lifetime of the polarization 3. Imaging of hyperpolarized 13C‐labeled substrates requires an extra RF transmitter and receiver, in addition to the proton channel, and a more powerful gradient setup because the gyromagnetic ratio of 13C is one fourth that of 1H. In addition, the smaller gyromagnetic ratio of 13C means that the signal‐to‐noise ratio (SNR) is lower than for 1H and the transient nature of the 13C hyperpolarization means that signal averaging cannot compensate for this. Detection of hyperpolarized methyl protons in lactate would give, for the same level of polarization, a significant increase in SNR compared with direct detection of the 13C‐labeled C1 carbon. However, direct hyperpolarization of 1H is difficult due to its relatively short T1; the T1 of the lactate methyl protons in vivo at 4.7 T is about 1.7 s 4. Therefore, the feasibility of transferring nuclear spin polarization from the hyperpolarized 13C nucleus to 1H has been explored. Frydman and colleagues 5, 6 used a spatially encoded ultrafast Heteronuclear Single Quantum Correlation experiment for 1H detection of hyperpolarized 13C nuclei in high‐resolution solution experiments in vitro. Sarkar et al. 7 used a reverse insensitive nuclei enhanced by polarization transfer (INEPT) sequence for proton detection of hyperpolarized 15N choline, and Harris et al. 8 used a spatially selective variant of this experiment to monitor the kinetics of choline phosphorylation catalyzed by choline kinase in vitro. Recently, Dzien et al. 9 used a reverse INEPT sequence to study pyruvate decarboxylase activity in cultures of S. cerevisiae following injection of hyperpolarized [U‐2H3, 2‐13C]pyruvate. Chekmenev et al. 10 used a refocused INEPT sequence in spectroscopic studies in solution to transfer hyperpolarization from 13C to 1H in [1‐13C]succinate‐d2 and in 2,2,3,3‐tetrafluoropropyl 1‐13C‐propionate‐d3, and Truong et al. 11 used the same sequence, in conjunction with 2D fast steady state free precession 1H imaging, to image hyperpolarized 2‐hydroxyethyl‐13C‐propionate‐d2,3,3 in a phantom. Mishkovsky et al. 12 described spectroscopic studies in vivo, in which a heteronuclear polarization transfer sequence was used to acquire localized 1H spectra of hyperpolarized [1‐13C]acetate in rat brain, in which polarization was transferred from the carboxyl carbon to the methyl protons. We demonstrate here dynamic imaging of the conversion of hyperpolarized [1‐13C]pyruvate to lactate in tumor‐bearing mice in which labeled lactate in the tumor was detected via its methyl protons using a modified reverse INEPT experiment, in which a double dual‐spin echo sequence ensured acquisition of a fully refocused echo (Fig. 1).

Figure 1

Lactate dehydrogenase catalyzes exchange of hyperpolarized 13C label between injected hyperpolarized [1‐13C]pyruvate and the endogenous lactate pool. Hyperpolarized [1‐13C]lactate is detected by transferring hyperpolarization from the C1 carbon to the spin‐coupled (J = 4.1 Hz) methyl protons in a reverse INEPT experiment.

METHODS

Transfer of Polarization from Lactate 13C1 to the Methyl Protons

We first discuss the use of a reverse INEPT sequence to transfer the longitudinal polarization of a single spin‐1/2 nucleus of isotopic species S to transverse polarization of a set of N magnetically equivalent spins‐1/2. The conventional reverse INEPT pulse sequence has the following form:

In the absence of relaxation, and assuming infinitely short pulses, the polarization transfer amplitude from the S‐spin to the N magnetically equivalent I‐spins is given by the functions 13, 14: where the delays are expressed as angles . If the initial 13C polarization level is denoted , the maximum level of I‐spin polarization and the optimal values of the time variables are given by

The case N = 1 is relevant to polarization transfer from lactate 13C1 to the C2 proton. In the best case, the initial 13C polarization level, p , is preserved upon transfer to the C2 proton, leading to an enhancement in hyperpolarized magnetization by a factor , taking into account the relative gyromagnetic ratios. The case N = 3 is relevant to polarization transfer from lactate 13C1 to the methyl protons. In the best case, the methyl protons acquire a polarization of 0.385 p . The hyperpolarized magnetization is therefore enhanced by the factor , taking into account the number of polarized protons and the relative gyromagnetic ratios. Furthermore, optimal transfer to the methyl protons occurs at a much shorter interval, assuming equal J‐couplings. In fact, the coupling constant between the C1 carbon and the C3 methyl protons in [1‐13C]lactate is larger than the coupling constant with the C2 proton (3.2 Hz versus 4.1 Hz) 15. In the absence of relaxation, the optimal value of is therefore approximately three times shorter, and the achievable 1H magnetization 15% larger, when the methyl protons are targeted, compared to the C2 proton. Because short pulse sequence intervals generally lead to smaller relaxation losses, the lactate methyl protons are a more promising target for polarization transfer than the C2 proton.

Pulse Sequence

The pulse sequence (Fig. 2A) starts with a saturation module on the proton resonances, so that unwanted signals from water and lipids are suppressed, followed by a reverse INEPT preparation module, after which prephasing gradients are applied on both readout and phase encoding axes, followed by a symmetric echo‐planar acquisition train 16. The transmission coil in our setup could not be used to pulse simultaneously on 1H and 13C and therefore there was a delay between the 1H and 13C pulses, which otherwise would happen at the same time in a conventional INEPT sequence. In the modified INEPT preparation sequence, the 1H and 13C coherences evolve with the same phase as in a conventional INEPT sequence at each of the 90 ° pulses and at the end of the preparation period (Fig. 2B). If relaxation effects are neglected, maximum polarization transfer occurs when where is the center‐to‐center delay between a pair of 13C and 1H pulses. In order for spin echoes to be formed at the time of the second 13C 90 ° pulse in the reverse INEPT module, when magnetization is flipped back along the z axis, and at the end of this module (at the end of ) (Fig. 2B), the timing must fulfill the following conditions:

Figure 2

(A) Pulse sequence for transferring hyperpolarization from the C1 carbon to the methyl protons of lactate and imaging of the resulting hyperpolarized proton signal. (B) Pulse sequence for the double dual‐spin echo INEPT (reverse INEPT) module shown in panel A. Shorter and longer bars refer to 90 ° and 180 ° pulses, respectively. The 90 ° pulse on 1H is the 1H excitation pulse. The first and second 90 ° pulses on 13C are the 13C excitation and flip‐back pulses, respectively. The phases of the RF pulses are, in the order as displayed, . (C) Evolution of 13C and 1H y magnetizations in [1‐13C]lactate during the reverse INEPT module. Three simulations are shown with: no relaxation, 1H and 13C T1 and T2 relaxation at 3T, and 1H and 13C T1 and T2 relaxation at 7T. The y component of the methyl group proton magnetization is shown.

Equations 2 and 3 determine the values of . The interval was kept to a minimum and was determined by the length of the 13C flip‐back and 1H excitation pulses. The total duration of the reverse INEPT module, from the first 13C pulse to the echo formed at the end of the module, was 278 ms.

The saturation module consisted of a 4‐ms 90° sinc pulse, with a bandwidth of 8 kHz, followed immediately by a spoiler gradient in the slice direction. A 4‐ms sinc‐shaped pulse was designed with the SLR algorithm 17 for both excitation and flip‐back of the 13C coherences (the first and second 90° pulses on 13C). The bandwidth was 600 Hz to allow selective excitation of [1‐13C]lactate without disrupting the [1‐13C]pyruvate polarization. A sinc‐shaped 1H pulse was used for selective excitation of the lactate C3 methyl protons (the 1st 90° pulse on 1H). The bandwidth was 1500 Hz to avoid excitation of the C2 and water protons. The 13C and 1H magnetizations were inverted using 10 ms adiabatic hyperbolic‐secant pulses 18. The bandwidth of the 13C pulses was 8 kHz, so that even far off‐resonance [1‐13C]pyruvate magnetization (far from the magnet isocenter) would experience full inversion and the hyperpolarization would not be destroyed by the pulses. For the 1H pulses, the bandwidth was only 1000 Hz to avoid inversion of the C2 proton resonance (approximately 850 Hz from the C3 proton resonance).

The dual‐spin echo design was required because an adiabatic pulse results in a non‐linear phase change across the swept frequency range, which can only be cancelled by another adiabatic pulse with the same waveform and RF power 19, 20. This sequence also ensures that the spin echo resulting from phase evolution induced by local field variations coincides with complete polarization transfer, under conditions where the 1H and 13C pulses cannot be applied simultaneously.

Simulation of the Effects of Relaxation

Evolution of the 13C and 1H polarizations during the reverse INEPT preparation block was simulated in the weak‐polarization limit using the SpinDynamica platform (available online at www.spindynamica.soton.ac.uk) in Wolfram Mathematica (version 10.4; Wolfram Research, Inc., Champaign, Illinois, USA). For simplicity, shaped pulses were treated as being infinitely short and relaxation losses during the pulses were neglected. A relaxation model of uncorrelated random fields was used.

Spectroscopic Experiments with [1‐13C]Lactate at Thermal Equilibrium

The validity of these simulations was tested experimentally by implementing the reverse INEPT experiment on a high‐field (14.1T), high‐resolution NMR spectrometer (Bruker Spectrospin Ltd., Coventry, United Kingdom), where the higher sensitivity allowed observation of transfer of thermal 13C polarization into 1H. Experiments were performed with 1 M [1‐13C]lactate in 100% D2O using a 5‐mm 1H/broadand inverse detection probe (Bruker Spectrospin Ltd.). To eliminate signal originating from direct proton excitation, the pulse sequence was phase cycled, wherein alternate acquisitions the phases of the initial 90° pulse and the receiver were shifted by 180°. T1 relaxation times were measured with an inversion recovery sequence (n = 1, TR1H = 25.6 s, TR13C = 300 s). The time between the 90 and 180 pulse was varied between 0.2 s to 25.6 s for the 1H acquisitions and from 2.34 s to 300 s for the 13C acquisitions. T2 relaxation times were measured with a Carr‐Purcell‐Meiboom‐Gill sequence (n = 1, TR1H = 15 s, TR13C = 90 s). The minimum echo time was 10.054 ms and over 16 acquisitions the number of echoes was increased to 1000 (TEmax = 10.054 s).

MR Scanner

Experiments were performed on a 7T Agilent scanner (Agilent, Palo Alto, California, USA) with a 42‐mm diameter 1H and 13C transmit/receive volume coil (Rapid Biomedical, Rimpar, Germany).

Phantom Experiments

A 60‐µL [1‐13C]lactate sample containing 58 mg 50% wt/wt [1‐13C]lactate solution (Sigma‐Aldrich, St. Louis, Missouri, USA), 15 mM OXØ63 (GE Healthcare, Amersham, United Kingdom), 1.2 mM Dotarem gadoterate meglumine (Dotarem; Guerbet, Roissy, France), and 20 µL 1/10 vol/vol dimethyl sulfoxide (Sigma‐Aldrich) was hyperpolarized for 2 h using a Hypersense polarizer (Oxford Instruments, Abingdon, United Kingdom) at 1.2 K in a magnetic field of 3.35T with microwave irradiation at 94.116 GHz. The hyperpolarized sample was then dissolved in 4 mL superheated phosphate‐buffered saline, and 0.5 mL was injected into an 18‐mm inner diameter sphere filled with water. Two spectra were acquired using the pulse sequence shown in Figure 2, but without the imaging gradients (Fig. 3A). The delay between the INEPT preparation module and the beginning of signal acquisition was 170 ms, calculated from the center of the 90° 1H excitation pulse, which was set at the C3 1H resonance frequency (Fig. 2B). Data were acquired into 2048 points covering a bandwidth of 12.5 kHz. In a second experiment, hyperpolarized [1‐13C]lactate was injected and a series of echo planar images were acquired from the C3 1H resonance, with a time resolution of 2 s and starting 2 s after the completion of the lactate injection (a single image is shown in Fig. 3B). The receiver bandwidth was 125 kHz and the echo spacing 400 µs. A field of view of 4 × 4 cm2 covered a 32 × 32 data matrix, and the k‐space center was acquired after only four echoes to minimize the echo time (173 ms). A 1H fast spin echo image was acquired (256 × 256, 4 × 4 cm2, slice thickness 80 mm) to provide a positional reference.

Figure 3

Polarization transfer from 13C to 1H in thermally polarized [1‐13C] lactate. (A) 90 ° pulse and acquire 1H spectrum. (B) Transfer of polarization from 1‐13C to 3,3,3‐1H3 using the reverse INEPT sequence. Phase cycling ensured that only transferred polarization was observed. (C) Spectrum acquired using the reverse INEPT sequence with no 13C pulses. All spectra are the sum of 32 transients. The reverse INEPT spectra were acquired with TR = 90 s to allow full 13C relaxation; the directly detected 1H spectrum was acquired with TR = 15 s to allow full 1H relaxation.

Tumor Model

Animal experiments were performed in compliance with a project license issued under the Animals (Scientific Procedures) Act of 1986. Protocols were approved by the Cancer Research UK, Cambridge Institute Animal Welfare and Ethical Review Body. EL4 lymphoma cells (5 × 105) were injected subcutaneously into the lower flank of female C57BL/6J mice, and the resulting tumor was allowed to grow for 8 days, when it was >1 cm in diameter.

Dynamic Imaging In Vivo

The mouse was fasted for 6 hours before imaging 21 and warmed at 32°C 1 h before induction of anesthesia using 1.5%–2.5% isoflurane. The [1‐13C]pyruvate sample contained 44 mg [1‐13C]pyruvic acid (CIL, Tewksbury, Massachusetts, USA), 15 mM OXØ63 and 1.4 mM Dotarem and was hyperpolarized using the Hypersense polarizer. Before injection, it was dissolved rapidly in 6 mL buffer containing 40 mM Tris, 185 mM NaOH, and 100 mg/L ethylenediaminetetraacetic acid heated to 180°C and pressurized to 10 bar. The injection took 8 s, and imaging started 10 s after completion of the injection when a substantial amount of [1‐13C]lactate had already been generated from the injected [1‐13C]pyruvate. Images were acquired every 2 s, and a total of 30 images were acquired, with the seventh acquisition used as a reference for echo planar imaging phase correction. Ninety‐degree pulses were used for 13C excitation and flip‐back so that all of the [1‐13C]lactate polarization produced from the injected [1‐13C]pyruvate during each 2 s interval was detected in the 1H image. A 90° flip angle was used for 1H excitation to make full use of the transferred polarization. The same acquisition parameters were used for in vivo and phantom imaging.

T2‐weighted proton fast‐spin echo images (16 slices, slice thickness = 2 mm) with a 128 × 128 data matrix covering a 4 × 4 cm2 field of view were acquired to provide a positional reference.

Image Reconstruction

Phase correction was performed using the reference image data, as described by Zhou 16. The partial k‐space was then zero‐filled from 20 × 32 to 32 × 32 before Fourier Transformation. Phase correction and image reconstruction were performed in MATLAB (MathWorks, Natick, Massachusetts, USA).

RESULTS

Hyperpolarized 13C label is exchanged between injected hyperpolarized [1‐13C]pyruvate and the endogenous unlabeled lactate pool in the reaction catalyzed by lactate dehydrogenase (Fig. 1A) 1. Polarization was transferred from the C1 carbon to the indirectly coupled C3 methyl protons (J = 4.1 Hz) using a reverse INEPT sequence (Fig. 2B) and the resulting hyperpolarized 1H signal imaged using an echo planar imaging readout (Fig. 2A). Simulations showed that evolution of the magnetization of the three magnetically equivalent methyl 1H spins in [1‐13C]lactate under the four‐spin coupling Hamiltonian, relative to the initial magnetization of the hyperpolarized 13C spin, enhances the hyperpolarized magnetization by a factor of 4.6 and that this is decreased by relaxation to a factor of 1.9 at 3T, and to 0.6 at 7T (Fig. 2C). The simulations that included relaxation were performed using the following published values for T1 and T2 at 3T and 7T: (7 T) = 300 ms 22, (3 T) = 520 ms 23, (7 T) = 100 ms 24, (3 T) = 256 ms 25, (3T) = 45 s 26, and (4.7T) = 1.73 s 4. These simulations were tested experimentally using thermally polarized 1M [1‐13C]lactate at a high field (14.1T). The T1 and T2 relaxation times of the 1‐13C and 3,3,3‐1H lactate resonances were measured using inversion recovery and CPMG sequences respectively, yielding  = 2.2 ± 0. 1 s,  = 1.6 ± 0.1 s,  = 15.8 ± 0.1 s, and  = 3.5 ± 0. 1 s. Simulation of the reverse INEPT experiment using these relaxation times yielded an enhancement of 0.082, which was in good agreement with a value of 0.084 measured experimentally (compare the methyl peak intensities in Fig. 3A and 3B).

1H spectra and images acquired using the reverse INEPT sequence, following injection of hyperpolarized [1‐13C]lactate into a phantom, are shown in Figure 4. 1H signal in the first acquisition (solid line in Fig. 4A and image shown in Fig. 4B) was approximately 10 times larger than in the second acquisition (dotted line in Fig. 4A and image shown in Fig. 4C) due to depletion of the 13C hyperpolarization by the 90° 13C excitation pulse. The methyl proton resonance had a peak width at half height of about 35 Hz and therefore splitting due to 1H and 13C coupling was not resolved (the methyl proton resonance of [1‐13C]lactate is split into a doublet by coupling to the C2 proton (J = 6.9 Hz) and these doublets are further split into doublets by coupling to the C1 13C (J = 4.1 Hz). In the image, this splitting of the methyl proton resonance will not compromise SNR if the k‐space center is acquired at the time when the spin echo is formed, where the image signal is then the integral of all the in‐phase peaks. The SNR of the spectrum from the first acquisition was 8618, which decreased to 1560 for the second acquisition. The SNR for the first image was 586.4 and only 56.9 for the second. The spectrum and image SNRs were measured as the ratios between maximum and mean signals, respectively, and the standard deviation of the background noise 27. There was no observable excitation of the water resonance, which should be about 1090 Hz away from the lactate methyl proton resonance. The residual signal observed in the second image (Fig. 4C) appeared to be water signal from the injection line. The B0 field was only shimmed over the spherical phantom. Water protons in the injection line may therefore have been off‐resonance and excited by the transition band of the proton inversion pulses. The residual signal was spatially displaced from the injection line in the phase encoding direction, consistent with it being from off‐resonance signal. The hyperpolarized [1‐13C]lactate solution (0.5 mL) was injected into the bottom of the sphere phantom, which contained 3 mL of water, and the first acquisition started only 2 s after completion of the injection. The methyl proton signals were concentrated, therefore, at the bottom of the phantom.

Figure 4

Phantom experiments with hyperpolarized [1‐13C]lactate. (A) 1H spectra acquired after injection of hyperpolarized [1‐13C]lactate into the phantom, where the 1H excitation was set to the methyl proton resonance frequency. The second spectrum (dotted line) was acquired 5 s later. (B, C) Sequential methyl group 1H images acquired after injection of hyperpolarized [1‐13C]lactate into the phantom. The lactate proton images, which are rendered in color, have been overlaid on a fast‐spin echo water 1H image, which has been rendered in grayscale.

Dynamic images of the methyl proton resonance of hyperpolarized [1‐13C]lactate were acquired using the reverse INEPT sequence following injection of hyperpolarized [1‐13C]pyruvate into an EL4 tumor‐bearing mouse (Fig. 5A). A series of images are shown in Figure 5A and an overlay of the first image, rendered in false color, on an anatomic image acquired using a 1H fast spin echo sequence is shown in Figure 5B. The 1H signals from hyperpolarized [1‐13C]lactate were observed at the base of the tumor and adjacent to the body of the animal. We have observed a similar distribution of labeled lactate in this tumor model using direct 13C detection (data not shown). Unlike in the phantom, the hyperpolarized 13C and 1H signals in the tumor are sustained over time by inflow of hyperpolarized [1‐13C]pyruvate into the tumor from the rest of the animal.

Figure 5

(A) Dynamic 1H images of the lactate methyl protons acquired using the reverse INEPT sequence at the indicated times following injection of hyperpolarized [1‐13C]pyruvate into a tumor‐bearing mouse. The first image (at 0 s) was acquired 2 s after completion of the injection, which took a total of 10 s. (B) The image in panel A acquired at 0 s and rendered in false color overlaid on a fast spin echo 1H image of tissue water, which is in grayscale. The tumor is outlined.

DISCUSSION

The reverse INEPT pulse sequence transfers 13C nuclear spin polarization in hyperpolarized [1‐13C]lactate from the 13C1 carbon to the spin‐coupled methyl protons. The SNR for the lactate 1H image acquired in vivo was 17 (Fig. 5B), which is comparable to SNR values obtained previously in this tumor model with direct 13C detection, where the SNR of 13C images of [1‐13C]lactate acquired using a 90° pulse and summed over a 20‐mm thick slab were between 13 and 20 28. However, these 13C images were acquired using a 20‐mm‐diameter surface coil placed around the tumor, whereas the 1H images shown here were acquired using a 42‐mm‐diameter 1H and 13C transmit/receive volume coil. Given the difficulty in ensuring equal coil efficiencies and to generalize the relevance of the measurements shown here for other field strengths, the experiment was simulated. Simulation using published values for the T1 and T2 of the 1H and 13C nuclei in lactate in vivo showed that transferring 13C hyperpolarization into the methyl protons enhances the hyperpolarized magnetization by a factor of 4.6, and that this is decreased by relaxation to 1.9 at 3T, and to 0.6 at 7T, which was the field strength used here. The amplitude of the detected NMR signal depends on this polarization but is also proportional to the precession frequency, because it is generated by electromagnetic induction in the receiver coil. Assuming identical coil efficiencies, then detecting spin polarization in 1H rather than 13C is beneficial due to the higher gyromagnetic ratio of the proton, γ1H; for a given level of polarization, signal increases as ∼γ2 29 as magnetization is proportional to γ and, given the same magnetization, the current induced in the receiver coil is also proportional to γ. Hence, for the same level of polarization, 1H will generate a signal in the receiver coil that is approximately 16 times larger than that for 13C. With the simulated values for the magnetizations, which includes T1 and T2 relaxation of the 13C and 1H spins, detection of the 13C1 polarization via the methyl protons will increase the signal 7.6 fold at 3T and 2.4 fold at 7T. These numbers were obtained by multiplying the simulated magnetizations by four (the same operation was performed for all the SNR calculations shown below). However, noise also increases with frequency. Noise due to coil resistance, , is proportional to the square root of the frequency of the induced alternating current whereas noise due to sample resistance, , increases quadratically with frequency. Because the overall noise signal is proportional to , the SNR that takes account of sample and coil noise can be calculated as 30 where is the Larmor frequency, a and b are coil geometry parameters, and α and β are weightings for the two sources of noise, where α represents coil noise and β sample noise. Assuming the same coil geometry, the SNR for 1H is 11.3 ( ) times that for 13C when sample noise is neglected (α = 1, β = 0). With the calculated 13C and 1H magnetizations this will give an SNR benefit when detecting 13C hyperpolarization via the methyl protons of 5.4 times at 3T and 1.7 times at 7T (magnetization enhancement ×  , where the denominator is determined by coil noise, which is proportional to the fourth root of the Larmor frequency, as shown in Equation 6). If sample noise dominates (α = 0, β = 1) detection via 1H gives an SNR benefit of only 4 times that of 13C detection if relaxation is ignored and, given the calculated 13C and 1H magnetizations, the SNR benefit would decrease to 1.9 times at 3T and 0.6 times at 7T (4 × magnetization enhancement/4, where the denominator comes from the fact that sample noise is proportional to the Larmor frequency, as shown in Eq. 6). Although sample noise is thought to dominate with relatively large imaging objects at high magnetic fields 31, this is evidently not the only source of noise, because superconducting coils show an increased 1H SNR of 2‐ to 5‐fold at fields between 1.5T and 9.4T when compared with room temperature copper coils 32, 33, 34. Therefore, even at 7T, there may still be a SNR benefit in detecting hyperpolarized [1‐13C]lactate via the spin‐coupled methyl protons. There was some evidence for this in the measurements made here.

The dynamic images (Fig. 5A) showed rapid signal decay as each acquisition sampled effectively all of the hyperpolarized signal from [1‐13C]lactate generated from hyperpolarized [1‐13C]pyruvate in the preceding 2 s. Such a rapid decay has been observed previously in saturation‐recovery experiments, where following injection of hyperpolarized [1‐13C]pyruvate the [1‐13C]lactate produced was sampled with repeated spectrally selective 90° 13C pulses 35. This problem could be addressed by using a preparation module that allows partial transfer of the polarization 36. This would also allow serial observations of the pyruvate resonance, which is not possible with the reverse INEPT sequence, because all of the polarization is effectively transferred following the first application of the pulse sequence. The simulation shown in Figure 2C shows that there is also the potential for shortening the reverse INEPT module, and thus reducing signal loss due to T2 decay, because this may be effected without significantly reducing polarization transfer.

The longer 1H and 13C T2 relaxation times at lower magnetic field strengths will improve the efficiency of polarization transfer and there may be a benefit in going to very low fields because there is evidence that these may be more sensitive for hyperpolarized contrast agents 37. The four‐fold higher 1H gyromagnetic ratio means that there is a four‐fold lower demand on the gradient system, which, with the availability of high‐quality proton receive coils, makes this an attractive technique for clinical translation.

In conclusion, we have demonstrated a reverse INEPT sequence that allows 1H detection of hyperpolarized 13C label exchange between injected hyperpolarized [1‐13C]pyruvate and the tumor lactate pool. Further incorporation of a spectrally selective 1H 90° pulse that flips the magnetization back along the z axis at the end of the reverse INEPT preparation module would allow any 1H imaging sequence to be used for signal detection. The sequence is fully compatible with clinical scanners that are already equipped for hyperpolarized 13C imaging, where the lower field strengths and consequently longer relaxation times should improve sensitivity.