Increasing the sensitivity of hyperpolarized [15N2]urea detection by serial transfer of polarization to spin‐coupled protons

Purpose Hyperpolarized 15N‐labeled molecules have been proposed as imaging agents for investigating tissue perfusion and pH. However, the sensitivity of direct 15N detection is limited by the isotope's low gyromagnetic ratio. Sensitivity can be increased by transferring 15N hyperpolarization to spin‐coupled protons provided that there is not significant polarization loss during transfer. However, complete polarization transfer would limit the temporal window for imaging to the order of the proton T1 (2‐3 s). To exploit the long T1 offered by storing polarization in 15N and the higher sensitivity of 1H detection, we have developed a pulse sequence for partial polarization transfer. Methods A polarization transfer pulse sequence was modified to allow partial polarization transfer, as is required for dynamic measurements, and that can be implemented with inhomogeneous B1 fields, as is often the case in vivo. The sequence was demonstrated with dynamic spectroscopy and imaging measurements with [15N2]urea. Results When compared to direct 15N detection, the sequence increased the signal‐to‐noise ratio (SNR) by a factor of 1.72 ± 0.25, where both experiments depleted ~20% of the hyperpolarization (>10‐fold when 100% of the hyperpolarization is used). Simulations with measured cross relaxation rates showed that this sequence gave up to a 50‐fold increase in urea proton polarization when compared to spontaneous polarization transfer via cross relaxation. Conclusion The sequence gave an SNR increase that was close to the theoretical limit and can give a significant SNR benefit when compared to direct 13C detection of hyperpolarized [13C]urea.


| INTRODUCTION
Magnetic resonance imaging of hyperpolarized isotopically labeled substrates has enabled measurements of metabolic fluxes, pH, and tissue perfusion in vivo. The most commonly used label has been 13 C because of its relatively long polarization lifetime and the availability of 13 C-labeled substrates suitable for investigating metabolism. 1 Hyperpolarized 15 N-labeled substrates have also been investigated, as agents for assessing tissue perfusion (urea 2 and glutamine 3 ) and as pH probes (pyridine derivatives). 4 15 N labeled substrates have the advantage of very long hyperpolarization lifetimes, up to 200 s, and more when kept in 2 H 2 O. 2 However, the 2.5-fold lower gyromagnetic ratio when compared to 13 C (10-fold lower when compared to 1 H) results in lower magnetization and precession frequency and therefore lower sensitivity of detection. For imaging there is also the requirement for larger gradients.
Detection sensitivity can be improved, while still benefiting from the long 15 N polarization lifetime, using sequences such as insensitive nuclei enhanced by polarization transfer (INEPT) to transfer hyperpolarization from 15 N to 1 H immediately before signal acquisition. 5 Reverse INEPT-type sequences have been used previously with hyperpolarized 13 C-labeled substrates to produce hyperpolarized proton spectra [6][7][8][9] and images 10,11 and with hyperpolarized 15 N labeled substrates to produce spectra. [12][13][14] In all of these INEPT-based experiments 100% of the available polarization was used in a single acquisition. To obtain dynamic information the hyperpolarization of the low γ nucleus must be sampled in discrete packets in order to allow repeat measurements, which in the case of direct 15 N or 13 C detection is achieved using small flip angle (FA) pulses. For example, in the case of dynamic perfusion measurements with hyperpolarized [ 15 N 2 ]urea only a portion of the 15 N polarization should be transferred to the urea protons at each measurement. The same is true for measurements of flux in an enzyme-catalyzed reaction, for example exchange of hyperpolarized 13 C label between injected [1-13 C]pyruvate and the endogenous lactate pool.
Several approaches have been taken to achieve partial transfer of polarization. Harris et al 13 used spatially selective coherence transfer to probe different regions of the sample at different times. Barb et al exploited chemical exchange of deuterons in hyperpolarized 15 ND 2 -amido-glutamine with solvent protons to acquire a series of proton spectra from the protonated isotopologue. 12 Dzien et al 7 utilized spontaneous 13 C → 1 H cross-relaxation to detect, in a series of dynamically acquired proton spectra, the production of acetaldehyde from hyperpolarized [U-2 H 3 ,2-13 C]pyruvic acid, in the reaction catalyzed by pyruvate decarboxylase. We have previously described a spectrally selective reverse INEPT sequence in which 13 C hyperpolarization in lactate, which had been produced in a tumor from injected hyperpolarized [1-13 C]pyruvate, was transferred to the methyl protons and imaged. 11 In this case the fully depleted [1-13 C]lactate hyperpolarization was replenished after each transfer by further labeled lactate production from the injected pyruvate. However, to the best of our knowledge, no one has yet demonstrated experimentally partial transfer of hyperpolarization from a low γ to a high γ nucleus, while maintaining the majority of the hyperpolarization in the low γ nucleus. We demonstrate here partial transfer of 15 N hyperpolarization in [ 15 N 2 ]urea to urea protons ( Figure 1) in consecutive acquisitions and subsequent imaging of these protons, as would be required for dynamic imaging of tissue perfusion. The sensitivity of urea proton detection in this experiment was compared with direct 15 N detection and, in simulations using measured cross relaxation rates, with proton detection where polarization is transferred spontaneously from 15 N to 1 H via the nuclear Overhauser enhancement (NOE).

| Solvent exchange of [ 15 N 2 ]urea protons
The exchange rate in a 100 mM [ 15 N 2 ]urea solution was measured at neutral pH using a 14.1 T nuclear magnetic resonance (NMR) spectrometer equipped with a 5 mm BBI probe (Bruker Spectrospin Ltd., Coventry, UK). The water proton resonance was saturated for between 0.1 and 2.6 s and then spectra acquired using a 90° pulse and a bandwidth of 6000 Hz into 8192 complex points. The exchange rate was calculated as described in Ref. 15 15 N and the directly bonded protons is 90 Hz. These protons are in exchange with solvent water between the 90° and 180° pulses was varied between 0.25 and 8 s for the 1 H measurements and between 2.5 and 80 s for the 15 N measurements. T 2 relaxation times were measured with a Carr-Purcell-Meiboom-Gill (CPMG) sequence (n = 1, TR 1H = 10 s, TR 15N = 100 s). The minimum echo time for the 1 H measurements was 0.0125 s, which was increased by iteratively adding more spin echo sandwiches while leaving the inter echo spacing the same until, over six acquisitions, the maximum echo time of 0.4 s was reached. For 15 N T 2 measurements the echo time was varied between 0.0624 and 2 s.

| Polarization measurements
Spectra were acquired using a 90° pulse and a sweep width of 20 kHz into 16 384 complex points from 4 mL of hyperpolarized urea using a 14.1 T NMR spectrometer and a 10 mm BBO probe (Bruker Spectrospin Ltd.) at room temperature. The signal-to-noise ratio (SNR) was compared to that in spectra of the same solution after decay of the hyperpolarization. For these experiments, measurements at thermal equilibrium from [ 13 C]urea were acquired with a pulse repetition time (TR) of 225 s and were the sum of 237 averages. For [ 15 N 2 ]urea the TR was 1000 s and 32 averages were acquired. The thermal polarization: was calculated to be 4.87 × 10 −6 for 15 N and 12.08 × 10 −6 for 13 C, assuming a temperature of 300 K, where ℏ is the reduced Planck's constant, ɣ the gyromagnetic ratio, k B Boltzmann's constant, and B 0 = 14.1 T. The hyperpolarization, P hyp , was calculated from the SNRs of the hyperpolarized and thermal measurements using: The measured polarization was 6.2% for [ 13 C]urea and 2.3% for [ 15 N 2 ]urea. The value for 15 N was lower than the 5% reported previously, 2 and the value for 13 C was between a value of 3% reported in vivo 17 and a value of 10% estimated at the time of injection. 18

| Measurement of coil performance
A cylindrical phantom containing 2 mL of 4 M [ 15 N 2 ]urea was placed through the loop of the dual-tuned resonator and 1 H and 15 N spectra acquired with a sweep width of 10 kHz into 2048 complex points with one average using a 2 ms BIR4 90° pulse, with pulse shape parameters as described by Merkle et al. 19 Coil performance at the 1 H and 15 N frequencies was assessed by comparing the SNRs of the two spectra.

| Pulse sequence
The INEPT pulse sequence, 5 which transfers polarization from S to I spins via J-coupling, can be written as: where full transfer of polarization is achieved when τ 1,2 = 1/(4 J). Simultaneous application of 90° pulses to the I and S spins converts an antiphase state of the S spins to an observable antiphase state in the I spins. A later version of this sequence 20 refocuses the I spin magnetization.
In an IS spin system full transfer occurs when all the delays τ 1,2,3,4 are 1/(4 J). Merkle et al 19 later described a sequence that used composite pulses to compensate for B 1 inhomogeneity: These composite pulses were later replaced with modified BIR4 pulses to produce the BINEPT sequence, which is the basis of the pulse sequence described here. (1)

KREIS Et al.
A BIR4 pulse 21 is composed of three sections: adiabatic half-passage in reverse, adiabatic inversion, and adiabatic half-passage. The FA is controlled by two phase jumps Δϕ 1 and Δϕ 2 = −Δϕ 1 before and after the adiabatic inversion segment respectively. The transformation induced by a BIR4 pulse can be described using a composite pulse analogy, where (90 • y 180 • (y+ + ∕2) 90 • y ) is analogous to a BIR4 pulse with phase jumps Δϕ 1 = −Δϕ 2 = π+δ/2. Both the BIR4 pulse and this composite pulse execute a rotation of δ rad about the x axis, although a composite pulse requires a considerably more uniform B 1 field to achieve this transformation. With this simplification, the BINEPT sequence can be written as: where for this example the I spin is proton and the S spin 15 N. For δ = π/2 this is identical with the composite pulse sequence and is analogous to the BINEPT sequence using adapted BIR4 pulses in terms of net rotations; however, the paths taken by the magnetization vectors differ. In this simplified sequence, the phase offset δ of the first 180° pulse on the S spin must be 90° and τ 1-4 must be 1/(4 J) in order to fully transfer polarization from the S to the I spin in a two-spin system. When τ 1 and τ 2 are shortened and the phase offset δ adjusted, some of the magnetization can be returned to the z axis while still transferring some of the polarization. For a simple IS spin system the transferred polarization (P I ) is equal to sin(δ)sin(π J τ)P 0 and the polarization returned to the z axis (P S ) is equal to −cos(δ)cos(π J τ)P 0 , where P 0 is the original polarization, J is the coupling constant between the I and S spins and τ = 2τ 1 = 2τ 2 (see product operator analysis in the Supporting Information Text S1). In all cases, τ 3 = τ 4 = 1/(4 J). For an IS N spin system these terms are P I = N sin(δ)sin(π J τ) and P S = −cos(δ)[cos(π J τ)] N . A similar approach has been described previously in the HINDER sequence (hyperpolarized insensitive nucleus delivers enhancement repeatedly), 22 where spin order is divided between I or S spin polarization by changing the phase, δ, of the second 90° pulse on the S spin and shortening the inter pulse delays in the classical INEPT sequence. To summarize, we have combined the BINEPT and HINDER sequences to give an ImpeRfection RobUst Partial Transfer (IRRUPT) sequence, where δ and τ 1 and τ 2 in the BINEPT sequence can be adjusted to achieve partial polarization transfer. An additional 180 degrees was added to δ to return the remaining magnetization to the positive instead of the negative axis (making P I = −cos(δ) [cos(π J τ)] N positive). The delays between the pulses (τ 1-4 ) and the additional phase offset δ in the segmented BIR4 pulse on the S spin were chosen as described for the HINDER sequence. 22 For two protons coupled with a 90 Hz coupling constant (J) to one low γ nucleus: τ 1 + τ 2 = 0.442/(2 π J) = 782 µs, τ 3 + τ 4 = 1/(2 J) = 5555 µs, δ = 18.050°. The delays were not corrected for the fact that relatively long adiabatic pulses were used instead of hard pulses. With adiabatic pulses in the simulations these parameters resulted in ~20% of the 15 N hyperpolarization being transferred to the coupled protons (J NH = −90 Hz) ( Figure 2). Each adiabatic half passage segment in the BIR4 pulse was 500 μs, giving a total pulse duration of 2 ms The last BIR4 pulse in the sequence flips the proton magnetization onto the z axis. Then, either a simple excitation pulse or a sliceselective 2D-single-shot echo-planar imaging (EPI) sequence was used for proton acquisition. In both cases a 90° pulse was used in order to make maximum use of the transferred polarization.

| Simulation of the IRRUPT pulse sequence
Evolution of the 15  where J NH is the negative heteronuclear coupling constant, 24 Ŝ is the product operator for the 15 N spin, and Î 1 and Î 2 the operators for the two equivalent protons. To simulate the effect of off-resonance excitation, the term was added to the Ĥ 0 Hamiltonian. v 0,15N and v 0,1H are the excitation frequency offsets in Hz. To estimate relaxation losses the PhenomenologicalRelaxationSuperoperator function in SpinDynamica was used with the measured T 1 and T 2 times. An uncorrelated relaxation model was assumed. We define the proton polarization P 1H divided by the depleted 15 N polarization (1-P 15N ) at the end of the transfer block as the efficiency of polarization transfer (efficiency = P 1H /(1−P 15N )), which was typically >80% (Figure 3).

| Polarization transfer in phantom experiments
Polarization transfer experiments were performed on the 7 T scanner using the dual-tuned home-built surface coil. A spherical phantom filled with 3 mL water was positioned in the magnet isocenter. For hyperpolarized acquisitions, 1.5 mL of water were removed and replaced with 1.5 mL hyperpolarized [ 15 N 2 ]urea solution. Spectra were acquired with a 2 ms 90° BIR4 excitation pulse after the polarization transfer block, with TR = 2s, sweep width = 100 kHz, number of points = 4096. Images were acquired with a 2D EPI sequence after the polarization transfer block. Six water presaturation pulses with crusher gradients were used prior to the polarization transfer block for both imaging and spectroscopy experiments. Images were acquired with FOV = 32 × 32 × 1 mm, TR = 1s, bandwidth = 250 kHz, matrix size = 32 × 32.

| Interleaved direct and indirect detection
The SNR benefits of indirect over direct detection were determined by interleaving the two acquisition strategies. After injection of 1.5 mL hyperpolarized [ 15 N 2 ]urea three direct detection 15 N spectra were acquired using 30° pulses followed by three indirect polarization transfer measurements (with no water pre saturation), then three direct detection measurements followed by another three indirect detection measurements. Each spectrum was phase and baseline corrected and then normalized to the standard deviation of the noise in the first 400 points at the downfield end of the spectrum. The indirect detection 1 H spectra were further corrected to account for depletion of polarization due to the prior direct 15 N detection. This was achieved by multiplying spectra 4, 5, and 6 (indirect detection) by three times the reciprocal of the average signal loss between spectra 1 and 2 and between spectra 2 and 3 (direct detection). Spectra 10, 11, and 12 were corrected in a similar way but using a factor calculated from the signal loss between spectra 7 and 8 and between spectra 8 and 9. The 1 H spectra were recorded with a sweep width of 10 kHz into 2048 complex points, an acquisition time of 204.8 ms and a TR of 500 ms The directly detected 15 N spectra were acquired with a nominal 30° FA pulse, sweep width 10 kHz, 2048 complex points, TR = 500 ms, which were the same acquisition parameters as used for the indirect measurements. The SNRs were calculated by integrating a 60 point-wide region containing the peak and dividing it by the standard deviation of the noise in the 400 point-wide region at the downfield end of the spectrum. For this boundary condition the system of differential equations (Equations 5-6) has a solution of the form f (t) = a e −Bt − e −Ct , where, from an initial rate approximation, the cross relaxation rate between 1 H and 15 N ( . 25 This describes the combined cross relaxation between water (intermolecular NOE) and urea protons (intramolecular NOE) and the nitrogen-15 in [ 15 N 2 ]urea and represents an upper limit for the intramolecular relaxation rate. From this an upper limit for the reverse rate ( 15 N to 1 H), σ 21 , was calculated using the relation σ 21 = N 1 N 2 σ 12 , where N 1 /N 2 is the concentration ratio of the nuclei. 26 We were interested in only intramolecular cross relaxation, because this would give the greatest enhancement, N 1 /N 2 = 0.5. In the case of intermolecular cross relaxation the 15 N hyperpolarization would be diluted among the many participating water protons and give a much smaller 1 H enhancement. 26 The measured cross relaxation rates σ 12 and σ 21 were then used to calculate the spontaneous transfer of polarization from 15 N to urea protons that would occur in hyperpolarized [ 15 N 2 ]urea. The degree of transfer was compared with that produced by repeated application of the IRRUPT sequence, calculated using SpinDynamica.

| RESULTS
Implementation of polarization transfer sequences using a surface coil requires the use of inversion pulses that compensate for B 1 inhomogeneity. Furthermore, for dynamic measurements, polarization should be transferred in discrete packets from the hyperpolarized lower γ nucleus, in this case 15 N, to the detected high γ nucleus, in this case 1 H. We have modified the previously described BINEPT sequence, 19 which uses BIR4 adiabatic pulses, 21 for partial and sequential transfer of polarization from 15 N to 1 H (see Methods section for details).

| Simulations of the IRRUPT pulse sequence
The sequence was simulated with the delays and phases used experimentally (τ 1 + τ 2 = 0.442/(2 π J NH ) = 782 µs, τ 3 + τ 4 = 1/(2 J NH ) = 5555 µs, δ = 18.050°), which resulted in 21% of the 15 N polarization being transferred to 1 H and a 1 H z-magnetization that was 1.78 times greater than the initial 15 N z-magnetization ( Figure 2B). Polarization loss due to relaxation is minimal because transfer via the strong coupling (J NH = −90 Hz) is fast (~12 ms) and the simulation ( Figure 2B) showed that this can be neglected. Simulations using measured relaxation rates (Table 1), showed that these values were reduced only slightly, from 79% to 74% for the remaining 15 N magnetization and from 1.78 to 1.67 for the 1 H magnetization. In further simulations the effect of the pulse sequence was simulated for a large range of excitation frequency offsets and pulse amplitudes (Figure 3). Transfer efficiency was preserved for large regions of parameter space, demonstrating the sequence's insensitivity to B 1 inhomogeneity.

| Effect of solvent exchange on urea proton hyperpolarization
The polarization transferred from 15 N to the urea protons will be diluted by exchange with solvent water protons, decreasing the sensitivity of detection. However, this effect is small. The proton exchange rate between urea and water was determined by fitting the peak integrals following saturation I(t sat ) to the equation given by Horska and Spencer 15 : where c is a dimensionless factor, T 1 is the urea proton relaxation time, t sat are the presaturation times, and k the exchange rate. This gave c = (1.14 ± 0.09), k = (1.56 ± 0.15) s −1 , and T 1 = (2.73 ± 0.38) s. The errors are those for the fitting. The measured lifetime for a proton in urea (1/k) was 0.64 ± 0.06 s, which is similar to that measured previously for 1 M urea at pH 7 (0.55 s). 27 The IRRUPT sequence, including the flipback pulse, takes only ~12 ms and therefore the effect of solvent exchange on the urea proton hyperpolarization can be ignored.

| Experimental implementation of the pulse sequence
Partial transfer of polarization from 15 N to urea protons in [ 15 N 2 ]urea using IRRUPT can be used for dynamic spectral acquisition ( Figure 5A) or for imaging ( Figure 5B). The 90 Hz splitting in the urea 1 H spectra is due to the 1 H-15 N coupling. 28 The remaining signal at the end of the dynamic (9) I t sat = c 1 + kT 1 e −  Figure 5A) is residual signal from water protons.

| Comparison of direct and indirect detection of [ 15 N 2 ]urea in interleaved acquisitions
The SNR in the indirect detection spectra recorded immediately after the direct detection spectra was significantly higher ( Figure 6A). There was an ~8 s delay in changing from one pulse sequence to the next. After correcting for depletion of polarization in the preceding direct detection experiment there was a 2.09 ± 0.31(SD)-fold improvement in SNR in the indirect detection experiment ( Figure 6B). Each indirect measurement led to 20% depletion of the 15 N polarization. Comparing spectrum 1 with 2 and 2 with 3, and similarly spectrum 7 with 8 and 8 with 9 shows that each of the direct acquisitions depleted 13 ± 7 (SD)% of the polarization, which corresponds to a FA of 30° (acos(0.87) = 30°). To compare the SNR of the directly and indirectly detected spectra, we corrected the SNR improvement factor of 2.09 ± 0.31 by sin30°/ sin37°, which corrects for the fact that the indirect experiment depleted 20% of the 15 N polarization (cos(37°) = 0.8) whereas the direct detection experiment depleted 13% of the polarization. This gives a corrected improvement in the SNR of 1.72 ± 0.25. The improvement in SNR was less than expected, reflecting a poorer than expected performance of the 1 H circuit in the dual-tuned 1 H/ 15 N transmit/receive surface coil. The ratio of the SNRs in 1 H and 15 N spectra acquired using this coil from thermally polarized 4M [ 15 N 2 ]urea was 59.9. When corrected for the number of contributing nuclei per molecule (four protons, two 15 N nuclei) and the different thermal polarizations calculated using Equation (1) this gave an effective SNR enhancement (ε) of 6.07 when detecting 1 H versus 15 N for a given level of polarization. For example, if the SNR of an 15 N acquisition is 10, the SNR of a 1 H acquisition at the same nucleus concentration and polarization will be 60.7. This value for ε was less than an expected value of 54.92, if coil noise dominates, and a value of 9.86 if sample noise dominates (Equation 10).

| Spontaneous transfer of polarization between 15 N and 1 H in hyperpolarized [ 15 N 2 ]urea
Several studies have reported spontaneous transfer of hyperpolarization from a low γ nucleus to protons via cross relaxation. 29-33 Using the measured cross relaxation rates ( Figure 4B) we simulated transfer of polarization via cross relaxation and compared it with that obtained via J-coupling using the IRRUPT pulse sequence. Polarization transfer via the spin coupling between 15 N and 1 H gave up to a 50-fold higher proton polarization than that obtained via cross relaxation, although inevitably, because it depleted the 15  more rapidly, this was sustained over a shorter period of time ( Figure 7).

| DISCUSSION
The signal detected by a receiver coil increases with the square of the detection frequency, ν 2 . However, coil and sample noise also increase as ν 1/4 to ν, depending on the source of the noise. Therefore, the overall increase in the SNR as a function of ν is given by Wang et al 11 and Hoult and Lauterbur 34 : where 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. Equation 10 can be used when comparing the SNR of a direct detection experiment with a 90° pulse with an indirect detection experiment in which all the available polarization is transferred with perfect efficiency to the coupled high γ nucleus. However, for dynamic measurements only small portions of the hyperpolarization should be used at any one time, either by using a small FA pulse in the direct detection experiment or by using partial polarization transfer in the indirect experiment. In this situation we also need to consider the residual polarization left following each excitation because this represents the longitudinal magnetization available for subsequent excitation. 35 The detectable transverse magnetization in a direct detection experiment depends on the FA thus: Expressing the detectable magnetization in terms of the z-magnetization used M hyp z used then: In an indirect detection experiment the detected transverse magnetization in the higher γ nucleus is proportional to the gain from the higher precession frequency (γ 1H /γ 15N ) multiplied by the z-magnetization used M hyp z used in the lower γ nucleus: Comparing Equations (12) and (13) shows, that if then transferring polarization to the higher γ nucleus will result in greater transverse magnetization than would be obtained by direct detection of the lower γ nucleus. However, the actual gain in SNR will depend on coil performance at the two different resonance frequencies. For indirect detection versus direct detection in a polarization transfer experiment with perfect transfer efficiency, which is effectively the case here (see Figure 3C,F), then the gain in SNR is given by: where is the coil performance given by Equation (10). This is illustrated in Figure 8A, where the SNR of direct 15 N detection with a 90° pulse is assumed to be 1. The SNR improvement for different detection strategies ( 15 N direct detection, 15 N → 1 H indirect detection, or 13 C direct detection) as a function of the magnetization used per acquisition are indicated. The upper and lower bounds for the improvement in SNR are determined by whether coil or sample noise dominates respectively. Direct experimental measurements of gave a value of 6.07, which was less than the lower bound given by Equation (10), reflecting poorer than expected performance of the 1 H circuit in the dual tuned coil. However, with this experimentally determined value for there was good agreement between the theoretical gain in SNR for indirect 15 N detection and that measured experimentally ( Figure 8B). Indirect detection of hyperpolarized [ 15 N 2 ]urea is an advantage over direct detection of [ 15 N 2 ]urea if more than 2% of the 15 N polarization is used in each transfer, and over direct detection of [ 13 C]urea if more than 11% of the 15 N polarization is transferred at each step ( Figure 8A), assuming that sample noise dominates and is only 9.86. With better coil performance these percentages are decreased such that if coil noise dominates, ie, the gain from detecting 15 N polarization via spin-coupled protons is fully realized, then indirect 15 N detection is always an improvement over direct detection and an improvement over direct detection of [ 13 C]urea if only 0.4% of the 15 N polarization is transferred to proton. However, in the comparison with detection of [ 13 C]urea there are other factors to consider. Although the [ 15 N 2 ]urea T 1 is over 200 s in 2 H 2 O, which allows the hyperpolarized material to be stored prior to injection, the T 1 in water measured here was only 24 s and values as low as 9.8-12.9 s have been measured in blood, 2 whereas we have measured a T 1 for [ 13 C] urea at 7 T in vivo of ~16 s 36 and values of 43 s (at 11.7 T) 37 and 78 s (at 3 T) 38  exchange with solvent protons where the exchange rate is fast compared to the coupling constant, is weak and not observed. The sequence described here could also be used to detect hyperpolarized 15 N via indirectly bonded protons, for example α-trideuteromethylglutamine via the C2 proton, where in the absence of directly bonded protons the 15 N T 1 is much longer, although the J-coupling will be weaker and therefore the efficiency of polarization transfer reduced. This glutamine derivative has been suggested as an alternative to [ 13 C]urea for imaging tissue perfusion. 3 The 15 N in this molecule can be polarized to ~10% and in the rat kidney in vivo has a T 1 of 146 s as compared to 18 s for [ 13 C]urea. Although polarization transfer sequences based on J-coupling have been used previously with hyperpolarized 13 C-labeled substrates to produce hyperpolarized proton spectra 6-9 and images 10,11 and with hyperpolarized 15 N labelled substrates to produce spectra, [12][13][14] this is the first experimental demonstration of partial transfer, which with urea would be required for dynamic imaging of tissue perfusion. Simulations using measured cross relaxation rates showed polarization transfer via spin coupling can give orders of magnitude higher proton polarization than that obtained via cross relaxation 26,29,[31][32][33] (Figure 7). Moreover, the degree of transfer can be controlled to balance proton signal enhancement with the duration of the 15 N hyperpolarization.
To summarize, we have shown that partial polarization transfer from 15 N to 1 H using a modified BINEPT sequence can be used for dynamic imaging of hyperpolarized [ 15 N 2 ]urea. If more than 10% of the 15 N polarization is used in each acquisition then the sequence can give better sensitivity than direct 13 C detection of [ 13 C]urea for similar levels of hyperpolarization, and with full transfer of polarization can give an SNR that is 8.0-22.4-fold greater, depending on coil performance and whether coil or sample noise dominates. However, although we have used adiabatic pulses, implementation of this sequence in vivo will likely require the much better B 1 field of a volume transmit coil, as was required for 1 H detection of hyperpolarized [1-13 C]lactate in vivo. 10,11