Proton NMR of ¹⁵N-Choline Metabolites Enhanced by Dynamic Nuclear Polarization

Abstract

Dynamic Nuclear Polarization (DNP) by the so-called 'dissolution' procedure 1 is rapidly gaining momentum as a novel method to enhance weak nuclear magnetic resonance (NMR) signals from molecular tracers, so that one can visualize their biodistribution and metabolism in ViVo. 2 The major limitation of the technique arises from the short lifetimes of hyperpolarized spin states in liquids. In particular, longitudinal relaxation times T 1 of protons in solutions of biomolecules are too short to allow for transport and in ViVo injection of hyperpolarized compounds. Most applications of the technique have therefore focused on 13 C NMR of 13 C-enriched tracers containing nonprotonated carbons with T 1 ( 13 C) ≈ 20-40 s. Choline (CH 3 ) 3 N + CH 2 CH 2 OH plays a key role in several critical biological processes, in particular in the synthesis and metabolism of phospholipids in cell membranes, and in cholinergic neurotransmission. Although the choline molecule does not contain any slowly relaxing carbons, it possesses a quaternary nitrogen with T 1 ( 15 N) > 120 s, which lends itself to hyperpolarization. 3 The conversion of choline to phosphocholine catalyzed by choline kinase has recently been monitored by 15 N NMR in Vitro, 3 employing hyperpolarization of 15 N spins. In ViVo measurements using this method may, however, be hampered by insufficient 15 N peak separation of choline metabolites (∼ 0.2 ppm for phosphocholine vs choline, i.e., only 6 Hz in a field B 0 ) 7 T) and by poor sensitivity of 15 N NMR. A sensitivity improvement by at least an order of magnitude would be required, e.g., to monitor phosphocholine accumulation in tumor cell cultures. 4 The above limitations may be overcome by transferring the hyperpolarization from 15 N to protons, as in recent heteronuclear 2D DNP-NMR experiments. 5 A similar concept was recently used for 13 C enhanced by PASADENA. 6 In this work, we show that one can transfer the long-lived 15 N hyperpolarization to remote methylene CH 2 O protons in choline across three bonds via 3 J( 15 N, 1 H), which significantly improves both the sensitivity and the spectral dispersion of choline metabolites. We also show that T 1 ( 15 N) in choline can be considerably increased by deuteration of the methyl groups.The conventional 1 H spectrum of 15 N-enriched choline is shown in Figure 1A. The peak at 3.19 ppm, which stems from the nine magnetically equivalent methyl protons, is commonly used for in ViVo quantification of choline-containing compounds, 7 whereas the multiplets due to the NCH 2 (3.50 ppm) and CH 2 O protons (4.05 ppm) 8 exhibit an AA′XX′ pattern. 9 The CH 2 O and methyl peaks have additional doublet structures due to 3 J( 15 N, 1 H) ≈ 3.7 Hz and 2 J( 15 N, 1 H) ≈ 0.8 Hz. (In nonenriched choline, one observes triplets due to 3 J( 14 N, 1 H) ) 2.7 Hz and 2 J( 14 N, 1 H) ) 0.6 Hz. 8,9 ) As shown in Figure 1B, the small n J( 15 N, 1 H) couplings in choline can be used to transfer hyperpolarization from 15 N to CH 2 O and methyl protons, using a reversed INEPT pulse sequence. 10,11...

Feasibility of in vivo15N MRS detection of hyperpolarized 15N labeled choline in rats

“…40,41 In vivo relaxation times showed a decrease compared with the in vitro values, which can be explained by the fact that the relaxation times of metabolite are likely to be influenced by the microenvironment. 43 In addition, the in vivo relaxation times exhibited a deviation from the standard mono-exponential decay, with a very fast decay (6 AE 1.5 s) during the first seconds, followed by a slower decay afterwards (126 AE 15 s).…”

“…38 To date, besides optically polarized 3 He and 129 Xe, 39 6 Li is the only hyperpolarized nucleus besides 13 C that has been reported to be detected in vivo. 38 Two recent studies reported remarkably long in vitro longitudinal relaxation times for 15 N Cho, 40,41 suggesting that hyperpolarized 15 N Cho could be a suitable candidate for in vivo experiments.…”

Localized in vivo hyperpolarization transfer sequences

“…

In vivo localized and fully adiabatic homonuclear and heteronuclear polarization transfer experiments were designed and performed in the rat brain at 9.4 T after infusion of hyperpolarized sodium [1,[2][3][4][5][6][7][8][9][10][11][12][13] C 2 ] and sodium [1-13 C] acetate. The method presented herein leads to highly enhanced in vivo detection of short-T 1 13 C as well as attached protons.

…”

“…This is particularly challenging in vivo, as the spectral resolution in tissues is more limited than in vitro. Recently, polarization transfer methods were proposed for in vitro DNP-enhanced high-resolution applications in liquid-state NMR allowing the enhancement of short-T 1 nuclear spins via J-coupling-mediated transfer from the large spin polarization of the long-T 1 nuclear spins in various molecules (7)(8)(9).In this article, we present an in vivo hyperpolarization transfer sequence to enhance the signal of short-T 1 nuclear spins. The proposed method allows for the in vivo localized detection of aliphatic carbons or even protons and thus offers the possibility to increase the spectral resolution or obtain additional information on the chemical environment of specific nuclear spins in biomedical hyperpolarized MR experiments.…”

Proton hyperpolarisation preserved in long-lived states

“…For instance, the long T 1 ( 15 N) E 189 s of partially-deuterated choline (CD 3 ) 3 15 N + CH 2 CH 2 OHÁCl À in D 2 O can be used to preserve the enhanced 15 N polarisation during the voyage, prior to transferring the magnetisation from 15 N to 1 H for detection. 6 Non-equilibrium populations of nuclear spins can be stored in the form of long-lived states (LLS) with lifetimes T LLS that can last much longer than the longitudinal 'spin-lattice' relaxation times T 1 . [10][11][12] These states are largely immune to relaxation effects because of the spin-permutation symmetry of the system.…”