Proton NMR of 15 N-Choline Metabolites Enhanced by Dynamic Nuclear Polarization Riddhiman Sarkar, † Arnaud Comment, ‡,§,| Paul R. Vasos, † Sami Jannin, | Rolf Gruetter, ‡,§,⊥ Geoffrey Bodenhausen, † He ´le `ne Hall, O Deniz Kirik, O,# and Vladimir P. Denisov* ,# Laboratory for Biomolecular Magnetic Resonance, Ecole Polytechnique Fe ´de ´rale de Lausanne, CH-1015 Lausanne, Switzerland, Laboratory for Functional and Metabolic Imaging, Ecole Polytechnique Fe ´de ´rale de Lausanne, CH-1015 Lausanne, Switzerland, Department of Radiology, UniVersity of Lausanne, CH-1015 Lausanne, Switzerland, Laboratory for Physics of Nanostructured Materials, Ecole Polytechnique Fe ´de ´rale de Lausanne, CH-1015 Lausanne, Switzerland, Department of Radiology, UniVersity of GeneVa, CH-1015 GeneVa, Switzerland, Department of Experimental Medical Science, Brain Repair and Imaging in Neural Systems, Lund UniVersity, SE-221 84 Lund, Sweden, and Lund UniVersity Bioimaging Center, Lund UniVersity, SE-221 84 Lund, Sweden Received March 18, 2009; E-mail: Vladimir.Denisov@med.lu.se Dynamic Nuclear Polarization (DNP) by the so-called ‘dissolu- tion’ 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 neurotrans- mission. 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 transfer- ring 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 While the NCH 2 signal is absent in the 1 H DNP spectrum because the relevant coupling constant is too small, the CH 2 O and N(CH 3 ) 3 signals are remarkably enhanced (>2 × 10 3 times), compared to the inverse-INEPT spectrum in thermal equilibrium (not shown). Among the major choline metabolites, the CH 2 O peak exhibits the largest 1 H chemical- † Laboratory for Biomolecular Magnetic Resonance, EPFL. ‡ Laboratory for Functional and Metabolic Imaging, EPFL. § Department of Radiology, University of Lausanne. | Laboratory for Physics of Nanostructured Materials, EPFL. ⊥ Department of Radiology, University of Geneva. O Department of Experimental Medical Science, Brain Repair and Imaging in Neural Systems, Lund University. # Lund University BioImaging Center, Lund University. Figure 1. (A) Conventional 1 H NMR of 15 N-enriched choline in H 2 O. (B) 1 H DNP-NMR of 15 N-choline, using inverse-INEPT. (C) 15 N-filtered 1 H spectrum of a mixture of 0.3 M natural-abundance acetylcholine (ACh) and phosphocholine (PC) and 1.3 mM 15 N-choline (Cho), 256 scans. (D) The inverse-INEPT 1 H DNP-NMR spectrum of a mixture of natural- abundance Cho and ACh. Left: Molecular structure of choline and inverse- INEPT pulse sequence. Proton signals that do not originate from 15 N magnetization were suppressed by two pulsed field gradients (PFG) in a 10:1 ratio. Published on Web 10/22/2009 10.1021/ja9021304 CCC: $40.75  2009 American Chemical Society 16014 9 J. AM. CHEM. SOC. 2009, 131, 16014–16015