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Enrico Chiavazza1,2, Eugen Kubala2,3, Concetta V. Gringeri2,4, Stephan Düwel2,5, Markus Durst2,5, Rolf F. Schulte2, and Marion I. Menzel2
1Molecular Biotechnology & Life Sciences, University of Turin, Turin, Italy, 2GE Global Research, Munich, Germany, 3Institute of Organic Chemistry, Johannes Kepler
University, 4Nuklearmedizinische Klinik im Klinikum Rechts der Isar and 5Institute of Medical Engineering, Technische Universität München, Munich, Germany
Hyperpolarization quenching in 13C nuclei bound to fast relaxing
quadrupolar 14N mediated by scalar coupling relaxation in amide groups
exposed to Earth's magnetic field
- 20 µL [13C]urea (14N/15N2– 8M), 25 mM OX063
Trityl radical, 2.5mM Dotarem;
- 100 µL [5-13C]glutamine (5-14N/15N – 0.6M), 45 mM
OX063 Trityl radical 5mM Dotarem;
- Glassing agent: Glycerol;
- Dissolution agent: 5ml Tris (30mM) buffered D2O;
- Final concentration: 32 and 12 mM, [13C]urea and [5-
13C]glutamine, respectively;
- Hypersense 3.35T polarizer for 1h at 94.115 and
94.105 GHz, [13C]urea and [5-13C]glutamine,
respectively;
- Transfer time: 16 - 18 sec;
- 3T GE Signa HDx scanner set up with a purpose-
built solenoid 13C coil;
- Small flip angle pulses sequence (5° for the
[13C]/[13C,15N] urea and 10° for the [5-13C]/[13C,15N]
glutamine samples);
- Thermal polarization: 2048 scan averaged
measurement on the sample after adding 4% v/v
Dotarem (90°, TR 1s);
- Liquid polarization calculated from the integrated
hyperpolarized and the thermal spectrum, a thermal
Boltzmann distribution was assumed for the thermal
measurement;
- Polarization values not corrected for the T1decay
since the T1at low field was markedly different from
the one measured at 3 T.
Experimental
The hyperpolarized signal is strongly enhanced by the
presence of an auxiliary magnetic field during the
transfer, as well as by the use of 15N labeled amides
(Fig. 1). No polarization preserving effect was
observed when a radical scavenger (sodium
ascorbate 5mM) was added to the dissolution agent
(Tab. 1). The observed low field relaxation behavior
for 14N-13C amides suggested that, in such conditions
(relatively strong J coupling, short 14N nucleus T1and
weak magnetic field), a new relaxation mechanism
becomes dominant. Scalar coupling (type II) is known
to be an efficient relaxation mechanism in closely
resonant nuclei (79Br-13C). Its contribution to
relaxation has been theoretically estimated3using eq.
1 and has been found to be equivalent to an averaged
R1of 1.5±0.1 s-1 (Fig. 2). This polarization quenching
has been successfully overcome by keeping the
hyperpolarized sample close to a permanent magnet
(0.2 T). Alternatively, 15N labeling of the substrates
appeared to be effective and may be a safer solution.
This phenomenon should be taken into account
during the design of a DNP-MRI laboratory, either by
locating the polarizer in the stray field of the MR
scanner or by connecting it to the MR scanner with a
suitable sustained magnetic field transfer system.
Magnetic resonance spectroscopic imaging (MRSI)
with hyperpolarized substances is one of the most
promising molecular imaging methods. This
approach has the potential to overcome the main
drawback of the 13C-MRS/MRI technique, namely
the low absolute sensitivity that results from the low
gyromagnetic ratio and low natural isotopic
abundance of 13C. The possibility of using
hyperpolarized agents in either MR spectroscopy or
in MR imaging is strictly dependent on their
relaxation time. Glutamine is an important
metabolite, its utilization is greatly enhanced and
linked to the energetic metabolism in tissues where
a proliferative state is activated (e.g injuries, tumor)1.
Working with [5- 13C] glutamine for this purpose, a
rapid polarization loss was observed after
completing the dissolution process, yielding an
almost zero signal in the resulting NMR spectrum
(Fig. 1). The same behavior has been observed in
[13C]urea. To the best of our knowledge no one has
described and explained a similar transient fast
relaxation phenomenon. The significant T1
shortening that is observed can be explained in
terms of the scalar coupling relaxation (2nd kind)
contribution to relaxation due to the fast relaxing
quadrupolar 14N nucleus coupling with the 13C
nucleus. This contribution is efficient at the low
environmental magnetic field present in the
laboratory. In fact, the use of an auxiliary magnetic
field of about 0.2 T from a permanent magnet during
sample transfer to the MRI scanner reduced the T1
shortening; using this method, a sufficient level of
liquid-state polarization was obtained for both
molecules to enable their use as DNP probes.
Introduction
1) Lehninger AL , Nelson DL, Cox MM , Principles of
Biochemistry 1993,Worth Publishers;
2) P. Mielville, S. Jannin, G. Bodenhausen ,, J. Magn. Res.
210 (2011) 137–140;
3) Becker J, Shoup RR, Far rar TC. Pure App. Chem. 32
(1972) 51-66;
This work has been published as E. Chiava zza et al. JMR
227 (2013) 35-38
Acknowledgements: co-funding by BMBF grant
number 01EZ1114
Contact: enrico.chiavazza@unito.it
References
Results & Discussion
The spin–lattice relaxation time T1is determined by
contributions from different and independent
mechanisms:
- Dipolar interaction, Quadrupolar interaction, Spin
rotation(Rd, Rq, Rsr)No Field Dependence
- Paramagnetic dipolar interaction
R1∝1/B0
but only when using nitroxide radicals2
- Chemical shift anisotropy relaxation (c.s.a)
R1∝B02
- Scalar coupling relaxation (s.c):
R1∝1/B02through ω=γB0
(1)
Methods
Fig. 2: Estimated SC contribution profile to 13C
relaxation rate obtained from the Eq. 1. T1
14N = 10-3 s
JC-N = 14 Hz B0 = 20µT 2mT during transfer
Table 1. Polarization values and relaxation times of [13C]urea and [5-13C]glutamine, measured at 3T.
T1(s)
at 3 T
Transport in earth’s
magnetic field
Sample attached to 0.2 T
permanent magnet during
transport
Transport in earth’s
magnetic field, ascorbate
added as radical scavenger
Liquid Pol (%) Liquid Pol (%) Liquid Pol (%)
[13C,14N2]urea 78±4 3·10-3±1·10-3 13±1 3·10-3±1·10-3
[13C,15N2]urea 85±7 30±2 25±1 - - -
[5-13C, 14N]glutamine 8.0±0.1 0.02±5·10-3 0.7±0.1 0.05±5·10-3
[5-13C, 15N]glutamine 7.7±0.4 0.7±0.2 0.8±0.1 - - -
Fig. 1: Hyperpolarized spectra of [5-13C]glutamine (above) and [13C]urea (below): first column, samples transferred at low
field magnetic field (<1mT); second column, samples transferred with a 0.2 T auxiliary magnetic field; third column 15N
labeled samples transferred at low magnetic field (<1mT). Glutamine signal is indicated as (a); (b) and (c) are assigned to
[5-13C] glutamate and [5-13C] pyroglutamate, respectively;
(a)
(b)
(c) (a)
(b) (c)
(a)
(b) (c)
0246810
0
2
4
6
8
10
12
Distance (m)
R1,SC (s-1)
[13C]urea
[5-13C]glutamine