Photogenerated Radical in Phenylglyoxylic Acid for in Vivo Hyperpolarized 13C MR with Photosensitive Metabolic Substrates

Article abstract of DOI:10.1021/jacs.8b09326

Whether for 13C magnetic resonance studies in chemistry, biochemistry, or biomedicine, hyperpolarization methods based on dynamic nuclear polarization (DNP) have become ubiquitous. DNP requires a source of unpaired electrons, which are commonly added to the sample to be hyperpolarized in the form of stable free radicals. Once polarized, the presence of these radicals is unwanted. These radicals can be replaced by nonpersistent radicals created by the photoirradiation of pyruvic acid (PA), which are annihilated upon dissolution or thermalization in the solid state. However, since PA is readily metabolized by most cells, its presence may be undesirable for some metabolic studies. In addition, some 13C substrates are photosensitive and therefore may degrade during the photogeneration of a PA radical, which requires ultraviolet (UV) light. We show here that the photoirradiation of phenylglyoxylic acid (PhGA) using visible light produces a nonpersistent radical that, in principle, can be used to hyperpolarize any molecule. We compare radical yields in samples containing PA and PhGA upon photoirradiation with broadband and narrowband UV?visible light sources. To demonstrate the suitability of PhGA as a radical precursor for DNP, we polarized the gluconeogenic probe 13C-dihydroxyacetone, which is UV-sensitive, using a commercial 3.35 T DNP polarizer and then injected this into a mouse and followed its metabolism in vivo.

Full text of DOI:10.1021/jacs.8b09326

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Article
Photo-generated radical in phenylglyoxylic acid for in vivo
13
hyperpolarized C-MR with photo-sensitive metabolic substrates
Irene Marco-Rius, Tian Cheng, Adam P. Gaunt, Saket Patel, Felix Kreis, Andrea
Capozzi, Alan Wright, Kevin M. Brindle, Olivier Ouari, and Arnaud Comment
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b09326 • Publication Date (Web): 12 Oct 2018
Downloaded from http://pubs.acs.org on October 12, 2018
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Photo-generated radical in phenylglyoxylic acid for in vivo hyperpolar-
ized 13C MR with photo-sensitive metabolic substrates
Irene Marco-Rius,a* Tian Cheng,a Adam P. Gaunt,a Saket Patel,b Felix Kreis,a Andrea Capozzi,c Alan J. Wright,a Kevin M. Brindle,a
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Olivier Ouari,b and Arnaud Comment a,d
a University of Cambridge, Cambridge, UK ; b Aix-Marseille University, CNRS, ICR, Marseille, France ; c Department of Electrical En-
gineering, Center for Hyperpolarization in Magnetic Resonance, Technical University of Denmark, Lyngby, Denmark; d General Elec-
tric Healthcare, Chalfont St Giles, UK.
Photolysis; Dynamic Nuclear Polarization; Hyperpolarization; Benzoylformic acid; Dihydroxyacetone
ABSTRACT: Whether for 13C magnetic resonance studies in chemistry, biochemistry or biomedicine, hyperpolarization methods based on dynamic
nuclear polarization (DNP) have become ubiquitous. DNP requires a source of unpaired electrons, which are commonly added to the sample to be
hyperpolarized in the form of stable free radicals. Once polarized, the presence of these radicals is unwanted. These radicals can be replaced by non-
persistent radicals created by photo-irradiation of pyruvic acid (PA), which are annihilated upon dissolution or thermalization in the solid state. How-
ever, since PA is readily metabolized by most cells, its presence may be undesirable for some metabolic studies. In addition, some 13C substrates are
photo-sensitive and, therefore, may degrade during photo-generation of PA radical, which requires ultraviolet (UV) light. We show here that photo-
irradiation of phenylglyoxylic acid (PhGA) using visible light produces a non-persistent radical that, in principle, can be used to hyperpolarize any
molecule. We compare radical yields in samples containing PA and PhGA upon photo-irradiation with broadband and narrowband UV-visible light
sources. To demonstrate the suitability of PhGA as a radical precursor for DNP, we polarized the gluconeogenic probe 13C-dihydroxyacetone, which is
UV-sensitive, using a commercial 3.35 T DNP polarizer and then injected this into a mouse and followed its metabolism in vivo.
been hyperpolarized using PA as a precursor molecule for photo-
INTRODUCTION
generation of radicals.12,13 However, when PA itself is not one of the
substrates of interest,10,13 metabolic interference caused by its pres-
ence may be undesirable. Furthermore, PA cannot be considered a
universal polarizing agent because some metabolic substrates are
photo-sensitive and can degrade during the exposure to UV light
when generating the radical. One example of this problem is [2-
13C]dihydroxyacetone (DHAc), which has been previously hy-
perpolarized with OX063 trityl radical and used to study gluconeo-
genesis, glycolysis and fatty acid synthesis in the liver14–16. DHAc ab-
sorbs light below 300 nm leading to sample degradation.17 Finally,
dimethyl sulfoxide (DMSO), which is frequently used as glassing
agent for the preparation of DNP samples, is also photo-sensitive.
The purpose of this work is to demonstrate that phenylglyoxylic
acid (PhGA) can also be used as an efficient radical precursor for
DNP. PhGA has an excellent safety profile and no reported effect on
metabolism on the timescale of the hyperpolarized 13C MR experi-
ments.18 Because of its extended absorption into the visible spec-
trum, a radical can be photo-generated in PhGA at longer wave-
lengths and can therefore be used in conjunction with molecules that
are sensitive to UV light. We show that [2-13C]DHAc can be hy-
perpolarized using the photo-generated radical of PhGA and a com-
mercial 3.35 T HyperSense polarizer, and that the resulting solution
can be used for 13C MR metabolic studies in vivo.
Hyperpolarization by dissolution dynamic nuclear polarization
(DNP) can enhance the magnetic resonance (MR) signals of mole-
cules in solution by up to 5 orders of magnitude.1 As the list of mol-
ecules that have been hyperpolarized increases every year, so do ap-
plications across organic and polymer chemistry,2 as well as biomed-
icine.3 DNP is based on transfer of spin polarization from unpaired
electrons of stable free radicals to nuclei at cryogenic temperatures.
Stable radicals such as trityls, nitroxides or 1,3-bisdiphenylene-2-
phenylallyl (BDPA) are admixed with the sample containing the
molecule to be hyperpolarized.4 A consequence of having to intro-
duce stable radicals is that they accelerate nuclear spin relaxation,
which may cause significant signal loss during the dissolution pro-
cess. A concern for applications in the biomedical field, where disso-
lution DNP has seen a rapid translation from the laboratory to the
clinic,5–7 is the potential radical toxicity. Medical regulatory bodies
currently demand that radicals are filtered out prior to injection into
a human subject,8,9 which adds to the complexity of the process and
becomes a potential failure point before the release of the solution
for injection.
Non-persistent photo-induced free radicals generated by ultravi-
olet (UV) light irradiation of pyruvic acid (PA) have been proposed
as an alternative to the persistent radicals used in dissolution DNP.10
Recently it has been demonstrated that these non-persistent radicals
can be annihilated inside the frozen sample by warming it to ~200 K.
After this point, the nuclear spin hyperpolarization can persist for
several hours,11 making it possible to transport the sample for use at
a distant location. Several 13C-labeled metabolic substrates have
EXPERIMENTAL SECTION
All chemicals were purchased from Sigma-Aldrich (Haverhill, UK)
and data were processed in MATLAB (Mathworks, Natick, MA, USA),
unless stated otherwise.
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Dynamic nuclear polarization
Page 2 of 17
UV-Vis absorption measurements
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The ultraviolet–visible (UV-Vis) absorption spectra of PhGA, PA,
DHAc and DMSO in water (146 mM, 600 µl) were measured using an
Ocean Optics USB2000+ spectrometer and DH-2000-BAL UV-VIS-
NIR light source (Halma PLC, Amersham, UK).
Four samples were prepared for dissolution DNP: (sample #1) 8 M
[2-13C]DHAc and 1 M PhGA in water; (sample #2) 8 M [2-13C]DHAc,
1 M PhGA and 1.2 mM gadoteric acid (Gd3+, Dotarem, Guerbet,
Roissy, France) in water; (sample #3) 8 M [2-13C]DHAc, 1 M d5-
PhGA and 1.2 mM Gd3+ in 2H2O; and (sample #4) 8 M [2-13C]DHAc,
21 mM OX063 trityl radical (Albeda Research Aps, Copenhagen, Den-
mark) and 1.2 mM Gd3+ in 1:3 DMSO/water (v/v). All solutions were
sonicated for 5 min at 50 ºC before addition of Gd3+. The choice of
PhGA concentration in samples #1, #2 and #3 was based on the fact
that hyperpolarized DHAc had previously been polarized with 21 mM
OX063,14–16 a radical with a similar ESR line width as the one obtained
after photo-irradiation of PhGA, and that a similar concentration of
photo-generated radical (18-20 mM) can be obtained if the samples
are doped with 1 M PhGA.
To estimate the efficiency of broadband and narrowband UV-Vis
light sources in generating photo-induced radicals in PA and PhGA, the
power profile of each source (provided by the manufacturer) was mul-
tiplied by the absorption spectrum of PA and PhGA between 300 nm
and 420 nm (Fig. 1). The integral of the multiplied spectra was used to
compare the effective light intensity provided by each source.
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Broadband and narrowband photo-irradiation
To investigate the effects of photo-irradiation on the selected radical
precursors, two solutions were prepared: neat PA diluted to 7.0 M in a
mixture of 1:1 glycerol/water (v/v) and sonicated for 5 min at 40 ºC;
and a sample containing 7.1 M PhGA in a mixture of 1:1 glycerol/water
(v/v) sonicated for 5 min at 40 ºC. To study the relationship between
PhGA concentration, light source and radical yield, the 7.1 M PhGA
sample was diluted to 50%, 25% and 7% of the original PhGA concen-
tration in a mixture of 1:1 glycerol/water (v/v). Additionally, the effect
of the light sources on two photo-sensitive solutions was tested: neat
dimethyl sulfoxide (DMSO); and a sample containing 8.0 M DHAc
dissolved in 2H2O sonicated for 10 min at 40 ºC.
Between 7 and 11 beads of samples #1, #2 and #3 were simultane-
ously irradiated for 200 s with the VisiCure 405 source. The beads were
then placed into a standard HyperSense sample holder in contact with
LN2 to preserve the radical and rapidly inserted into a HyperSense po-
larizer operating at 3.35 T and 1.25 K (Oxford Instruments, Abingdon,
UK).
A microwave (µ-wave) sweep between 94.07 GHz and
94.22 GHz (10 MHz steps, 10-min µ-wave irradiation/step) was per-
formed for samples #2, #3 and #4 and for a sample containing 7 M
[1-13C]PA in 1:1 glycerol/water (v/v) irradiated for 400 s with the
BlueWave 75 source. The background signal measured at each µ-wave
frequency was subtracted from all µ-wave sweeps. For dissolution
DNP, samples were polarized with the µ-wave source set to
94.110 GHz (except the sample used for the in vivo experiment, which
was polarized at 94.205 GHz) and 100 mW for a period of 1.5-2 h. The
samples were dissolved with 6 ml of a phosphate saline buffer (PBS).
Frozen beads of each solution were formed by dispensing droplets
from a syringe into an ESR quartz dewar flask (Wilmad-Lab Glass WG-
850-B-Q, Goss Scientific, Crewe, UK) filled with liquid nitrogen
(LN2). The beads were photo-irradiated using either a broadband
source (Dymax BlueWave 75, Dymax Europe GmbH, Wiesbaden,
Germany) or a narrowband source (Dymax BlueWave LED VisiCure
405 nm, Dymax Europe GmbH, Wiesbaden, Germany) operating at
maximum power. From this point onwards these two sources are re-
ferred to as “BlueWave 75” and “VisiCure 405”. The stand used for UV
irradiation is described in ref. 19. Note that transmission through the
quartz dewar was the same across the entire range of wavelengths used
in this study.20
Hyperpolarized 13C MR in a phantom at 7 T
Liquid-state 13C MR acquisitions were carried out in a small-animal
horizontal bore 7 T MR scanner (Agilent, Palo Alto, CA) using a 42-
mm-diameter bird-cage 1H/13C transmit volume coil and a 20 mm
outer diameter 13C receive surface coil (Rapid Biomedical GmbH,
Rimpar, Germany). The temporal evolution of the hyperpolarized 13
C
X-band ESR measurements and radical concentration estima-
tion
signal was recorded in a 3 mL phantom starting 16 1 s after dissolu-
tion using the following parameters: repetition time = 1 s, nominal flip
ESR spectra of single frozen beads inside the quartz dewar filled with
LN2 were acquired using a benchtop X-band ESR spectrometer
(MiniScope MS5000, Magnettech GmbH, Berlin, Germany). ESR pa-
rameters were optimized to resolve the hyperfine structure of the spec-
tra and then kept constant throughout all the experiments (number of
accumulations = 1, B = [323-353] mT, sweep time = 20 s, modulation
amplitude = 0.1 mT, modulation frequency = 100 kHz, microwave
power = 0.2 mW). Sequential ESR spectra were acquired for each bead
after different irradiation times to obtain a radical build-up curve (Sup.
Fig. 2). The effect of deuterating PhGA on the ESR line width was in-
vestigated by substituting protonated PhGA in water with perdeuter-
ated PhGA (d5-PhGA) in 2H2O. The synthesis of d5-PhGA is detailed
in the Supporting Information.
angle = 9 º, pulse width = 400 µs (sinc pulse truncated to 5 lobes), spec-
tral width = 32 kHz. T1 relaxation time constants were determined by
fitting an exponential decay function to the data and corrected for the
flip angle (see Suppl. Inf. for details on the mathematical formula).
The thermal equilibrium 13C MR signal was acquired with the same
parameters but a longer repetition time (~5´T1) and the 13C hyperpo-
larization at the time of the first acquisition was calculated as the ratio
between the hyperpolarized and thermal signals multiplied by the the-
oretical equilibrium polarization at 7 T and 298 K.
Following ESR signal acquisition, the frozen bead was extracted
from the quartz dewar, inserted into an Eppendorf tube and weighed to
estimate the volume of the bead. Radical concentration was deter-
mined by comparing double integration of the first derivative ESR
spectrum, corrected for bead volume (~4 µl), with a calibration curve
obtained from beads of known concentrations of 4-Hydroxy-2,2,6,6-
tetramethylpiperidine 1-oxyl (TEMPOL, Sup. Fig. 1). Each measure-
ment was repeated at least twice.
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Following the hyperpolarized 13C phantom experiments, the sam-
ples obtained after dissolution were mixed with 10% 2H2O and their
thermally-polarized 13C MR spectrum was measured in a 600 MHz
vertical-bore Bruker spectrometer (Bruker BioSpin GmbH, Rhein-
stetten, Germany). To further investigate the impact of photo-irra-
diation on the solutions containing PhGA, the 13C spectra of two ad-
ditional samples prepared by dissolving either a non-irradiated bead
or a 200-s-irradiated bead (using VisiCure 405) in 600 µl PBS (10%
2H2O) were recorded for comparison. All four samples were meas-
ured with the following parameters: repetition time = 6 s, nominal
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flip angle = 30º, pulse width = 2.6 µs (hard pulse), spectral width =
38 kHz. T1s were measured using an inversion recovery sequence.
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RESULTS
UV-Vis absorption and light source power profiles
The UV-Vis spectra of the two precursors studied here show that
PhGA has a broad absorption between 200 and 400 nm, while PA
has a weaker and narrower absorption around lmax = 320 nm corre-
sponding to the n-p* electronic transition characteristic of a-ke-
toacids.24 The metabolic substrate DHAc has an absorption peak
around lmax = 270 nm and the DMSO solvent one around lmax = 240
nm (Fig. 1).
Figure 1. UV-Vis absorption spectra (left axis) and light source power
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distributions (right axis). Power profiles were provided by the manufac-
turer (Dymax Europe GmbH, Wiesbaden, Germany).
Table 1. Maximum radical yield after photo-irradiation
The power dependence of the two light sources on wavelength
was obtained from the manufacturer’s manual and is overlaid on Fig-
ure 1. While the total output power density was nearly the same for
both light sources (19 W/cm2 for the BlueWave 75 and 16.8 W/cm2
for the VisiCure 405), the power distribution across the spectrum
was radically different. Based on the multiplied spectra calculated
from the UV-Vis absorption of PA and PhGA and the wavelength-
dependent power distribution of each light source, the BlueWave 75
light source was expected to be more effective for radical generation
in PA while the VisiCure 405 source should be better suited for rad-
ical generation in PhGA since the ratio Areabroadband / Area405nm is
much larger for PA than for PhGA (15 vs. 2.8).
Precursor
Radical yield (mM) Radical yield (mM)
BlueWave 75
25.7 3.7
VisiCure 405
40.4 5.8
PhGA (7.1M)
PA (7.0M)
77.7 11.2
23.8 3.4
In vivo hyperpolarized 13C MR at 7T
Procedures were performed in compliance with project and per-
sonal licenses issued under the United Kingdom Animals (Scientific
Procedures) Act, 1986 and were approved by the Cancer Research UK,
Cambridge Institute Animal Welfare and Ethical Review Body. A fe-
male C57B6 mouse (body weight = 32.2 g) was anesthetized with 2%
isoflurane, its body temperature maintained at 37ºC, and placed inside
the 7 T MR system. The coil setup was identical to the one used for the
Broadband and narrowband photo-irradiation
phantom experiments (a 42-mm-diameter bird-cage H/13C transmit
1
volume coil and a 20 mm outer diameter 13C receive surface coil; Rapid
Biomedical GMBH, Rimpar, Germany), with the receive surface coil
placed over the liver of the mouse. Positioning of the surface coil was
confirmed with sagittal, coronal and axial T2-weighted 1H images.
Photo-irradiated beads (~30 µl) from sample #2 were polarized as
PhGA and PA were irradiated with either the BlueWave 75 broad-
band light source or the VisiCure 405 narrowband light source. The
associated ESR spectra are displayed in Figures 2a and 2b. The
scheme of the photochemical pathway for radical production in
PhGA and PA at 77 K is shown in the Supplementary Information.
Frozen PA and PhGA beads turned a yellow-orange color after irra-
diation, which became darker as irradiation time increased (Fig. 3a).
Table 1 summarizes the radical yield for each precursor and light
source. BlueWave 75 yielded 3.3 times more radical and 3 times
faster than VisiCure 405 when PA was the precursor. Conversely, ir-
radiation of PhGA with VisiCure 405 resulted in a 1.6-fold increase
in radical yield compared to broadband irradiation, without a reduc-
tion in the build-up time. The radical yield increased with PhGA
concentration (Fig. 3c). It is noticeable that irradiation with either
BlueWave 75 or the narrowband light source produced similar radi-
cal yields in solutions with PhGA concentrations below 1.77 M.
However, irradiation with BlueWave 75 generated lower radical
yields at higher PhGA concentrations.
described above with µ-wave irradiation at 94.205 GHz and dissolved
in 6 ml of PBS. 400 µl of this solution was injected into the mouse via a
tail-vein over a period of 3 s. 13C MR acquisition started 12 s after the
injection. The parameters used for 13C MR acquisition were: repetition
time = 0.2 s, nominal flip angle = 15º, pulse width = 2 ms (sinc pulse
truncated to 5 lobes), spectral width = 6 kHz, total acquisition time =
80 s. The transmitter was centered at 72.3 ppm, and every 10th acquisi-
tion it was switched to 214 ppm for a single acquisition before returning
to 72.3 ppm. Therefore, the spectral region around 72.3 ppm was sam-
pled 360 times (every 0.2 s) and the region around 214 ppm was sam-
pled 40 times (every 2 s). This high temporal resolution acquisition
strategy was chosen to maximize the signal-to-noise ratio of the
summed spectra assuming an in vivo T1 of 10 s for the metabolites (see
analysis presented in ref. 21). A similar scheme has previously been used
for the detection of glucose metabolism in vivo.22,23 The volume coil ex-
cited the whole body, and reception was coil-selective.
The ESR line width of PhGA was about 3 times narrower than
that of PA. Perdeuteration of PhGA (d5-PhGA) resulted in a ~10%
narrower ESR line width than protonated PhGA (FWHM =
1.30 0.05 mT vs FWHM = 1.45 0.05 mT, Fig. 3b) but did not
change the radical yield.
Liquid-state 13C MR at 14.1 T
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Figure 2. X-band ESR spectra of frozen droplets in LN2, photo-irradiated with a broadband (BlueWave 75) or a narrowband (VisiCure 405) light
source. (a) PhGA (7.1 M) in 1:1 water/glycerol (v/v). (b) PA (7.0 M) in 1:1 water/glycerol (v/v). (c) DMSO (neat). (d) DHAc (8.0 M) in 2H2O.
The spectral structure in (b) and (c) are caused by the hyperfine coupling to the proton spins of the methyl groups. The units on the y-axis are the same
in all four plots.
Figure 4. Solid-state microwave spectra at 3.35 T and 1.25 K: (open red
circles) photo-irradiated [2-13C]DHAc solution (8 M) in H2O doped
with 1 M PhGA (18 mM radical); (black dots) [2-13C]DHAc solution
(8 M) in H2O/DMSO (v/v) doped with 21 mM OX063; (green trian-
gles) photo-irradiated [1-13C]PA solution (7 M) in H2O/glycerol
(65 mM radical).
Figure 3. Photo-irradiation of PhGA dissolved in 1:1 glycerol/water
The photo-sensitive metabolite DHAc and glassing agent DMSO
were also irradiated with both light sources. Irradiation of DMSO
and DHAc with BlueWave 75 produced radicals that were clearly ob-
served in the ESR spectra, while irradiation with VisiCure 405 did
not generate any observable radical (Fig. 2c and Fig. 2d).
(v/v). (a) 4-µl-bead of PhGA in LN2 before and after photo-irradiation.
(b) ESR spectrum of unlabeled PhGA and d5-PhGA. (c) Maximum rad-
ical yield vs. PhGA concentration using either the broadband (blue open
circle) or the narrowband light source (red dots). Radical yield is re-
ported as mean standard deviation across 3 measurements.
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A few impurities from the [2-13C]DHAc sample were observed at
DNP of [2-13C]DHAc with PhGA-derived radical
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chemical shifts of 91.6 ppm, 94.9 ppm, 108.1 ppm, 105.9 ppm and
112.7 ppm. The same small quantities of impurities were also de-
tected in the same solution before it had been UV-irradiated or po-
larized by DNP (Fig. 5b-iii). No additional peaks were detected in
the thermally polarized spectra of the samples that had undergone
photo-irradiation or photo-irradiation and subsequent DNP (Fig.
5b).
The samples containing 8 M [2-13C]DHAc and 1 M PhGA (or d5-
PhGA) in water were strongly acidic (pH = 0.5 0.5). A 200-s irra-
diation with VisiCure 405 was sufficient to reach a plateau in radical
production with a yield of 18.1 2.5 mM in the samples with and
without Gd3+ (the variability of 5 measurements was 0.5 mM). No
ESR signal originating from photo-irradiation of a 1 mM solution of
Gd3+ in water could be detected (data not shown).
In comparison, a sample prepared with [2-13C]DHAc doped with
21 mM OX063 and 1.2 mM Gd3+ polarized nearly 1.6 times slower
than the samples with PhGA-derived radical but reached 3 times
higher liquid-state polarization levels (Table 2). Interestingly, the T1
at 7 T after dissolution of the [2-13C]DHAc sample with OX063 was
29.5 0.5 s, ~5 s shorter than that of the sample with non-persistent
PhGA-derived radical. It was also slightly shorter at 14.1 T, where
the T1 was 17.3 1.4 s for [2-13C]DHAc with OX063. Note that the
The width of the µ-wave spectrum of samples containing radical
generated by photolysis of PhGA was noticeably broader than the
spectrum of the PA sample containing OX063 trityl radical but nar-
rower than that for photo-irradiated PA (Fig. 4). However, unlike
OX063, the µ-wave spectrum of the PhGA-derived radical was
strongly asymmetric, with approximately 1.7 times higher 13C polar-
ization when the µ-wave frequency is set to the value corresponding
to the optimal negative polarization, namely 94.205 0.005 GHz.
The build-up rate was however identical for both the negative and
positive optimal frequencies and there was no noticeable difference
when unlabeled PhGA was replaced by d5-PhGA (Sup. Fig. 3).
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presence of 6 µM of Gd3+ in the solution did not affect the T1 of [2-
13C]DHAc.
Hyperpolarized [2-13C]DHAc
The build-up time constants as well as the liquid-state 13C polari-
zation were measured for all the [2-13C]DHAc samples (Table 2).
The build-up time constant in samples containing PhGA was not af-
fected by the addition of Gd3+ or perdeuteration of PhGA. However,
the liquid-state polarization increased by 1.7-fold upon addition of
1.2 mM Gd3+. The polarization values shown in Table 2 were meas-
ured approximately 16 s after the start of the dissolution process,
which means that the estimated 13C polarization at the time of disso-
lution was ~8 % for the samples doped with Gd3+ and polarized with
the µ-wave frequency set to 94.110 GHz. Based on the solid-state
data, the 13C polarization at the time of dissolution should reach
~14 % if this sample was negatively polarized at 94.205 GHz.
After dissolution, the pH was neutral in all cases (pH = 7.0 0.5).
The 13C longitudinal relaxation time constant (T1) of [2-13C]DHAc
with PhGA was 35.2 1.8 s at 7 T and 19.9 1.0 s at 14.1 T. Radical
quenching was confirmed by making seven frozen beads from the
dissolved sample and averaging 100 ESR measurements to compen-
sate for the dilution following dissolution. As confirmation, no ESR
signal was observed.
Table 2. DNP results obtained with 8 M [2-13C]DHAc samples
polarized using a HyperSense polarizer with liquid-state polari-
zations measured 16 s after dissolution at 7 T.
Figure 5. (a) Representative 13C MR spectrum (sum of 180 spectra) of
a hyperpolarized solution containing 40 mM [2-13C]DHAc, 5 mM
PhGA and 6 µM Gd3+ in PBS. The MR acquisition started 16 s after the
beginning of the dissolution process with a repetition time = 1 s and
nominal flip angle = 9 º. (b) 13C MR spectra of thermally-polarized solu-
tions at 300 K and 14.1 T. All samples contained [2-13C]DHAc
(40 mM-70 mM) and PhGA (4.5-8.0 mM) in PBS with 10% 2H2O.
[13C]urea was added to samples (i) and (ii) after dissolution as a refer-
ence. These samples were made from frozen beads of 8 M [2-13C]DHAc
and 1 M PhGA in water. (i) Frozen beads had been irradiated using a
narrowband light source (VisiCure 405 nm) for 200 s and dissolution
DNP performed on them. (ii) 1.2 mM Gd3+ had been added to the sam-
ple prior to photo-irradiation and dissolution DNP. (iii) The frozen
beads were dissolved in PBS without photo-irradiation or DNP. (iv)
The frozen beads were irradiated for 200 s and melted in PBS, but they
did not undergo dissolution DNP.
[2-13C]
DHAc doped with
Liquid-state 13
C
polarization (%)
Build-up time
constant (s)
1033 82
1023 97
862 81
(1) PhGA
3.1 0.8
5.1 1.2
4.7 1.0
(2) PhGA & Gd3+
(3) d5-PhGA
&
Gd3+
(4) Trityl & Gd3+
1611 353
16.0 4.0
[2-13C]DHAc (213.9 ppm) and [2-13C]DHAc hydrate
(96.6 ppm) dominated the hyperpolarized 13C spectrum recorded in
the 7 T MR scanner (Fig. 5a). The two peaks at 199.4 ppm and 175.3
ppm correspond to the resonances of [1-13C]PhGA and [2-
13C]PhGA, respectively. No recombination products from the
quenched radical were detected in the hyperpolarized 13C spectrum.
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Figure 6. (a) Axial and sagittal T2-weighted images through the mouse with approximate position of the surface coil (red dots represent the cross
sections of the coil). (b) 13C MR spectrum acquired in vivo at 7 T following the intravenous injection of 400 µl of a hyperpolarized solution into a
mouse. The solution was composed of 40 mM [2-13C]DHAc, 5 mM PhGA and 6 µM Gd3+ in PBS. The previously reported resonance detected at ~89
ppm has not been assigned. DHAc region: sum of 40 spectra. Region with the metabolic products of DHAc and DHAc hydrate: sum of the first 70
spectra.
avoided by using a narrowband light source in the visible spectrum
in conjunction with PhGA as the radical precursor, which has an ab-
sorption spectrum that extends above 400 nm. The photochemistry
of the radical generation upon photo-irradiation of PhGA and PA at
77 K are shown in Supplementary Figure 1.
In vivo 13C MR of hyperpolarized [2-13C]DHAc metabolism
The 13C MR spectrum after the intravenous injection of 400 µl of
a hyperpolarized solution containing 40 mM [2-13C]DHAc, 5 mM
PhGA and 6 µM Gd3+ into a 32.2-g-mouse is shown in Figure 6. The
injection of this solution did not cause any observable changes in the
heart rate or respiratory pattern of the mouse. Since this sample was
negatively polarized at 94.205 GHz, the 13C polarization was esti-
mated to be 8.7% at the time of injection, which is 1.7 times higher
than the 13C polarization recorded for the same sample positively po-
larized at 94.110 GHz (Table 2). Besides the injected DHAc and its
hydrate, metabolic products were detected between 69 and 77 ppm.
Note that the resonance appearing at ~89 ppm has previously been
reported in earlier in vivo studies,15 but it has not been assigned. The
peaks at 73.8 and 71.8 ppm could be seen for the first 20 s (first 90
acquisitions) and DHAc could be monitored for approximately 60 s
(29 acquisitions). The T1 of [2-13C]DHAc was determined to be
15.8 0.3 s in vivo.
A comparison between the efficiency of the two light sources in
photo-generating radicals in PA and PhGA was made based on the
wavelength-dependent power profile of each light sources and the
absorption spectrum of the two precursors. This simple analysis as-
sumes that radical generation is proportional to the density of pho-
tons absorbed by the precursors at each wavelength. Such compari-
son is only qualitative and most likely depends on many other factors
such as light penetration and precursor concentration. Nevertheless,
it reproduces the features observed experimentally, namely that
while the broadband light source is more effective in PA radical
photo-generation, the difference between the narrowband and the
broadband light sources for PhGA radical photo-generation is rela-
tively small, especially for samples containing ~1 M precursor, which
was the concentration used to polarize [2-13C]DHAc.
The X-band ESR line width of photo-induced PhGA radicals is
1.4 mT, which is comparable to the line width of the OX063 trityl
radical and much narrower than the photo-irradiated PA and
TEMPOL line widths (see Sup. Fig. 4) – the latter nitroxyl radical
having also previously been used to hyperpolarized 13C-substrates by
dissolution DNP.21 The four peaks observed in the ESR spectrum of
PA (Fig. 2b) are due to coupling of the unpaired electron in C2 with
the methyl group. The PhGA-derived radical exhibits a narrower line
width than the PA-derived radical because the methyl group is sub-
stituted by a phenyl group, thereby increasing the distance between
the unpaired electron localized around the ketone carbon and the
neighboring protons, consequently reducing the hyperfine coupling.
However, the non-negligible g-anisotropy of the photo-induced
PhGA radical leads to a larger spread of the ESR absorption line
compared to OX063, especially at the high fields where DNP is per-
formed. Because narrow ESR line width radicals are generally more
efficient for DNP,25 especially at 3.35 T, PhGA was expected to be a
competitive candidate for hyperpolarized 13C MR experiments using
DISCUSSION
PhGA as a radical precursor for DNP
Non-persistent radicals can be created through UV-Vis light irra-
diation of a precursor molecule. Although this is a simple procedure,
the intense light used for the photo-generation of radicals may affect
and degrade other molecules that are present in the sample. This is
not the case for acetate or butyrate which can be used in conjunction
with PA,12,13 but some solvents (e.g. DMSO) and some metabolic
substrates that are used with hyperpolarized 13C MR are photo-sen-
sitive in the UV range. For example, most keto-acids - like PA - ab-
sorb light around 325 nm. DHAc, a metabolite that enters glucone-
ogenesis at the level of the trioses, has an absorption spectrum cen-
tered at 270 nm. We have shown that irradiation of DHAc and
DMSO with a broadband UV-Vis source leads to the creation of rad-
icals that are stable at 77 K. These radicals may not only affect the
DNP process but also represent the unwanted degradation of these
molecules. We have demonstrated that this degradation can be
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the HyperSense polarizer. Although, the maximum 13C polarization they can be annihilated in the solid-state,11 non-persistent photo-
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obtained with photo-induction was lower than the one obtained
with OX063, it was larger than what can typically be obtained with
TEMPOL using the Hypersense.26,27
The µ-wave spectrum acquired from samples polarized with
photo-irradiated PhGA was nearly as narrow as that of the sample
doped with OX063 but was clearly asymmetric because of the g-an-
isotropy of the PhGA-derived radical. Interestingly, no improve-
generated radicals can enable storage and transport, which would
certainly benefit future clinical studies. It is also likely that the 13C
polarization could be improved by performing DNP at higher mag-
netic fields and/or lower temperatures as well as by modulating the
µ-wave frequency, as was previously demonstrated with the photo-
induced radical in PA.12,19,31
An additional advantage of photo-generated radicals is the longer
lifetime of 13C hyperpolarization in liquid state. The 13C T1 of
[2-13C]DHAc measured at 7 T and at 14.1 T immediately after dis-
solution was 15-20% longer in the solution prepared with PhGA
than in the solution containing 0.1 mM OX063. This is a significant
but not large change because the dominant relaxation mechanism at
high magnetic fields is due to chemical shift anisotropy (CSA).
However, at lower magnetic fields, such as in the stray magnetic field
experienced by the solution during transfer between the polarizer
and MR system, paramagnetic relaxation dominates32 and the differ-
ence in T1 between the two types of solutions is expected to be much
greater. Therefore, in a clinical setting where the transfer time is
much longer and the solution might be located in a magnetic field as
low as earth’s magnetic field, quenching the radical will help main-
tain the 13C polarization until the solution is injected.33 Removing
paramagnetic relaxation of the hyperpolarized nuclei will also bene-
fit other research fields that use dissolution DNP as a signal enhance-
ment method, such as low field NMR or the study of long-lived sin-
glet states.34,35
ment in 13C polarization or significant change in µ-wave spectrum
(Sup. Fig. 3) could be detected using d5-PhGA, despite its 10% nar-
rower ESR line width at X-band as compared to unlabeled PhGA.
Again, this is most likely due to the non-negligible g-anisotropy of
the PhGA-derived radical, which is exacerbated at 3.35 T as com-
pared to X-band ESR measurements typically performed at 0.3 T.
At 3.35 T, the optimal concentration for OX063 trityl radical is
15-21 mM15,28 and that of TEMPOL is 30-50 mM.29 Hence, since the
ESR line width of the PhGA-derived radical is only slightly broader
than that of OX063, it was assumed that 15-25 mM of radical should
be photo-generated in the PhGA samples in order to polarize 13C-
enriched substrates using a HyperSense polarizer. This radical con-
centration can be achieved by irradiating 1 M PhGA for 150-200 s
with a light source. Increasing the PhGA concentration in solution
increased the photo-generated radical concentration up to 40 mM.
Previous studies showed that by adding ~1 mM Gd3+ to a trityl-
doped sample, 13C polarization could be greatly improved, possibly
because Gd3+ causes a shortening in the T1 of the DNP-active elec-
tron spin in the low temperature regime.26,27 We observed a similar
trend in [2-13C]DHAc samples prepared with PhGA-derived radical
and doped with Gd3+, with a nearly 2-fold increase in 13C liquid-state
polarization.
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The impurities observed in the hyperpolarized 13C MR spectra of
[2-13C]DHAc were also observed in a thermally-polarized sample
that had neither been photo-irradiated nor hyperpolarized. This
shows that the molecule of interest stays intact during both the
photo-irradiation and the dissolution DNP processes.
In analogy with what has been observed with photo-irradiated
beads of frozen PA, the only expected recombination products of the
PhGA-derived radical are CO2 and benzoic acid.10 Electrospray ion-
ization mass spectrometry analysis showed benzoic acid as the sole
product of PhGA photo-irradiation (Sup. Fig. 5). This is further sup-
ported by the results of Lippert et al. who showed that, when reacted
with hydrogen peroxide, the only product of hyperpolarized [2-
13C]PhGA is [1-13C]benzoic acid.18
The radical yield with photo-irradiated PhGA depended on pH.
The highest radical yield was obtained when the solution containing
the precursors was acidic, below the pKa of PhGA (pKa = 2.3), and it
decreased as pH was increased. A similar effect has been observed
for UV-irradiated aqueous solutions of PA.30 Therefore, PhGA is a
radical precursor that is better adapted for acidic formulations of
DNP samples. Conversely, TEMPOL cannot be used to polarize
acidic samples because it spontaneously degrades.
PhGA has itself been shown to be an interesting probe for detect-
ing hydrogen peroxide using hyperpolarized 13C MR.18 The pro-
posed method herein could obviously be used to hyperpolarize 13C-
PhGA samples following photo-irradiation with visible light. The
presence of a 13C spin on the ketone carbon will broaden the ESR
spectrum through an additional hyperfine coupling and hence most
likely reduce its efficiency for DNP at 3.35 T, but as already shown
with 13C-PA this can be mitigated by increasing the magnetic field of
13C MR measurements of hyperpolarized [2-13C]DHAc metab-
olism in vivo
PhGA is a moderate acid (pKa = 2.3) with LD50 = 180 mg/kg in
mice, 36 and is excreted via urinary elimination.37 In fact, a pharmaco-
kinetic study where PhGA was injected intravenously into rats con-
firmed that PhGA did not metabolize and that it was excreted via uri-
nary elimination within ~50 min (apparent first-order rate of elimi-
the polarizer and using µ-wave frequency modulation.27
nation of PhGA is ~ 0.1025 min-1).38 The injection of 400 µl of the
hyperpolarized [2-13C]DHAc solution (50 mM) containing 5 mM
of PhGA (~LD50/20) into a 32-g mouse did not affect monitored
physiological parameters. Nevertheless, daily exposure to large
doses of PhGA (above 200 mg/kg) should be avoided because of
potential neurotoxicity due to its amination product, a-phenlygly-
cine, which leads to depletion of striatal dopamine.39 Less than 100
µM of CO2 and benzoic acid were expected to be present in the so-
lution following recombination of the PhGA-derived radical. The
CO2 most likely escaped during the dissolution process and such a
low concentration of benzoic acid is below the doses used therapeu-
tically in humans.40
Hyperpolarization of [2-13C]DHAc with PhGA-derived radical
The highest liquid-state 13C polarization – measured in a phantom
16 s after dissolution and obtained after having positively polarized
a [2-13C]DHAc sample containing photo-generated PhGA radical –
was 5%. The estimated liquid-state 13C polarization in the sample
negatively polarized that was injected in the mouse for in vivo appli-
cation was 8.7% 16 s after dissolution. This is approximately 2 times
lower than the maximum 13C polarization obtained with OX063rad-
ical. In the framework of a potential clinical study, part of this differ-
ence could be recovered thanks to the fact that no filtration and qual-
ity control for residual radicals would be required, possibly allowing
for a more rapid release of the dose for injection. In addition, because
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Many liver pathologies, such as non-alcoholic fatty-liver disease,
Page 8 of 17
Mathematical description of the hyperpolarized signal corrected by
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are characterized by an aberrant glucose metabolism. Hyperpolar-
ized [2-13C]DHAc – which follows gluconeogenesis, glycolysis and
fatty acid synthesis – has been previously used to study hepatic me-
tabolism. For example, the metabolic products of hyperpolarized [2-
13C]DHAc in perfused mouse livers reported on differences between
fed and fasted metabolic states.14 Hepatic metabolic changes after a
fructose injection were also detected in vivo using hyperpolarized
[2-13C]DHAc.16
Here, the main metabolic product of hyperpolarized
[2-13C]DHAc in the liver, glycerol 3-phosphate, was clearly seen in
the 13C MR spectrum as a doublet (75.7 and 71.8 ppm), with glycer-
aldehyde 3-phosphate in the middle at 73.8 ppm. Smaller peaks were
also detected, which are probably metabolites of the gluconeogenic
and glycolytic pathways, including lactate, glucose and glycerol, as
reported in a 1H-decoupled spectrum of perfused liver.14 Although it
has been observed in previous in vivo studies,16 the [2-13C]phos-
phoenolypyruvate resonance (~152 ppm) was not observed in this ex-
periment because it was outside the excitation bandwidth of the pulse
used for radiofrequency excitation.
flip angle and T1 relaxation.
Pg. S3:
Supp. Fig. 1. ESR spectrometer calibration curve.
Pg. S4:
Supp. Fig. 2. Radical yield as a function of photo-irradiation time of
PhGA, PA and DHAc.
Supp. Fig. 3. Solid-state microwave spectra of [2-13C]DHAc doped
with unlabeled and perdeuterated PhGA.
Pg. S5:
9
Supp. Fig. 4. First derivative and absorption ESR spectra of solutions
containing OX063, TEMPOL, photo-irradiated PhGA, or photo-ir-
radiated PA.
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Pg. S6:
Supp. Fig. 5. Electrospray ionization mass spectrometry analysis of
the mixture after 2h of photoirradiation of PhGA.
Pgs. S6-S9:
Synthesis data for the synthesis of deuterated PhGA.
AUTHOR INFORMATION
Corresponding Author
The 13C T1 of [2-13C]DHAc in vivo was ~16 s at 7 T. This is sig-
nificantly shorter than the ~25 s previously reported in a 3 T MRI
scanner,15 supporting the conclusion derived from our phantom ex-
periments that CSA is an important relaxation mechanism at high
field. The apparent signal decay time constants of the metabolic
products and DHAc hydrate were similar to that reported in rats in
vivo.16 The DHAc-to-metabolite ratio was ~70, which is about 2.5
times higher than that reported in perfused liver.14 This is consistent
with the fact that regions outside the liver contribute to the DHAc
signal, while its metabolites are mainly produced in the liver. These
data show that the method proposed herein can be used for meta-
bolic studies of the liver in vivo.
* Irene Marco-Rius; University of Cambridge, UK; irene.marco-
rius@cruk.cam.ac.uk
Author Contributions
All authors have given approval to the final version of the manuscript.
Funding Sources
European Union’s Horizon 2020 European Research Council (ERC
Consolidator Grant) under grant agreement No 682574 (ASSIMILES),
a Cancer Research UK Programme grant (17242) and by the CRUK-
EPSRC Imaging Centre in Cambridge and Manchester (16465). FK
and SP received founding from a Marie Curie ITN studentship under
grant agreement No 642773 (EUROPOL). AC received funding from a
Marie Sklodowska-Curie grant agreement no. 713683 (COFUNDfellows-
DTU).
CONCLUSIONS
We have described a method to generate a photo-induced radical
for dissolution DNP in samples containing photo-sensitive mole-
cules. The method was used to hyperpolarize [2-13C]DHAc with the
radical precursor PhGA using the commercial HyperSense polarizer.
The non-persistent radical generated by photo-irradiation of PhGA
was quenched after dissolution, increasing the 13C longitudinal relax-
ation time. The liquid-state 13C polarization was only half of the max-
imum polarization obtained with OX063 trityl radical but was nev-
ertheless sufficient to report on the metabolism of the gluconeogenic
and glycolytic probe [2-13C]DHAc in vivo. The 13C polarization
could be improved by using a higher field hyperpolarizer and opti-
mizing the photo-generated radical concentration. Photo-irradiated
PhGA could also become an attractive polarizing agent for the re-
mote production of hyperpolarized 13C-molecules since it was
demonstrated that non-persistent photo-generated radicals can be
annihilated in the solid state.11
ACKNOWLEDGMENT
The authors would like to thank Dr. De-en Hu, Dominik McIntyre and
Madhu Basetti for technical help; Dr. Michael Tayler (University of
Cambridge) and Mark Van Criekinge (University of California San
Francisco) for fruitful discussions. This work is part of a project that has
received funding from the European Union’s Horizon 2020 European
Research Council (ERC Consolidator Grant) under grant agreement
No 682574 (ASSIMILES). FK and SP received founding from the Eu-
ropean Union’s Horizon 2020 research and innovation program under
the Marie Sklodowska-Curie grant agreement No 642773
(EUROPOL). AC received funding from the European Union's Horizon
2020 research and innovation program under the Marie Sklodowska-Curie
grant agreement no. 713683 (COFUNDfellowsDTU).
ABBREVIATIONS
ASSOCIATED CONTENT
Supporting Information
MR, magnetic resonance; PhGA, phenylglyoxylic acid; PA, pyruvic
acid, DMSO, dimethyl sulfoxide; DHAc, dihydroxyacetone; G3P,
glycerol 3-phosphate; LN2, liquid nitrogen; DNP, dynamic nuclear
polarization; PBS, phosphate saline buffer; µ-wave, microwave.
PDF containing supporting figures, mathematical description of the
hyperpolarized signal corrected by flip angle and T1 relaxation, and
details on the perdeuteration of phenylglyoxylic acid.
Pg. S2:
Scheme 1. Radical production upon photo-irradiation of phenylgly-
oxylic acid and pyruvic acid at 77 K.
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Hyperpolarized [13C] Pyruvate Documentation Page
Imaging Program
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Cancer
(CIP)
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Figure 1. UV-Vis absorption spectra (left axis) and light source power distributions (right axis). Power
profiles were provided by the manufacturer (Dymax Europe GmbH, Wiesbaden, Germany).
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Figure 2. X-band ESR spectra of frozen droplets in LN2, photo-irradiated with a broadband (BlueWave 75) or
a narrowband (VisiCure 405) light source. (a) PhGA (7.1 M) in 1:1 water/glycerol (v/v). (b) PA (7.0 M) in
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are caused by the hyperfine coupling to the proton spins of the methyl groups. The units on the y-axis are
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Figure 3. Photo-irradiation of PhGA dissolved in 1:1 glycerol/water (v/v). (a) 4-μl-bead of PhGA in LN
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yield vs. PhGA concentration using either the broadband (blue open circle) or the narrowband light source
(red dots). Radical yield is reported as mean ± standard deviation across 3 measurements.
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Figure 4. Solid-state microwave spectra at 3.35 T and 1.25 K: (red open circles) photo-irradiated [2-
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C]DHAc solution (8 M) in H O doped with 1 M PhGA (18 mM radical); (black dots) [2- C]DHAc solution (8
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M) in H O/DMSO (v/v) doped with 21 mM OX063; (green triangles) photo-irradiated [1- C]PA solution (7
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M) in H O/glycerol (65 mM radical).
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Figure 5. (a) Representative C MR spectrum (sum of 180 spectra) of a hyperpolarized solution containing
3+
40 mM [2-13C]DHAc, 5 mM PhGA and 6 μM Gd
in PBS. The MR acquisition started 16 s after the
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beginning of the dissolution process with a repetition time = 1 s and nominal flip angle = 9 º. (b) C MR
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spectra of thermally-polarized solutions at 300 K and 14.1 T. All samples contained [2- C]DHAc (40 mM-70
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mM) and PhGA (4.5-8.0 mM) in PBS with 10% H O. [ C]urea was added to samples (i) and (ii) after
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dissolution as a reference. These samples were made from frozen beads of 8 M [2- C]DHAc and 1 M PhGA
in water. (i) Frozen beads had been irradiated using a narrowband light source (VisiCure 405 nm) for 200 s
and dissolution DNP performed on them. (ii) 1.2 mM Gd
irradiation and dissolu-tion DNP. (iii) The frozen beads were dissolved in PBS without photo-irradiation or
DNP. (iv) The frozen beads were irradiated for 200 s and melted in PBS, but they did not undergo dissolution
DNP.
3+
had been added to the sample prior to photo-
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Figure 6. (a) Axial and sagittal T -weighted images through the mouse with approximate position of the
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surface coil (red dots represent the cross sections of the coil). (b) C MR spectrum acquired in vivo at 7 T
following the intravenous injection of 400 μl of a hyperpolarized solution into a mouse. The solution was
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in PBS. The previously reported resonance
detected at ~89 ppm has not been assigned. DHAc region: sum of 40 spectra. Region with the metabolic
products of DHAc and DHAc hydrate: sum of the first 70 spectra.
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