Imaging of Hydroxyl-Radical Generation Using Dynamic Nuclear Polarization-Magnetic Resonance Imaging and a Spin-Trapping Agent

Article abstract of DOI:10.1021/acs.analchem.0c02331

Reactive oxygen species (ROS) play an important role in cell metabolism, but they can cause oxidative damage to biomolecules. Among ROS, the hydroxyl radical (·OH) is one of the most reactive molecules in biological systems because of its high reaction rate constant. Therefore, imaging of ·OH could be useful for evaluation of the redox mechanism and diagnosis of oxidative diseases. In vivo dynamic nuclear polarization-magnetic resonance imaging (DNP-MRI) is a noninvasive imaging method to obtain spatiotemporal information about free radicals with MRI anatomical resolution. In this study, we investigated the visualization of hydroxyl radicals generated from the Fenton reaction by combining DNP-MRI with a spin-trapping agent (DMPO: 5,5-dimethyl-1-pyrroline N-oxide) for ·OH. Additionally, we demonstrated the radical-scavenging effect using four thiol-related reagents by DNP-MRI. We demonstrated that DNP enhancement could be induced by the DMPO-OH radical using the DNP-MRI/spin-trapping method and visualized ·OH generation for the first time. Maximum DNP enhancement was observed at an electron paramagnetic resonance irradiation frequency of 474.5 MHz. Furthermore, the radical-scavenging effect was simultaneously evaluated by the decrease in the DNP image value of DMPO-OH. An advantage of our methods is that they simultaneously investigate compound activity and the radical-scavenging effect.

Full text of DOI:10.1021/acs.analchem.0c02331

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Imaging of Hydroxyl-Radical Generation Using Dynamic Nuclear
Polarization-Magnetic Resonance Imaging and a Spin-Trapping
Agent
Shinichi Shoda, Fuminori Hyodo,* Yoko Tachibana, Mamoru Kiniwa, Tatsuya Naganuma, Hinako Eto,
Norikazu Koyasu, Masaharu Murata, and Masayuki Matsuo
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ABSTRACT: Reactive oxygen species (ROS) play an important role in cell metabolism, but they can cause oxidative damage to
biomolecules. Among ROS, the hydroxyl radical (·OH) is one of the most reactive molecules in biological systems because of its
high reaction rate constant. Therefore, imaging of ·OH could be useful for evaluation of the redox mechanism and diagnosis of
oxidative diseases. In vivo dynamic nuclear polarization-magnetic resonance imaging (DNP-MRI) is a noninvasive imaging method
to obtain spatiotemporal information about free radicals with MRI anatomical resolution. In this study, we investigated the
visualization of hydroxyl radicals generated from the Fenton reaction by combining DNP-MRI with a spin-trapping agent (DMPO:
5
,5-dimethyl-1-pyrroline N-oxide) for ·OH. Additionally, we demonstrated the radical-scavenging effect using four thiol-related
reagents by DNP-MRI. We demonstrated that DNP enhancement could be induced by the DMPO-OH radical using the DNP-
MRI/spin-trapping method and visualized ·OH generation for the first time. Maximum DNP enhancement was observed at an
electron paramagnetic resonance irradiation frequency of 474.5 MHz. Furthermore, the radical-scavenging effect was simultaneously
evaluated by the decrease in the DNP image value of DMPO-OH. An advantage of our methods is that they simultaneously
investigate compound activity and the radical-scavenging effect.
INTRODUCTION
Therefore, monitoring of ·OH could be useful for evaluation
of the redox mechanism and diagnosis of oxidative diseases.
Many studies have reported the detection of ·OH generation
with high sensitivity using methods such as the fluorescent
■
Reactive oxygen species (ROS) are produced during both
normal and abnormal cellular metabolism and can play both
beneficial and harmful roles in cell physiology. The beneficial
effect of ROS is that they act as messengers in cell-signaling
pathways and inflammatory responses, such as infusion of
11−13
probe method and chemiluminescence.
However, these
methods are difficult to use for in vivo studies because of
limitations surrounding the permeability of fluorescence
wavelengths (less than 1 mm), and they also provide no
direct evidence of free-radical detection. Electron paramagnetic
resonance (EPR) spectroscopy with a spin-trapping agent is a
1
neutrophils for production of superoxide via NADPH oxidase.
However, ROS also have harmful effects, including oxidative
damage to biomolecules such as lipids, proteins, nucleic acids,
2
,3
and sugars. This oxidative stress might induce when the
balance between ROS generation and the antioxidant defenses
is shifted toward more oxidizing conditions. Oxidative stress
has been implicated in a variety of diseases, such as
neurodegenerative disorders, atherosclerosis, diabetes, inflam-
Received: June 1, 2020
Accepted: October 6, 2020
4
−7
mation, and cancer.
Among ROS, the hydroxyl radical (·
OH) is one of the most reactive molecules in biological
8
−10
systems because of its high reaction rate constant.
©
XXXX American Chemical Society
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convenient and useful technique to detect free radicals and
commercially available and of reagent-grade quality. All
reagents and solvents were of analytical grade, unless otherwise
specified, and used without further purification. Water was
purified with a Milli-Q system from Millipore (Nihon
Millipore K.K., Tokyo, Japan).
14,15
identify ROS by their spectral signals.
In this technique,
short-lived oxygen-derived radicals are trapped by a spin-
trapping agent to form long-lived radicals and generate an EPR
spectrum. One of the most recognized spin-trapping agents is
5
,5-dimethyl-1-pyrroline N-oxide (DMPO), whose spin adduct
Fenton Reaction and Spin-Trapping Assay. Hydroxyl
generates a hydroxyl radical-specific EPR spectrum as DMPO-
OH (Figure 1B). Therefore, imaging of hydroxyl-radical
radicals were generated by the Fenton reaction and trapped
1
6
24,25
with DMPO.
In the Fenton reaction, iron(II) was oxidized
by H O to iron(III), generating a hydroxyl radical and a
2
2
hydroxide ion. The DMPO-OH adduct was obtained by
mixing a DMPO solution with H O and FeSO to a final
2
2
4
volume with PBS. The DMPO, H O , and FeSO final
2
2
4
concentrations used ranged 10−400 mM, 1−400 mM, and 4−
5
mM, respectively.
Detection of Hydroxyl Radicals with DMPO by X-
Band EPR Experiments. EPR spectra were obtained with an
X-band EPR spectrometer (JEOL Ltd. Tokyo, Japan) at room
temperature under the following conditions: microwave
frequency = 9.4 GHz (336 mT); microwave power = 1 mW;
modulation width = 0.06 mT; sweep time = 1 min; sweep
width = ±5 mT; and time constant = 0.03 s. The reaction
mixture contained the following reagents and final concen-
trations: 10 mM DMPO, 5 mM H O , and 5 mM FeSO The
Figure 1. Chemical structure of 5,5-dimethyl-1-pyrroline N-oxide
2
2
4.
(
DMPO) and DMPO-OH and the EPR spectrum of DMPO-OH. (A)
EPR spectrum was recorded 1 min after the reagents were
mixed.
Chemical structure of DMPO and its spin adduct, DMPO-OH. (B)
EPR spectra of DMPO-OH produced by the hydroxyl-radical
generation system (Fenton reaction).
Phantom Imaging of DMPO-OH Using DNP-MRI. In
vitro free-radical imaging was performed with a low-field
DNP−MRI system (Keller) obtained from Japan Redox Inc.
generation enables monitoring of ROS generation and the
antioxidant activity of chemical drugs in oxidative disease.
However, because the image resolution of EPR imaging is
directly dependent upon the linewidth of free-radical
compounds, EPR images’ spatial resolution is relatively low.
Additionally, fast imaging is needed for visualization of spin-
trapped free radicals because of the instability of spin-trapped
adducts. Therefore, EPR imaging technology has not yet
achieved this type of visualization. Recently, Saito et al.
(Fukuoka, Japan). The external magnetic field (B ) for EPR
0
irradiation and MRI was fixed at 15 mT, and the radio-
frequency of the EPR irradiation ranged 460−483 MHz. As a
high-sensitivity local detector, a rectangular round surface coil
(longitudinal direction 20 mm, lateral direction 20 mm) was
used for EPR irradiation.
In DNP-MRI, the DNP phenomenon is induced by
irradiating a resonant microwave adjusted to the resonant
frequency of the electron spin of the free-radical solution.
Therefore, it is necessary to determine the suitable resonant
frequency of the DMPO-OH radical for induction of DNP. A
phantom (200 μL, 5.4 mm inner diameter, and 9 depth) was
prepared for comparison of the DNP phenomenon. In the left
tube of the phantom, DMPO solution was added after H O
13
performed biological imaging using C-labeled DMPO using
1
7
hyperpolarized NMR. They clearly visualized DMPO
metabolism, but they did not show the distribution of
DMPO-OH.
In vivo dynamic nuclear polarization-magnetic resonance
imaging (DNP-MRI) is a noninvasive imaging technology to
obtain spatiotemporal information about free radicals and
2
2
and FeSO were mixed. The final concentrations of DMPO,
4
H O , and FeSO were 400, 400, and 4 mM, respectively.
2
2
4
18,19
redox status with the anatomical resolution of MRI.
The
Another tube inside the phantom was filled with PBS. A
surface coil for EPR irradiation and a DNP−MRI system were
used to obtain the phantom images. To determine the
optimum EPR-resonant frequency of DMPO-OH, DNP-MRI
measurements were performed while changing the EPR
irradiation frequency in steps of 0.5 MHz or 1 MHz from
460 to 483 MHz. The phantom images were obtained by
DNP-MRI 30 s after mixing the H O and FeSO . The
proton signal in tissues includes free radicals, as the DNP effect
can be dramatically enhanced by EPR irradiation at the
resonant frequency of the free radical prior to applying the
MRI pulse sequence. Therefore, DNP-MRI has been used to
investigate the functional status of tissues, such as their redox
2
0,21
22
23
state,
oxygen partial pressure, and malignant ascites
using redox-sensitive nitroxyl radicals. In this study, we
investigated the visualization of hydroxyl radicals by combining
DNP-MRI with spin trapping of hydroxyl radicals using
DMPO and visualization of the radical-scavenging effects by
DNP imaging of DMPO-OH.
2
2
4
scanning conditions for the DNP-MRI were as follows: power
of EPR irradiation = 7 W; flip angle = 90°; repetition time
(TR) × echo time (TE) × TEPR = 500 × 25 × 250 ms;
number of acquisitions = 10; and number of phase-encoding
steps = 32. The image’s field of view (40 × 40 mm) was
represented by 64 × 64 matrix after image reconstruction.
Effects of H O Concentration on DNP-MRI. Various
MATERIALS AND METHODS
■
Chemicals. We purchased DMPO from Dojindo Labo-
ratories, and ferrous sulfate heptahydrate and phorbol 12-
myristate13-acetate (PMA) were purchased from Wako.
Suplatast tosilate (ST) was purchased from Tokyo Chemical
Industry Co., Ltd. (Tokyo Japan) All other chemicals were
2
2
concentrations of H O (1−400 mM) and a constant
2
2
concentration of FeSO (4 mM) were mixed in a four-tube
4
phantom (200 μL, 5.4 mm deep, and 9 mm depth) to
investigate the effect of H O , and then DMPO (0.4 M) was
2
2
B
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added. The EPR irradiation frequency was set to 474.5 MHz
for DNP−MRI measurement. DNP−MRI measurements were
performed 90 s after mixing of DMPO. The scanning
conditions for the DNP−MRI experiment were as follows:
EPR irradiation power = 7 W; flip angle = 90°; TR × TE ×
TESR = 500 × 25 × 250 ms; and number of acquisitions = 10.
Imaging of Neutrophil-Derived DMPO-OH. A total of
1
.2 mL of 4% thioglycollate (TGC) medium (Nissui
Pharmaceutical) was injected into the peritoneum of female
2
6
Slc: ICR mice. Peritoneal cells were harvested by lavage with
Hanks Balanced Salt Solution at 4 h after injection. Cells were
collected by centrifugation at 1000 × g for 5 min at 4 °C, and
7
the supernatant was removed. The neutrophils (4.0 × 10
cells/mL) were mixed with DMPO (700 mM) and PMA (0.01
mM) and incubated at 37 °C for 1 min. Imaging was
performed by DNP-MRI 3 min after mixing.
Imaging of the Hydroxyl Radical-Scavenging Effect.
As antioxidants for hydroxyl-radical generation, we employed
thiols such as dimethyl sulfoxide (DMSO), dimethyl sulfone
(
DMS), dimethyl sulfoniopropionate (DMSP), and ST. The
27
known hydroxyl-radical-scavenging effect of DMSO was
compared with the inhibitory effects of the other thiols above
by EPR, and we confirmed the visualization of those effects
using DNP-MRI. Various concentrations of those compounds
were mixed with Fenton reagents, and the EPR signal and
image intensity were observed by DNP-MRI. The thiol
compounds were added to a mixed solution of DMPO,
H O , and FeSO in a four-tube phantom.
Figure 2. Determination of suitable EPR frequencies of DMPO-OH
for DNP enhancement. (A) DNP images at various EPR irradiation
frequencies at 30 s after mixing the DMPO and Fenton reagents. In
each case, the left tube contains DMPO-OH, and the right tube was
filled with PBS. (B) Plots of DNP image intensity at various EPR
frequencies. DNP-MRI was performed at various EPR frequencies
from 460 to 483 MHz. Image intensity of DMPO-OH including tubes
with (closed circle) or without (open circle) EPR irradiation. Image
intensity of the PBS tube with (closed square) or without (open
square) EPR irradiation.
2
2
4
The concentrations of the solutions subjected to EPR
measurement were 10, 5, and 5 mM for DMPO, H O , and
2
2
FeSO , respectively. The corresponding concentrations were
4
4
00, 400, and 5 mM for DMPO, H O , and FeSO ,
2 2 4
460 to 469 MHz, enhancement was increased above 470 MHz
respectively. The concentrations of all other thiol compounds
were 2.5 mM. The ESR and DNP-MRI measurements were
performed 30 s after mixing of H O and FeSO . The scanning
(
Figure 2B). Maximum enhancement was observed at 474.5
MHz, and then the enhancement decreased at higher
frequencies. The enhancement factor (DNP on/DNP off)
was 3.07 at 474.5 MHz. In contrast, in DMPO-OH solutions
that did not receive EPR irradiation, the MRI signal did not
change. In tubes filled with PBS, the MRI signal always showed
no enhancement regardless of the frequency or presence of
EPR irradiation. The image intensity of DMPO-OH solutions
without EPR irradiation was higher than that of PBS solutions
because of the T shortening of FeSO in the ·OH generation
2
2
4
conditions for the DNP−MRI experiment were as follows:
EPR irradiation frequency = 474.5 MHz; EPR irradiation
power = 7 W; flip angle = 90°; TR × TE × TESR = 500 × 20
×
250 ms; number of acquisitions = 1; slice thickness = 100
mm; field of view = 40 mm; and sampling number = 64. The
inhibition map was calculated using the following formula:
Inhibition map = (1 − Image intensity (DNP on) /Image
Intensity (PBS)) × 100.
1
4
system. These results suggested that the DMPO-OH radical
induces the DNP and generates an enhancement image using
the DNP−MRI system and that the optimal frequency is 474.5
MHz.
RESULTS AND DISCUSSION
■
Figure 1 shows the chemical structure and EPR spectrum of
DMPO-OH, which is the spin-trapping adduct of DMPO and ·
OH. It is known that the spin-trapping agent, DMPO, could
react with the ·OH produced by the Fenton reaction and be
converted to DMPO-OH, which produces the EPR spectrum.
The EPR spectrum of DMPO-OH has four peaks (ratio of
peak heights is 1:2:2:1); therefore, the production of ·OH
generation could be identified by the form of the spectrum. To
determine the appropriate EPR-resonant frequency of the
DMPO-OH free radical for obtaining the DNP effect, DNP-
MRI was performed at various EPR frequencies from 460 to
Next, we examined the dependence of DNP−MRI signal
enhancement on the H O concentration (Figure 3A). As
2
2
expected, enhancement was positively associated with the
H O concentration (Figure 3B). We also confirmed negative
2
2
enhancement at lower H O concentrations, which we also
2
2
19
observed previously. In these DNP−MRI experiments, we
could not use high concentrations of FeSO to increase the
4
generation of ·OH because FeSO decreases the T relaxation
4
1
time and also decreases the image intensity on regular MRI at
higher concentrations. No effect of FeSO on DNP-MRI at 16
4
4
83 MHz (Figure 2 and Figure S1). Figure 2A shows typical
mT was observed at concentrations up to 4 mM. Therefore, we
examples of DNP images of DMPO-OH at various EPR
frequencies. Imaging with DMP-MRI clearly visualized the
enhancement of phantom tubes, including the hydroxyl-radical
generation system between 471 and 480 MHz. Although the
DNP enhancement in the tube including the DMPO-OH
showed no enhancement at EPR irradiation frequencies from
used a 4 mM FeSO solution in the Fenton reaction. In
4
addition, we observed enhancement by the activated
neutrophil-derived DMPO-OH on EPR and DNP−MRI
studies (Figure 3C). DNP enhancement on the DNP ON
image in phantom including activated neutrophils and DMPO
was observed compared to the DNP OFF image. The EPR
C
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Figure 4. Visualization of the radical-scavenging effect of DMPO on
the hydroxyl-radical generation system using DNP-MRI. (A) EPR
spectra of DMPO-OH and its inhibition rate after treatment with
various concentrations of DMSO. (B) Enhanced DNP images and
inhibition rates of DMPO-OH treated with various concentrations of
DMSO obtained by DNP-MRI. All tubes included 10 mM DMPO, 5
mM H O , and 5 mM FeSO . DMSO (0, 0.1, 0.25, 0.5, 0.75, 1, 2.5, 5,
Figure 3. DNP enhancement by modulation of H O concentration
in DNP-MRI and imaging of neutrophil-derived DMPO-OH. (A)
2
2
DNP images of phantoms filled with various concentrations of H O ,
2
2
2
2
4
acquired by DNP-MRI at 475 MHz. (B) Plots of DNP image
intensity with various H O concentrations. (C) Imaging of activated
or 10 mM) was added as a radical scavenger.
2
2
neutrophil-derived DMPO-OH. Neutrophils were collected from
mouse abdomen 4 hr after TGC treatment. DNP-MRI and EPR
spectroscopy were performed 1 min after incubation of neutrophils
with PMA and DMPO.
redox biology has become more important in the development
of understanding about the redox regulation system and signal
processes. Thiol compounds have antioxidant capabilities to
31
reduce oxidative stress. In this study, we selected the thiol
compounds to simultaneously observe their radical-scavenging
effects by DNP-MRI. Figure 5A shows the thiol compounds
used for EPR and DNP-MRI in this study. First, the radical-
scavenging effects of these thiol compounds were compared by
EPR spectroscopy.
In the EPR spectroscopy experiment (Figure 5B), ST
showed the strongest inhibition effect against hydroxyl-radical
generation. The inhibition rates of DMSO, DMS, DMSP, and
ST were 60, 2, 40, and 90%, respectively. DMSP and DMS
showed lower inhibition effects than DMSO. In the DNP−
MRI experiment (Figure 5C), the inhibitory effects were
compared in the images acquired by DNP-MRI, and the
difference images clearly showed the inhibition-dependent
manner of the thiol compounds’ action. Enhancement by
DMPO-OH on ST images including phantom was significantly
lower than that of the other thiols. This suggests that ST
suppressed the production of DMPO-OH and reduced the
effects of DNP in DNP-MRI.
spectrum of DMPO-OH in a neutrophil suspension with
DMPO was also observed. It is known that DMPO-OH is
generated from DMPO-OOH decomposition in the neutro-
28,29
phil-induced ROS and DMPO system.
In this study, we
demonstrated that the endogenously produced DMPO-OH
from the cells could be imaged by DNP-MRI.
To investigate the imaging of the scavenging effect by DNP-
27
MRI, we used DMSO, which has an ·OH-scavenging effect in
the ·OH generation system. At first, an EPR study was
performed to confirm the inhibition effect of DMSO against ·
OH generation (Figure 4A). The EPR signal decreased with
increasing DMSO concentration, and it was completely
inhibited by 10 mM DMSO. In the DNP−MRI experiments,
DMSO had a similar effect (Figure 4B). Although the image
intensities of the phantom were almost the same value with
DNP off, the image intensity with DNP on decreased with the
addition of DMSO. At 5 mM DMSO, enhancement was
completely suppressed. These results suggest that DNP-MRI
can detect the radical-scavenging effect and might be useful for
simultaneous imaging of various samples to confirm radical-
scavenging ability.
It is possible for DNP-MRI to simultaneously visualize
radical-scavenging effects in multiple samples, including
various thiol compounds. Although DMSO has a strong ·OH
2
7
The sulfur biochemistry of the thiol group has unique
features, and highly abundant low-molecular-weight thiol
counterparts are carefully balanced to maintain the redox
homeostasis in various cellular compartments, protecting
radical-scavenging effect, in this experiment, ST more
effectively suppressed DMPO-OH production. In Japan, ST
is widely used as an oral asthma control drug and has been
prescribed to adults and pediatric patients with allergic rhinitis
30
32−34
organisms from oxidative stress. Therefore, the role of thiol
and atopic dermatitis for more than 20 years.
Recently,
D
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Figure 5. Simultaneous imaging of the hydroxyl-radical-scavenging effect of various thiol compounds using EPR and DNP-MRI. (A) Chemical
structures of thiol compounds. (B) EPR spectra and inhibition rates of DMPO-OH by various thiol compounds. All tubes included 10 mM DMPO,
5
mM H O , and 5 mM FeSO . The concentration of each thiol compound was 2.5 mM. (C) Simultaneous DNP imaging of DMPO-OH and the
2 2 4
inhibition rate map in DMPO-OH tubes including various thiol compounds (*, P < 0.05). All tubes included 400 mM DMPO, 400 mM H2O2,
and 4 mM FeSO4. The concentration of each thiol compound was 2.5 mM.
Izumi et al. reported that administration of ST reduced
oxidative stress in radiation-induced lung injury and sup-
and could evaluate the antioxidant efficacy in multiple samples.
However, we used a high concentration of H O to investigate
2
2
35
pressed inflammation and fibrosis in mice. They suggested
that ST might also be useful as an antioxidant in the treatment
of oxidative stress-mediated diseases. Additionally, Fukuhara et
al. reported that ST protected the lungs against oxidative stress
induced by hyperoxic exposure and prolonged the survival of
the possibility of detection of hydroxyl-radical imaging by the
DNP−MRI system. Therefore, it is necessary to increase the
sensitivity of DNP-MRI or increase the averaging using long-
live spin-trapping agents for application in living organisms.
A relatively high dose of H O has been used in the Kochi
2
2
36
mice exposed to hyperoxia. They demonstrated that ST has
an ·OH-scavenging effect using an EPR spin-trapping method
with DMPO as a reference trapping agent. In our experiments,
ST showed a strong radical-scavenging effect, even better than
that of DMSO. An advantage of our methods is that they
simultaneously evaluate the activity of compounds needed to
investigate the radical-scavenging effect in this case.
oxydol-radiation therapy for treatment of the unresectable
37,38
carcinomas (KORTUC) II radiosensitizer.
Linear accel-
erator-based stereotactic radiotherapy has little effect on most
advanced neoplasms. The novel KORTUC II radiosensitizer,
which contains H O and sodium hyaluronate, was developed
2
2
to address this shortcoming. The effectiveness of KORTUC II
for the treatment of chemotherapy-resistant supraclavicular
lymph node metastases, recurrent breast cancer, and stage-IV
In this study, we demonstrated that DNP-MRI could
visualize the generation of ·OH as a DMPO-OH free radical
39−41
primary breast cancer has previously been demonstrated.
E
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Because KORTUC II administers radiation therapy after
injecting a drug containing H O into the tumor tissue, it is
Labour Sciences Research Grant (Research on Publicly
Essential Drugs and Medical Devices) from the Ministry of
Health, Labour and Welfare of Japan; and Special Coordina-
tion Funds for Promoting Science and Technology (SCF
funding program “Innovation Center for Medical Redox
Navigation”). This work was also supported by JSPS
KAKENHI (Grant Numbers 18H02765 and 19H03358). We
thank Richard Lipkin, PhD, from Edanz Group (www.
edanzediting.com/ac) for editing a draft of this manuscript.
2
2
assumed that there is a high concentration of H O in the
2
2
tumor region. Therefore, our methodology might be used to
understand the localization of radicals within the treatment site
by administration of DMPO. The clinical application of this
method is expected to expand to new theranostics that
sensitizes the effects of radiation therapy and simultaneously
visualizes the treatment area.
In conclusion, we succeeded in visualizing hydroxyl radicals
using DNP-MRI and further showed that the hydroxyl-radical-
scavenging effect could be evaluated using DNP-MRI.
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AUTHOR INFORMATION
Corresponding Author
Fuminori Hyodo − Department of Radiology, Frontier Science
for Imaging, School of Medicine, Gifu University, Gifu 501-
(10) Reyhani, A.; McKenzie, T. G.; Fu, Q.; Qiao, G. G. Macromol.
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1
194, Japan; Innovation Center for Medical Redox Navigation,
Kyushu University, Fukuoka 812-8582, Japan; orcid.org/
000-0002-9266-9937; Phone: +81-58-230-6437;
Email: hyodof@gifu-u.ac.jp; Fax: +81-58-230-6440
(
0
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Authors
(16) Ranguelova, K.; Mason, R. Magn. Reson. Chem.: MRC 2011, 49,
Shinichi Shoda − Department of Radiology, Gifu University,
Gifu 501-1194, Japan
Yoko Tachibana − Innovation Center for Medical Redox
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.analchem.0c02331
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1
Notes
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The authors declare no competing financial interest.
(
ACKNOWLEDGMENTS
■
This work was supported by the Medical Research and
Development Programs Focused on Technology Transfer,
Development of Advanced Measurement and Analysis Systems
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