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3-O-Methyl-D-glucose mutarotation and proton exchange rates assessed by 13C, 1H NMR and by chemical exchange saturation transfer and spin lock measurements


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3-O-Methyl-d-glucose (3OMG) was recently suggested as an agent to image tumors using chemical exchange saturation transfer (CEST) MRI. To characterize the properties of 3OMG in solution, the anomeric equilibrium and the mutarotation rates of 3OMG were studied by ¹H and ¹³C NMR. This information is essential in designing the in vivo CEST experiments. At room temperature, the ratio of α and β 3OMG anomers at equilibrium was 1:1.4, and the time to reach 95% equilibrium was 6 h. The chemical exchange rates between the hydroxyl protons of 3OMG and water, measured by CEST and spin lock at pH 6.14 and a temperature of 4 °C, were in the range of 360–670 s⁻¹.
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Journal of Biomolecular NMR
ISSN 0925-2738
Volume 72
Combined 1-2
J Biomol NMR (2018) 72:93-103
DOI 10.1007/s10858-018-0209-y
3-O-Methyl-d-glucose mutarotation and
proton exchange rates assessed by 13C, 1H
NMR and by chemical exchange saturation
transfer and spin lock measurements
Michal Rivlin & Gil Navon
1 23
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Journal of Biomolecular NMR (2018) 72:93–103
3-O-Methyl-d-glucose mutarotation andproton exchange rates
assessed by13C, 1H NMR andbychemical exchange saturation transfer
andspin lock measurements
MichalRivlin1· GilNavon1
Received: 24 June 2018 / Accepted: 5 September 2018 / Published online: 10 September 2018
© Springer Nature B.V. 2018
3-O-Methyl-d-glucose (3OMG) was recently suggested as an agent to image tumors using chemical exchange saturation
transfer (CEST) MRI. To characterize the properties of 3OMG in solution, the anomeric equilibrium and the mutarotation
rates of 3OMG were studied by 1H and 13C NMR. This information is essential in designing the invivo CEST experiments.
At room temperature, the ratio of α and β 3OMG anomers at equilibrium was 1:1.4, and the time to reach 95% equilibrium
was 6h. The chemical exchange rates between the hydroxyl protons of 3OMG and water, measured by CEST and spin lock
at pH 6.14 and a temperature of 4°C, were in the range of 360–670s−1.
Keywords 3-O-Methyl-d-glucose (3OMG)· Mutarotation kinetics· Anomeric equilibrium· NMR spectroscopy· Chemical
exchange saturation transfer (CEST)· Spin lock (SL)
α0 The initial concentration of alpha 3OMG
α Alpha anomer of 3OMG
β Beta anomer of 3OMG
BM Bloch–McConnell equations
c The time elapsed before the measurements had
CESL Chemical exchange spin lock
CEST Chemical exchange saturation transfer
CW Continuous wave
The fraction of the labile proton
HSQC Heteronuclear single quantum correlation
K Equilibration constant
k1 The forward mutarotation rate constant
k2 The reverse mutarotation rate constant
k k1 + k2
kb The exchange rate from the labile proton to
M0 The water resonance intensity at a very far irra-
diation offset
MTRasym Magnetization transfer asymmetry ratio
NMR Nuclear magnetic resonance
NOE Nuclear overhauser effect
3OMG 3-O-Methyl-d-glucose
QUESP Quantification of exchange rate using varying
saturation power
The water longitudinal relaxation rate
R NMR spin relaxation in the rotating frame
SL Spin lock
T1 The longitudinal relaxation time
The transverse relaxation time of water in the
absence of chemical exchange
ω1 CW irradiation amplitude
The frequency difference from the water peak
The normalized Z magnetization after satura-
tion at the CEST resonance
The normalized Z magnetization after satura-
tion without CEST effect (estimated from the
opposite frequency)
Interest is growing in chemical exchange saturation trans-
fer (CEST) MRI techniques as a means of producing new
contrast in magnetic resonance images with potential
usefulness in the clinic (Jones etal. 2018). CEST effect
obtained by functional exchangeable hydroxyl protons
groups of glucose and its analogues has been shown to be
* Gil Navon
1 School ofChemistry, Tel Aviv University, TelAviv, Israel
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a promising modality for the noninvasive diagnosis of can-
cer (Chan etal. 2012; Walker-Samuel etal. 2013; Rivlin
etal. 2013, 2014; Xu etal. 2015; Rivlin and Navon 2016,
2018; Wang etal. 2016; Longo etal. 2017). Also promis-
ing is the use of the non-metabolized (Hwang etal. 1992;
Xu and Krnjevic̀ 2001; Csaky and Glenn 1957; Jay etal.
1990) glucose analogue 3-O-methyl-d-glucose (3OMG) as
a CEST and CESL MRI agent for the detection of tumors
and the effects of stroke (Rivlin etal. 2014; Rivlin and
Navon 2018; Jin etal. 2018; Zu etal. 2018). 3OMG is
generally considered nontoxic, but detailed studies of its
toxicity are lacking. The present invitro work examines
the equilibria and kinetics of 3OMG required to assure
proper and effective use of this innovative product. Like
other cyclic forms of glucose (Los etal. 1956; Yamabe and
Ishikawa 1999) and its analogues, in water, 3OMG under-
goes a mutarotation reaction (Fig.1a) until equilibrium
between its α and β anomeric forms is reached (1H NMR
spectrum of 3OMG in D2O at 4°C is shown in Fig.1b).
The knowledge of the rate of mutarotation and the value
of the anomeric ratio at different temperatures and pH val-
ues is essential for the design of the MRI experiments.
Determination of the proton exchange rates can help in the
estimation of the tissue pH.
Fig. 1 a 3OMG mutarotation equilibrium, characterized by forward (k1) and reverse (k2) mutarotation rate constants. b 1H NMR spectrum of
0.5M 3OMG in D2O at T = 4°C, B0 = 11.7T
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Mutarotation kinetics
Because the mutarotation reaction is very slow, and the ratio
of the α and β anomeric forms may affect the results of the
CEST measurements in invivo experiments, it is essential
to determine the precise rate of mutarotation as a function
of temperature and pH.
3OMG crystalizes in the α anomer form and the mutaro-
tation reaction takes place upon its dissolution, reaching an
equilibrium between the two anomers. If α and β are the
concentrations of the α and β-anomers at time t, respectively,
is the initial concentration of α-3OMG and
is the
time elapsed from the dissolution until the beginning of the
The equilibration constant is defined as K = βeqeq.
In the present work 1H and 13C NMR spectroscopy were
used to follow the mutarotation process in freshly prepared
3OMG solutions.
α-3OMG in powdered form and D2O (99.9% isotopically
enriched) were purchased from Sigma-Aldrich Chemical
(Rehovot, Israel).
Determination of3OMG mutarotation reaction
Mutarotation was initiated by dissolving samples of 3OMG
in D2O solution. Kinetic measurements were begun as
quickly as possible with a Bruker DRX 500MHz spectrom-
eter, set to the desired temperature.
1H NMR spectra were recorded every few minutes and
α and β anomeric peaks were determined by their intensi-
ties and integrals. The curve plots and the kinetics calcula-
tions were made using Matlab software (Matlab R2017a, the
MathWorks, Inc). At steady state, α and β peaks’ intensities
were normalized according to the ratio of their integrals.
To select relaxation delay (d1), longitudinal relaxation
times, T1, were measured using the ‘inversion-recovery’
pulse sequence. The longest T1 for the high temperature
(T = 42°C) was 13.8, 3.6 and 2.2s for the HDO, α and β
anomeric peaks, respectively (for deuterated 3OMG solu-
tion) and thus, the relaxation delay was selected as 3–5T1.
Acquisition parameters were as follows: spectral width
3kHz, data size 32K, pulse width 3µs, relaxation delay
30–60s (depending on the temperature setting), acquisition
time 5.5s.
13C NMR spectra were recorded at 125.76MHz using
a Bruker DRX 500MHz spectrometer. Each sample was
scanned overnight with 1H decoupling. Some of the 13C
NMR measurements were performed without 1H decoupling.
Acquisition parameters were as follows: spectral width 7.5
kHz, data size 16K, pulse width 7.7µs, relaxation delay
12s, acquisition time 2.2s. The longest relaxation time T1
was determined for T = 45 °C as 1.5 and 1.7s for the α
and β anomeric peaks, respectively (for deuterated 3OMG
The conventional heteronuclear single quantum correla-
tion (HSQC) spectra were recorded with 256 increments,
32 scans, a recovery delay of 9s and an acquisition time
of 0.6s.
All data were recorded and analyzed with TopSpin 3.1
software (Bruker).
CEST measurements
CEST NMR experiments were performed on 20mM 3OMG
solutions (containing 5mM PBS and 5% D2O for lock) at pH
values of 6.14–7.47, and measured at 4°C and 37°C using
a Bruker DRX 500MHz spectrometer. The water intensity
was measured after long pre-saturation pulse (3–5s) at a
series of offset frequencies in the range of ± 5ppm. B1 power
was varied in the range of 0.5–5.9µT. The CEST effect is
described here in terms of a magnetization transfer asym-
metry ratio (MTRasym), a parameter that is defined as:
is the difference between the irradiation frequency
and the water resonance,
are the water
intensities after a long pre-saturation pulse at the offset
frequency and the offset frequency on the opposite side of
the water signal, respectively. M0 is the water resonance
intensity at a very far irradiation offset (at which the water
resonance intensity is unaffected by functional groups).
To define the CEST signal by the MTR asymmetry of the
normalized Z magnetization, two Z values were used: the
normalized Z magnetization after saturation at the CEST
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Zlab =Z
and the reference scan which rep-
resents the saturation without CEST effect that can be esti-
mated from the opposite frequency
Zref =Z(−Δ
CEST quantification
The exchange rates between the 3OMG hydroxyl protons and
water were estimated using the dependence of the water Z
magnetization on the CW irradiation amplitude ω1, identified
as quantification of exchange rate using varying saturation
power (QUESP) (McMahon etal. 2006) equation. The inverse
Z-spectrum analysis (Zaiss etal. 2014, 2018; Wu etal. 2015)
that allows for the correction of spillover effects was used:
is the fraction of the labile proton,
is its exchange
is the frequency difference from the water peak, and
is the water longitudinal relaxation rate. The term
that was neglected in a previous publication (Zaiss etal.
2018) was included in the present analysis because of the
proximity of the hydroxyl peaks to the water peak. Nonlinear
fit was used rather than the linear omega plot (Wu etal.
2015; Meissner etal. 2015), since in the latter, excessive
weight is given to low values of ω1.
Spin lock measurements
For on-resonance
dispersion experiments, nuclei
were excited by a 90° pulse and then spin locked by a long CW
pulse and variable B1 fields.
Spin lock quantification
For the calculation of the exchange rate from R dispersion
measurements, data were fitted to Eq.(8) given by Trott and
Palmer (2002):
is the transverse relaxation time of water in the
absence of chemical exchange, and the definitions of
are the same as above. In the fitting,
were kept as free parameters.
Results anddiscussion
The assignment of the aliphatic and the hydroxyl protons
of 3OMG in aqueous solution was based on the known
assignment of α-3OMG in DMSO (http://www.sdbs. t_frame _disp.cgi?sdbsn
o=13453 ; Onche etal. 2013). 1H NMR spectra at 18°C
before and after reaching anomeric equilibrium are pre-
sented in Fig.2a, b, respectively. Both α-H1 and β-H1
resonances are doublets with JH−H of 3.70 and 7.56Hz,
respectively. While the 13C NMR is not expected to differ
greatly between the two solvents, this is different for 1H
spectra. Hence the 13C–1H HSQC experiment was used for
the assignments of the aliphatic 3OMG protons (Fig.3).
Since the spectral peaks of the hydroxyl protons are well
separated at low pH and low temperature (Fig.4), their
identification was based on the known assignment in
t_frame _disp.cgi?sdbsn o=13453 ) as well as in analogy
to the known assignment of glucose (Onche etal. 2013).
The hydroxyl protons of the 3OMG are observable only
at low pH and low temperature and therefore are not suit-
able for the study of the mutarotation rates. Since the large
water signal interferes with following the aliphatic pro-
tons, the experiments were run in D2O solutions. It should
be noted that the mutarotation is presumed to be somewhat
slower in D2O than in H2O due to a combination of sol-
vent and kinetics isotope effects (Isbell and Pigman 1969;
Drake and Brown 1977). While at low temperatures both
1α and 1β peaks are well separated from the residual HDO
peak, at temperatures higher than 25°C the HDO peak
moves closer to the 1β peak. Thus, at these temperatures
the signal of the 2, 3β was used to follow the buildup of
the β anomer.
Time course of the mutarotation process was monitored
by running a set of 1H NMR single pulse experiments at
several different temperatures and pH values. For that pur-
pose, fresh solutions of 0.5M 3OMG were prepared in
D2O and immediately transferred to a 5mm NMR tube;
NMR measurements were started when the temperature
stabilized. Spectra were collected every few minutes for
several hours until equilibrium was reached. We simul-
taneously monitored the decrease of the α anomer signal
(peak at 5.26ppm) and the increase of the β anomer sig-
nals (peaks at 4.70ppm and 3.34ppm) that characterized
the mutarotation reaction. The rate of mutarotation for the
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α anomer was determined by measuring the kinetics of the
α-H1 peak. The kinetics of the β anomer was determined
by measuring the β-H1 peak at the low temperatures, w
hen it was well separated from the HDO peak, and the
β-H2, 3 peak at temperatures above 25°C.
The kinetics constants of mutarotation were observed by
optimizing the fit between Eqs.(2) and (3), and the data.
Examples are given in Fig.5.
The dependence of the rate constants on temperature
(Fig.6) yielded an activation energy of 35.3 (kJ/mol). The
inset shows the time required to reach 95% equilibrium
between the α and β anomer, in the temperature range of
Figure7 illustrates the dependence of the reaction rates
on the pH of the solutions of 0.5M 3OMG in D2O at 37°C,
in the pH range of 4.53–8.15. k was determined by fitting
the signal intensities to Eqs.(2) and (3). As expected in a
base-catalyzed exchange process, k increases with pH. The
reaction rates for both the temporal plots of the α and β ano-
mers were very similar. This was expected since they are the
Fig. 2 1H NMR spectra of 0.5 M 3OMG in D2O solution and its
anomeric configurations (α- and β-forms) at 18 °C. Spectra were
recorded with B0 = 11.7 T on a a fresh sample, b and 22 h after
3OMG dissolution. The net spectra of the α and β anomers are pre-
sented in c, d, respectively. They are obtained by subtracting spec-
trum b from spectrum a and the reverse, respectively
Fig. 3 2D 13C–1H HSQC spectrum (absolute value) of 0.5M 3OMG in D2O solution before (a) and after (b) anomeric equilibrium was reached,
recorded at 7°C (a) and 25°C (b). The anomeric 13C–1H cross peaks are indicated
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Fig. 4 1H NMR spectra of the hydroxyl protons of 0.5M 3OMG in
D2O solution at 4°C. Spectra were recorded with B0 = 11.7T (a) sev-
eral days following the sample preparation, and on a b fresh sample.
The net spectra of the α and β hydroxyl peaks are presented in c, d,
respectively. They are obtained by subtracting spectrum b from spec-
trum a and the reverse, respectively
Fig. 5 Kinetics of α and β anomers as determined by 1H NMR meas-
urements during the mutarotation of 0.5 M 3OMG in D2O at 13 °C
(a, b), 25°C (c, d) and 37°C (e, f). The kinetics of α anomer was fol-
lowed by the α-H1 peak and that of the β anomer by the β-H2, 3 peak.
Solid lines represent the fit adjustment to Eqs.(2) and (3) for α and β
anomers according to their evolution with time, respectively
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sums of the forward and reverse conversion rates: k = k1 + k2.
The reported results are the averaged of the two. The rate of
mutarotation for 3OMG at 25°C, k = 2.75E−04s−1, can be
compared to the value of k = 4.13 × E−04s−1 reported for
d-glucose (Dona etal. 2009).
13C NMR spectroscopy, which shows the unique features
of the α and β anomers, can be used to identify the two ano-
meric forms and the contribution of each at equilibrium. As
described earlier, signals at 98.8 and 88.2ppm are assigned
to β anomeric carbons of C1 and C3 of 3OMG, respectively,
and signals at 95.0 and 85.5ppm are assigned to α anomeric
carbons of C1 and C3 of 3OMG, respectively. Based on
13C NMR studies, (β/α)eq ratio of equilibrium population of
3OMG anomers is slightly temperature dependent, with an
average (β/α)eq ratio of 1.39 ± 0.01 in the temperature range
of 4–50°C (calculation based on the average of two sets
of intensities of the C1 and C3 of 3OMG). To verify that
there was no contribution from NOE, some of the 13C NMR
experiments were recorded without proton decoupling and
the intensity ratio of α to β anomeric forms was compared
with the 1H decoupled spectrum giving identical results.
The anomeric ratio was also evaluated from the 1H NMR
kinetic studies. The results at 12 pH values in the range of
4.53–8.15, showed no pH dependence giving an average
(α/β)eq ratio of 1.39 ± 0.09at 37°C. Hence, at equilibrium
the β anomer is favored (58%) compared to the α anomer
(42%), as illustrated in Fig.8.
CEST NMR studies
Earlier studies (Rivlin etal. 2014) have shown the 3OMG
MTRasym at 1.2ppm, 25°C, and pH range of 6.34–8.02,
exhibits maximum MTRasym effect at pH = 7.1. A significant
MTRasym effect is also detected at the same examined OH
group (1.2ppm) at 37°C (Fig.9).
Analyzing the Z spectrum at physiological condition,
where the hydroxyl protons are not resolved, requires the use
of the Bloch–McConnell (BM) equations (Zaiss and Bachert
Fig. 6 The dependence of
reaction rate on temperature dis-
played as Arrhenius plot (main
plot) and the time required to
reach 95% equilibrium (inset) in
fresh solutions of 0.5M 3OMG
in D2O at different tempera-
tures. Data were obtained from
1H NMR measurements of α
and β signals evolution during
mutarotation reaction
Fig. 7 The dependence of reaction rate on pH in fresh solutions of
0.5 M 3OMG in D2O at 37 °C [reaction rates are presented as an
average of α and β anomers and were obtained from their fit adjust-
ment to Eqs. (2) and (3), based on 1H NMR mutarotation measure-
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2013; Zaiss etal. 2015), which is beyond the scope of this
work. Our calculations were based on the analytical expres-
sions for the inverse Z-spectrum analysis (Zaiss etal. 2014,
2018; Wu etal. 2015). As mentioned in the “Experimental
section, this analysis (based on QUESP formula) enables the
elimination of spillover effects. To simplify its use, the case
of slow exchange was examined (all hydroxyls’ protons are
well observed). Hence, a series of multi B1 experiments with
a solution of 20 mM 3OMG (5 mM PBS, 5% D2O,
pH = 6.14, B0 = 11.7T) at 4°C were performed with satura-
tion pulse duration of 5s. Applying the inverse asymmetry
method (Fig.10) led to exchange rates of the hydroxyl pro-
tons, kb, of: 414, 390, 670 and 581s−1 for the hydroxyl peaks
at 0.75, 1.3, 2.0 and 2.7ppm, respectively. The same values
were obtained for the negative intercepts on the X axis in the
linear (“omega”) plots of
which is independent of the values of the fractions,
but as is mentioned in the “Experimental”section above, in
the omega plots excessive weight is given to low values of
ω1. The fractions obtained from the fitting (Fig.10) (as well
as from the intercept on the Y axis in the omega plots) were
3.68E−04, 1.96E−04, 0.46E−04 and 0.54E−04 for the
hydroxyl peaks observed at 0.75, 1.3, 2.0 and 2.7ppm from
water signal, respectively. These value deviate from the
actual hydroxyl concentrations of 3OMG solution, i.e.
1.8E−04, 3.6E−04, 0.75E−04 and 1.05E−04. Apparently
such deviation is inherent in the analysis, as it occurred also
in the analysis of the simpler system of the paramagnetic
species with single resonance far from the water peak (Zaiss
etal. 2018).
Quantification of the proton exchange rates can also be
made by using the on-resonance spin lock (SL) (Jin etal.
2011) method. However, it should provide in principal a
weighted average exchange rate for all the exchangeable
protons. Figure11 shows the on-resonance R dispersions
for the same 3OMG solution that was used for the CEST
measurements. The R curve pattern is in agreement with
previously published results by Jin etal. (2018). The result
of the fitted total fraction is in agreement with 3OMG con-
centration (fb = 7.2E−04, R2 = 0.9976). The fitted
1.67s, in agreement with the independently measured value.
The result of the exchange rate, kb, was 362s−1, which is in
the same range to those found with CEST experiments but
is slightly lower than the weighted average of the exchange
rates of the hydroxyl protons obtained in the CEST experi-
ments namely:
= 453s−1. However, the
fitted value of the frequency shift from the water peak,
was 0.78ppm rather than the expected weighted average
Fig. 8 The dependence of anomeric composition (β/α) on tempera-
ture in a sample of 0.5M 3OMG in D2O. Filled blue circle based on
13C NMR peaks’ intensities at equilibrium (each point represents an
average of two sets of intensities for 13C-3 and 13C-1); red color times
symbol based on 1H NMR kinetics measurements
Fig. 9 MTRasym plots of 20 mM 3OMG solution at 37 °C (con-
taining 5 mM PBS and 5% D2O) with a pH values from 6.14 to
7.49 measured at a frequency offset of 1.2 ppm from water signal
(B1 = 2.4 µT), and b pH value of 7.1at frequency offsets of 1.2, 2.1
and 2.8 from water signal and at different B1 values
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shift. The SL results were also tried to fit to the following
expression, which takes into account the independent contri-
butions of the individual hydroxyl protons, using the known
values of fi and Δωi:
However, the fitting to the experimental results was not
good and the resulted fitted parameters were unrealistic.
Thus we can conclude that the SL method gives a correct
rough estimate of the average exchange rates of the mul-
tiple exchange sites but cannot give information about the
individual exchange rates.
Fig. 10 The QUESP fitting (dots) to the inverse Z-spectrum analysis
(Eq.7, blue solid lines) for the hydroxyl peaks observed at a 0.75, b
1.3, c 2.0 and d 2.7ppm from the water peak, as obtained from exper-
imental CEST data of 20mM 3OMG solution (containing 5mM PBS
and 5% D2O, at pH = 6.14, T = 4°C, B0 = 11.7T)
Fig. 11 On-resonance R
dispersion versus spin lock
amplitude for 20mM 3OMG
solution (containing 5mM
PBS and 5% D2O, at pH 6.14,
T = 4°C, B0 = 11.7T)
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Since 3OMG was proposed for the imaging of tumors
using CEST–MRI, it was important to establish the ano-
meric ratio at equilibrium and the time needed to reach
this equilibrium. Indeed, this time was found to be very
long, and at low temperatures can last for several days.
This should be taken into consideration when perform-
ing MRI experiments. For instance, doing the experiments
using fresh 3OMG samples can give considerable errone-
ous and irreproducible results. The determination of the
anomeric ratio at equilibrium is essential for future cal-
culation by BM of the proton exchange rates, which can
help determine the pH in tumors. The determination of the
exchange rates of the hydroxyl protons by CEST and by SL
gives order of magnitude values but points to difficulties
in treating systems of multiple exchange sites.
Acknowledgements The study was supported by the Israel Science
Foundation (ISF) and the European Union’s Horizon 2020 research and
innovation program under Grant Agreement No. 667510.
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.
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... Since the CEST method provides orders of magnitude higher sensitivity than traditional proton MR spectroscopy ( 1 H MRS), it enables detection of subtle changes in the level of metabolite of interest 14,17 . Various groups have reported the use of glucose and its analogues as CEST contrast agent to study cancer and neurodegeneration 18,20,[24][25][26][27][28][29][30][31][32][33][34] . ...
... D-glucose and its analogues have shown potential as CEST contrast agents for the non-invasive detection of various cancers in preclinical and clinical studies 18,[28][29][30][31]33,[35][36][37][38][39][40] . Dynamic glucose-enhanced (glucoCEST) MRI has been used to study perfusion in cancer which corroborated with DCE MRI utilizing GBCAs. ...
... This study reported a higher glucoCEST contrast from tumor post-treatment with rapamycin. In addition, non-metabolized glucose analogues such as 3-O-methylglucose (3-OMG), have been exploited as CEST contrast agents for studies in cancer and stroke [24][25][26][28][29][30]32 . Further, another study has shown the use of a non-caloric sweetener, sucralose, as a CEST contrast agent to detect glioma in a preclinical model 41 . ...
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Clinical imaging is widely used to detect, characterize and stage cancers in addition to monitoring the therapeutic progress. Magnetic resonance imaging (MRI) aided by contrast agents utilizes the differential relaxivity property of water to distinguish between tumorous and normal tissue. Here, we describe an MRI contrast method for the detection of cancer using a sugar alcohol, maltitol, a common low caloric sugar substitute that exploits the chemical exchange saturation transfer (CEST) property of the labile hydroxyl group protons on maltitol (malCEST). In vitro studies pointed toward concentration and pH-dependent CEST effect peaking at 1 ppm downfield to the water resonance. Studies with control rats showed that intravenously injected maltitol does not cross the intact blood-brain barrier (BBB). In glioma carrying rats, administration of maltitol resulted in the elevation of CEST contrast in the tumor region only owing to permeable BBB. These preliminary results show that this method may lead to the development of maltitol and other sugar alcohol derivatives as MRI contrast agents for a variety of preclinical imaging applications.
... 21,38 Additionally, because of the mutarotation process in which both the molecules are involved, the ratio of α and β anomers at equilibrium is different between 3OMG (β:α = 58:42) and glucose (β:α = 64:36). 39,40 This different ratio might affect the exchange rate, because the β anomer shows faster exchange rates. 41 In an in vivo setting, single i.v. ...
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D‐Glucose and 3‐O‐Methyl‐D‐glucose (3OMG) have been shown to provide contrast in magnetic resonance imaging‐chemical exchange saturation transfer (MRI‐CEST) images. However, a systematic comparison between these two molecules has yet to be performed. The current study deals with the assessment of the effect of pH, saturation power level (B1) and magnetic field strength (B0) on the MRI‐CEST contrast with the aim of comparing the in vivo CEST contrast detectability of these two agents in the glucoCEST procedure. Phosphate‐buffered solutions of D‐Glucose or 3OMG (20 mM) were prepared at different pH values and Z‐spectra were acquired at several B1 levels at 37°C. In vivo glucoCEST images were obtained at 3 and 7 T over a period of 30 min after injection of D‐Glucose or 3OMG (at doses of 1.5 or 3 g/kg) in a murine melanoma tumor model (n = 3–5 mice for each molecule, dose and B0 field). A markedly different pH dependence of CEST response was observed in vitro for D‐Glucose and 3OMG. The glucoCEST contrast enhancement in the tumor region following intravenous administration (at the 3 g/kg dose) was comparable for both molecules: 1%–2% at 3 T and 2%–3% at 7 T. The percentage change in saturation transfer that resulted was almost constant for 3OMG over the 30‐min period, whereas a significant increase was detected for D‐Glucose. Our results show similar CEST contrast efficiency but different temporal kinetics for the metabolizable and the nonmetabolizable glucose derivatives in a tumor murine model when administered at the same doses. This study investigated the CEST properties of D‐Glucose and 3‐O‐Methyl‐D‐glucose (30MG) in vitro and after intravenous injection in a murine melanoma model. A marked and opposite pH dependence of the CEST contrast was observed in vitro for D‐Glucose and 3OMG, whereas both molecules showed a similar CEST enhancement following the in vivo administration at 7 T, but a reduced detectability at 3 T.
... A more promising molecule that has been intensively studied is the non-metabolizable 3-O-methyl-D-glucose (3OMG), that is considered non-toxic. Several studies tested 3OMG in different breast cancer models and showed higher uptake and CEST contrast in the more aggressive tumors, in according with the results obtained by FDG-PET (64)(65)(66). Beyond 3OMG, glucosamine (GlcN) and Nacetyl glucosamine (GlcNAc) can accumulate in tumors that overexpress the glucose transporters GLUT1 and GLUT2. These molecules were exploited as CEST contrast agents in breast and melanoma murine cancer models with different aggressiveness showing diverse accumulation inside the tumor (67,68). ...
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Altered metabolism is considered a core hallmark of cancer. By monitoring in vivo metabolites changes or characterizing the tumor microenvironment, non-invasive imaging approaches play a fundamental role in elucidating several aspects of tumor biology. Within the magnetic resonance imaging (MRI) modality, the chemical exchange saturation transfer (CEST) approach has emerged as a new technique that provides high spatial resolution and sensitivity for in vivo imaging of tumor metabolism and acidosis. This mini-review describes CEST-based methods to non-invasively investigate tumor metabolism and important metabolites involved, such as glucose and lactate, as well as measurement of tumor acidosis. Approaches that have been exploited to assess response to anticancer therapies will also be reported for each specific technique.
... The monobasic bicarbonate, thus, promotes the deprotonation of acac but also the ring-opening (mutarotation) of the RE, which can be both acid-and base-catalyzed. 33,34 Standard 1 H NMR analysis of the product in D 2 O ( Figure S4) already revealed the introduction of a strong acetyl signal (2.28 ppm −CH 3 ) together with an almost complete disappearance of the RE's β-hemiacetal signal (4.66 ppm) as a consequence of the ketone functionalization. For detailed structure elucidation, multiplicity-edited HSQC (heteronulear single quantum correlation) and HSQC-TOCSY (total correlation spectroscopy; with short-range/15 ms mixing time and long-range/120 ms mixing time) were performed at 65°C on the model compound, dissolved in [P 4444 ]-[OAc]:DMSO-d 6 (1:4 w/w, see SI Section S4). ...
Herein, we demonstrate an effective approach toward functionalization of cellulose nanocrystal (CNC) reducing ends by means of a Knoevenagel condensation reaction with a reactive β- diketone (acetylacetone). The end-wise modification was elucidated by advanced NMR analysis, which was facilitated by dissolving the CNCs in ionic liquid electrolyte and by the concomitant assignment of a model compound derived from D-cellobiose. The diffusion-edited 1H experi- ment afforded a simple method to identify the assigned model resonances in the reducing end-modified CNCs. The condensations can be carried out in aqueous bicarbonate solutions, avoiding the use of hazardous solvents. Under these preliminary aqueous conditions, end- group conversion of up to 12.5% could be confirmed. These results demonstrate the potential of β-diketone chemistry and the Knoevenagel condensation for functionalizing cellulose reducing ends. Application of this liquid-state NMR method for confirming and quantifying reducing end conversion is also shown to be invaluable. Extension of this chemistry to other 1,3-dicarbonyl compounds and solvation conditions should allow for the topochemical and (axially) chirotopic installation of functional moieties to CNCs, paving the way to asymmetric cellulose-based nanomaterials with unique properties.
... Another non-phosphorylated glucose analog that was investigated in our lab is 2-O-Methyl-D-glucose (2OMG). Unlike D-Glc, it has less β anomer at equilibrium in natural conditions of pH (77) [essential information for designing in vivo CEST experiments (78)], and there are no detailed studies of its pharmacokinetics. Thus, we performed highresolution 31 P NMR spectroscopy studies to explore its metabolism. ...
Early detection of the cancerous process would benefit greatly from imaging at the cellular and molecular level. Increased glucose demand has been recognized as one of the hallmarks of cancerous cells (the "Warburg effect"), hence glucose and its analogs are commonly used for cancer imaging. One example is FDG-PET technique, that led to the use of chemical exchange saturation transfer (CEST) MRI of glucose ("glucoCEST") for tumor imaging. This technique combines high-resolution MRI obtained by conventional imaging with simultaneous molecular information obtained from the exploitation of agents with exchangeable protons from amine, amide or hydroxyl residues with the water signal. In the case of glucoCEST, these agents are based on glucose or its analogs. Recently, preclinical glucoCEST studies demonstrated the ability to increase the sensitivity of MRI to the level of metabolic activity, enabling identification of tumor staging, biologic potential, treatment planning, therapy response and local recurrence, in addition to guiding target biopsy for clinically suspected cancer. However, natural glucose limits this method because of its rapid conversion to lactic acid, leading to reduced CEST effect and short signal duration. For that reason, a variety of glucose analogs have been tested as alternatives to the original glucoCEST. This review discusses the merits of these analogs, including new data on glucose analogs heretofore not reported in the literature. This summarized preclinical data may help strengthen the translation of CEST MRI of glucose analogs into the clinic, improving cancer imaging to enable early intervention without the need for invasive techniques. The data should also broaden our knowledge of fundamental biological processes.
Glucosamine (2-amino-2-deoxy-d-glucose, GlcN) is a naturally occurring amino monosaccharide that is essential for a variety of biological functions, it is mainly involved in the formation of polysaccharide structures. It was recently reported to enable the imaging of cancerous tumors as an exogenous contrast agent using the MRI technique of chemical exchange saturation transfer (CEST). In preparation for the clinical use of GlcN, its anomeric equilibrium and mutarotation rate constants were directly investigated in this study utilizing high resolution ¹H and ¹³C NMR spectroscopy. The effects of GlcN concentration, temperature, pH and buffer on the mutarotation rate constant and mutarotation equilibrium were measured. The mutarotation rate constant increased markedly with increasing GlcN concentrations. The rate constant of mutarotation of GlcN at room temperature was 2.2 × 10⁻⁴ - 5.0 × 10⁻⁴ s⁻¹ at concentrations of 0.02–0.5 M, corresponding to a time of 3.8–1.7 h hours to reach 95% equilibrium. The anomeric ratio was strongly pH-dependent. The influence of phosphate buffer on the apparent rate constant of GlcN mutarotation was investigated. For phosphate buffer saline values between 0 and 50 mM, there was a six-fold increase in rate at pH 7.0. The mutarotation rate constant rose rapidly with pH at a phosphate concentration of 50 mM: from 0.4 × 10⁻³ s⁻¹ at pH 5.0 to 7.8 × 10⁻³ s⁻¹ at pH 9.4, suggesting that the catalysis is due to the HPO4²⁻ and PO4³⁻ ions. These findings might help researchers design the experimental setting for employing GlcN for cancer detection using GlcN-CEST MRI.
The mutarotation equilibrium, by which reducing carbohydrates exist in solution as the α and β anomers of cyclic (furanoid and pyranoid) structures, along with open-chain (aldehyde and hydrate) forms, and whose ratios are depending on factors such as temperature, pH and solvent, portraits a phenomenon involved in numerous processes of chemical and biological importance. Herein, we have developed a DFT-based rationale that provides a broader landscape for anomerizations and ring-open chain interconversions, together with the pivotal role exerted not only by the aldehyde intermediate (essentially the only acyclic structure taken into account so far), but also the hydrate form (often more abundant at the equilibrium). These calculations reveal a more complex and richer scenario than was thought, and identify different mutarotation mechanisms that hinge on every monosaccharide. It is noteworthy that pyranose-furanose interconversion may actually occur without the intermediacy of open-chain forms. For the aldoses evaluated, namely d-glucose, d-ribose, and d-xylose, all structures involved in mutarotation undergo interconversion pathways, whose energy barriers calculated at the M06-2X/6-311++G(d,p) level, are in good agreement with previous experimental measurements.
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The efficacy of glucosamine (GlcN) and N-acetyl glucosamine (GlcNAc) as agents for chemical exchange saturation transfer (CEST) magnetic resonance molecular imaging of tumors is demonstrated. Both agents reflect the metabolic activity and malignancy of the tumors. The method was tested in two types of tumors implanted orthotopically in mice: 4T1 (mouse mammary cancer cells) and MCF7 (human mammary cancer cells). 4T1 is a more aggressive type of tumor than MCF7 and exhibited a larger CEST effect. Two methods of administration of the agents, intravenous (IV) and oral (PO), gave similar results. The CEST MRI observation of lung metastasis was confirmed by histology. The potential of the clinical application of CEST MRI with these agents for cancer diagnosis is strengthened by their lack of toxicity as can be indicated from their wide use as food supplements.
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Imaging metabolic dysfunction, a hallmark of solid tumors, usually requires radioactive tracers. Chemical exchange saturation transfer (CEST) imaging can potentially detect and visualize glucose uptake and metabolism, without the need for radioisotopes. Here, we tested the feasibility of using glucose CEST (glucoCEST) to image unlabeled glucose uptake in head and neck cancer by using a clinical 3T magnetic resonance imaging (MRI) scanner. The average CEST contrast between tumors and normal tissue in 17 patients was 7.58% (P = 0.006) in the 3–4 ppm offset frequency range and 5.06% (P = 0.02) in 1–5 ppm range. In a subgroup of eight patients, glucoCEST signal enhancement was higher in tumors than in normal muscle (4.98% vs. 1.28%, P < 0.021). We conclude that glucoCEST images of head and neck cancer can be obtained with a clinical 3T MRI scanner.
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Recent animal studies have shown that D-glucose is a potential biodegradable MRI contrast agent for imaging glucose uptake in tumors. Here, we show the first translation of that use of D-glucose to human studies. Chemical exchange saturation transfer (CEST) MRI at a single frequency offset optimized for detection of hydroxyl protons in D-glucose (glucoCEST) was used to image dynamic signal changes in the human brain at 7T during and after infusion of D-glucose. Dynamic glucose-enhanced (DGE) image data from four normal volunteers and three glioma patients showed strong signal enhancement in blood vessels, while the enhancement varied spatially over the tumor. Areas of enhancement differed spatially between DGE and conventional Gd-enhanced imaging, suggesting complementary image information content for these two types of agents. In addition, different tumor areas enhanced with D-glucose at different times post-infusion, suggesting a sensitivity to perfusion-related properties such as substrate delivery and blood-brain barrier (BBB) permeability. These preliminary results suggest that DGE MRI is feasible to study glucose uptake in humans, providing a time-dependent set of data that contains information regarding arterial input function (AIF), tissue perfusion, glucose transport across the BBB and cell membrane, and glucose metabolism.
Purpose: To evaluate the ability of spin-lock imaging to detect the uptake of 3-o-methyl-D-glucose (3oMG) in normal brain and brain tumors in animals. Methods: Measurements of the longitudinal relaxation rate in the rotating frame (R1ρ ) were made over a range of spin-lock powers in rat brains bearing 9L tumors. The dispersion of R1ρ values was quantified by ΔR1ρ , the difference of R1ρ values acquired with low and high locking powers. The glucose analogue 3oMG was administered intravenously and the differences of ΔR1ρ values ( ΔR1ρdiff) before and as a function of time after administration were calculated to isolate the contribution of 3oMG to the dispersions, which at high fields primarily reflects chemical exchange effects. In addition, the ratio of image signals from low and high locking fields (the spin-lock ratio, SLR), which requires fewer acquisitions and varies directly with ΔR1ρ , was computed as an alternative measure of the variation with locking power, and changes in SLR (SLRdiff ) after 3oMG were evaluated. Results: Both ΔR1ρdiff and SLRdiff in tumors increased rapidly after injection, whereas intact brain showed a gradual increase up to 1 h. The ΔR1ρdiff and SLRdiff were significantly different between tumors and contralateral normal tissues. Conclusion: Spin-lock methods can be used to detect 3oMG in vivo after injection, and appropriate analyses of MRI signals allow tumors to be distinguished from normal brain.
Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) has been developed and employed in multiple clinical imaging research centers worldwide. Selective radiofrequency (RF) saturation pulses with standard 2D and 3D MRI acquisition schemes are now routinely performed, and CEST MRI can produce semiquantitative results using magnetization transfer ratio asymmetry (MTRasym ) analysis while accounting for B0 inhomogeneity. Faster clinical CEST MRI acquisition methods and more quantitative acquisition and analysis routines are under development. Endogenous biomolecules with amide, amine, and hydroxyl groups have been detected during clinical CEST MRI studies, and exogenous CEST agents have also been administered to patients. These CEST MRI tools show promise for contributing to assessments of cerebral ischemia, neurological disorders, lymphedema, osteoarthritis, muscle physiology, and solid tumors. This review summarizes the salient features of clinical CEST MRI protocols and critically evaluates the utility of CEST MRI for these clinical imaging applications. Level of evidence: 5 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2018;47:11-27.
Purpose: Chemical exchange saturation transfer (CEST) NMR or MRI experiments allow detection of low concentrated molecules with enhanced sensitivity via their proton exchange with the abundant water pool. Be it endogenous metabolites or exogenous contrast agents, an exact quantification of the actual exchange rate is required to design optimal pulse sequences and/or specific sensitive agents. Methods: Refined analytical expressions allow deeper insight and improvement of accuracy for common quantification techniques. The accuracy of standard quantification methodologies, such as quantification of exchange rate using varying saturation power or varying saturation time, is improved especially for the case of nonequilibrium initial conditions and weak labeling conditions, meaning the saturation amplitude is smaller than the exchange rate (γB1 < k). Results: The improved analytical 'quantification of exchange rate using varying saturation power/time' (QUESP/QUEST) equations allow for more accurate exchange rate determination, and provide clear insights on the general principles to execute the experiments and to perform numerical evaluation. The proposed methodology was evaluated on the large-shift regime of paramagnetic chemical-exchange-saturation-transfer agents using simulated data and data of the paramagnetic Eu(III) complex of DOTA-tetraglycineamide. Conclusions: The refined formulas yield improved exchange rate estimation. General convergence intervals of the methods that would apply for smaller shift agents are also discussed. Magn Reson Med, 2017. © 2017 International Society for Magnetic Resonance in Medicine.
Glucose transport is important for understanding brain glucose metabolism. We studied glucose transport with a presumably non-toxic and non-metabolizable glucose analog, 3-O-methyl-d-glucose, using a chemical exchange-sensitive spin-lock MRI technique at 9.4 Tesla. 3-O-methyl-d-glucose showed comparable chemical exchange properties with d-glucose and 2-deoxy-d-glucose in phantoms, and higher and lower chemical exchange-sensitive spin-lock sensitivity than Glc and 2-deoxy-d-glucose in in vivo experiments, respectively. The changes of the spin-lattice relaxation rate in the rotating frame (ΔR1ρ) in normal rat brain peaked at ∼15 min after the intravenous injection of 1 g/kg 3-O-methyl-d-glucose and almost maintained a plateau for >1 h. Doses up to 4 g/kg 3-O-methyl-d-glucose were linearly correlated with ΔR1ρ. In rats with focal ischemic stroke, chemical exchange-sensitive spin-lock with 3-O-methyl-d-glucose injection at 1 h after stroke onset showed reduced ΔR1ρ in the ischemic core but higher ΔR1ρ in the peri-core region compared to normal tissue, which progressed into the ischemic core at 3 h after stroke onset. This suggests that the hyper-chemical exchange-sensitive spin-lock region observed at 1 h is the ischemic penumbra at-risk of infarct. In summary, 3-O-methyl-d-glucose-chemical exchange-sensitive spin-lock can be a sensitive MRI technique to probe the glucose transport in normal and ischemic brains.
Chemical Exchange Saturation Transfer (CEST) approach is a novel tool within magnetic resonance imaging (MRI) that allows visualization of molecules possessing exchangeable protons with water. Many molecules, employed as excipients for the formulation of finished drug products, are endowed with hydroxyl, amine or amide protons, thus can be exploitable as MRI-CEST contrast agents. Their high safety profiles allow them to be injected at very high doses. Here we investigated the MRI-CEST properties of several excipients (ascorbic acid, sucrose, N-acetyl-d-glucosamine, meglumine and 2-pyrrolidone) and tested them as tumor-detecting agents in two different murine tumor models (breast and melanoma cancers). All the investigated molecules showed remarkable CEST contrast upon i.v. administration in the range 1–3 ppm according to the type of mobile proton groups. A marked increase of CEST contrast was observed in tumor regions up to 30 min post injection. The combination of marked tumor contrast enhancement and lack of toxicity make these molecules potential candidates for the diagnosis of tumors within the MRI-CEST approach.
Chemical exchange saturation transfer (CEST) allows the indirect detection of dilute metabolites in living tissue via MRI of the tissue water signal. Selective radio frequency (RF) with amplitude B1 is used to saturate the magnetization of protons of exchanging groups, which transfer the saturation to the abundant water pool. In a clinical setup, the saturation scheme is limited to a series of short pulses to follow regulation of the specific absorption rate (SAR). Pulsed saturation is difficult to describe theoretically, thus rendering quantitative CEST a challenging task. In this study, we propose a new analytical treatment of pulsed CEST by extending a former interleaved saturation-relaxation approach. Analytical integration of the continuous wave (cw) eigenvalue as a function of the RF pulse shape leads to a formula for pulsed CEST that has the same structure as that for cw CEST, but incorporates two form factors that are determined by the pulse shape. This enables analytical Z-spectrum calculations and permits deeper insight into pulsed CEST. Furthermore, it extends Dixon's Ω-plot method to the case of pulsed saturation, yielding separately, and independently, the exchange rate and the relative proton concentration. Consequently, knowledge of the form factors allows a direct comparison of the effect of the strength and B1 dispersion of pulsed CEST experiments with the ideal case of cw saturation. The extended pulsed CEST quantification approach was verified using creatine phantoms measured on a 7 T whole-body MR tomograph, and its range of validity was assessed by simulations. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.