Application of Good's buffers to pH imaging using hyperpolarized 13C MRI

Article abstract of DOI:10.1039/c5cc05348j

N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), one of Good's buffers, was applied to pH imaging using hyperpolarized ¹³C magnetic resonance spectroscopy. Rapid NMR- and MRI-based pH measurements were obtained by exploiting the sensitive pH-

Full text of DOI:10.1039/c5cc05348j

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Von Morze, J. E. Blecha, D. Korenchan, M. Van Criekinge, R. Sriram, J. Gordon, H. Chen, S. Subramaniam,
R. Bok, Z. J. Wang, D. Vigneron, P. E. Z. Larson, J. Kurhanewicz and D. M. Wilson, Chem. Commun., 2015,
DOI: 10.1039/C5CC05348J.
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Application of Good’s buffers to pH imaging using hyperpolarized
13
C MRI
a
a
a
a
Robert R Flavell , Cornelius von Morze , Joseph E. Blecha , David Korenchan , Mark Van
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
a
a
Criekinge , Renuka Sriram , Jeremy Gordon , Hsin-Yu Chen , Sukumar Subramaniam ,
a
a
a
a
a
Robert Bok , Zhen J. Wang , Daniel Vigneron , Peder Larson , John Kurhanewicz , and
DOI: 10.1039/x0xx00000x
a, *
David M Wilson
www.rsc.org/
3
1 15-17
P,
1
18-20
19 21, 22
and F.
N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), one of
H,
While these techniques have provided
Good’s buffers, was applied to pH imaging using hyperpolarized important insights in animal model systems, they have various
1
3
C magnetic resonance spectroscopy. Rapid NMR- and MRI-based potential limitations, including long scan time, requirement for high
pH measurements were obtained by exploiting the sensitive pH- dose of probe administration, and in some cases, poor spatial
1
3
dependence of its C chemical shift within the physiologic range.
resolution.
1
3
Hyperpolarized C MRS is an emerging technology which relies
Metabolic reprogramming in cancer results in increased secretion
of acid into the extracellular space. This results in an interstitial
microenvironment that is slightly acidic, ranging from pH 6.5 – 7.0
for many cancer subtypes, in contrast with normal healthy tissue
which characteristically has pH 7.2 -7.4. This property has been
upon dramatic NMR signal enhancement provided via dynamic
2
3
nuclear polarization (DNP). This method has been applied to
2
4
numerous chemical substrates, and successfully translated to the
2
5
clinic for use in men with prostate cancer. An elegant pH imaging
1
3
strategy using hyperpolarized C MRS to study the equilibrium
2
6, 27
characterized
measurements, magnetic resonance, and fluorescence.
Moreover, acidification of the extracellular microenvironment
by
techniques
including
microelectrode
between bicarbonate and CO
this technique has practical limitations, including 1) low signal to
noise ratio of the CO resonance under typical physiologic
2
has been reported.
However,
1
-3
2
4
-6
accompanies local invasion and metastasis in a variety of tumors.
conditions (since pKa,bicarbonate = 6.17), potentially propagating
significant errors to pH estimates derived from this ratiometric
Treatment of solid tumors with sodium bicarbonate increases
7
tumor pH and reduces metastasis. Interstitial acidity is thought to
1
approach, 2) the short effective T relaxation time of bicarbonate in
26
induce resistance to chemotherapeutic agents, and many small
molecule agents targeting acid transporters are currently under
vivo, 3) the relatively low solubility of bicarbonate in glassing
agents resulting in difficulty producing sufficient polarized material,
8, 9
2
8
development.
Thus, acidic extracellular pH represents both a
and 4) the possibility for inhibition of carbonic anhydrase, which is
required for rapid equilibration between bicarbonate and CO
therapeutic target as well as a potential biomarker for the presence
of aggressive cancer with higher risk of metastases.
2
.
Since relative chemical shifts can in general be measured very
accurately using MRS, spectroscopic, or frequency specific imaging
Several magnetic resonance techniques, have been developed for
the purpose of imaging acidic interstitial pH, including methods
based on magnetic resonance spectroscopy (MRS) and chemical
1
3
techniques, we reasoned that a hyperpolarized C approach
employing a pair of probes with and without pH-dependent
chemical shift could address some of the limitations of this prior
approach. Specifically, this approach combines the robustness and
high accuracy of chemical shift based MRS methods with the high
6
, 10-14
exchange saturation transfer (CEST).
A central requirement for
these techniques is accuracy, since the pH change between healthy
and diseased tissues is small. In the MRS strategy, a probe that has
a known chemical shift response to pH changes is administered
systemically. The chemical shift is then measured using MRS, and by
comparison to a standard curve, the pH can be determined. This
approach has been studied using several different nuclei, including
1
3
signal to noise ratio inherent to hyperpolarized C imaging.
Initially, a small library of compounds was tested with thermal
1
3
equilibrium NMR, comparing C chemical shifts of long-T
pH 6.5 and 7.4 (Supplemental fig. 1). These compounds were
selected based on the following key criteria: 1) pK near the
1
nuclei at
a
physiologic range (between 6.0 and 8.0), 2) presence of a long T1
nucleus, and 3) feasibility of isotopic synthesis. Agents likely to be
toxic were excluded. The classes of compounds examined included
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previous synthesis of the natural abundance material combined
with a previously described technique for the preparation of
2
9, 30
isotopically enriched amino acids (Figure 2).
Secondary labelling
15
with N was incorporated in order to eliminate low field
14
quadrupolar T
increase the T
1
relaxation effects associated with N as well as to
31
2
relaxation time for imaging purposes. This came at
1
3
15
the expense of splitting of the C peak by the spin-1/2 N nucleus,
which could potentially limit the accuracy of chemical shift
measurement and signal to noise.
1
3
Since endogenous C nuclei are not abundant enough to allow for
1
3
in vivo imaging, we added a second compound, C urea, as a
13
chemical shift standard. Urea was selected since its C chemical
1
3
shift is close to that of the ACES carbonyl, and hyperpolarized C,
1
5
32
N urea is a well characterized imaging agent. A titration curve
was developed comparing the chemical shifts of ACES and urea
Figure 3). Values for δmin, δmax, and pK were obtained by iteratively
(
a
fitting the data, yielding the following equation linking chemical
1
5
Fig. 1. Structures of Good’s buffers. Long T
1
nuclei are indicated in red. The difference
nuclei. A positive δ ppm
shift difference to pH, in a manner similar to previously described:
in chemical shift between pH 6.5 and 7.4 is shown for long T
1
13
indicates a downfield C chemical shift. pKa are based on prior literature values.
ꢆꢇ ꢈ 13.92ꢉ
ꢀ
ꢁ ꢂ 6.58 ꢃ logꢄꢅ
ꢊ
ꢆꢃ5.51 ꢃ ꢇꢉ
biological buffers, dicarboxylic acids, and imidazole/histidine
derivatives.
In his landmark 1966 paper, Dr. N. Good described criteria for an The pK
a
obtained using this technique, 6.58 (at 37 degrees), is in
2
9
optimal biological pH buffer, and synthesized a small series of agreement with the previously published value of 6.56.
compounds which have since become the standard buffers used in
2
9
biochemistry. The criteria included pK
a
between 6 and 8, water
-6
solubility, impermeability to biological membranes, minimum
influence of temperature, concentration, or ionic strength on
dissociation of the buffer, lack of binding to other biological cations,
resistance to degradation, and easy synthetic preparation. We
rationalized that these properties would also yield optimal pH
imaging probes. Moreover, these compounds have been used
widely in the 50 years since their initial description, reducing the
likelihood of novel interactions which could block pH dependent
chemical shift change.
-
8
Change
in
ppm
-
-
-
10
12
14
4.5
6.5
pH
8.5
Four of Good’s buffers with long T
1
nuclei, and Tris, another
commonly used biological buffer, are depicted in Figure 1. Of these,
the compounds with the largest chemical shifts were ADA and ACES Figure 3. Titration curve depicting the difference in chemical shift between urea and
Figure 1, Supplemental figures 2-6). In initial studies, we found that ACES as a function of pH.
(
the pH dependent chemical shift change of ADA was blocked by the
presence of calcium, consistent with its known chelation properties
1
3
15
A method for hyperpolarization of C, N ACES was developed
and optimized with respect to microwave frequency and
concentration of Gd-DOTA (Supplemental table 2). The optimized
2
9
(
Supplemental table 1).
Thus, we selected ACES as the most promising overall compound
for imaging, due to the large pH dependant chemical shift, synthetic
13 15
prep was obtained by dissolution of C, N ACES in 0.95 equivalents
NaOH (using a 10M solution), and addition of Gd-DOTA to 0.5 mM,
3
3
with 20 mM OX63 trityl radical. Using this method, 12.5 ± 2.7% (n
3) average liquid state polarization, back calculated to the time of
dissolution was obtained. The T was 18 s at 11.7T and 25 s at 3T.
The polarization time constant was 2490 ± 396 (s.d., n= 10). A
=
1
3
4
copolarization method was developed to simultaneously polarize
1
3
15
13 15
13 15
C, N ACES and C, N urea. By copolarizing C, N ACES and
1
3
15
C, N urea, we were able to accurately determine the pH of
solutions in a 500 MHz NMR (Figure 4). There was a slight but
significant change in the measured pH by variation of temperature
1
3
15
13
Fig 2. Synthesis of C, N, C ACES.
(
Supplemental figure 8) and by variation of concentration of the
accessibility, predicted lack of toxicity, and known lack of chelation
probe (Supplemental figure 9). Furthermore, there was a slight
1
3
15
of abundant biological cations. C, N ACES was synthesized in 4 change in the pH measured at the earliest time points in
steps starting from 1- C glycine in 54% overall yield, based on the comparison to those measured at the latest time points
1
3
(
Supplemental figure 10). This change may be related to initial
2
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variation in temperature, and limitations of the kinetics of the acid
base equilibria (although these are known to be very rapid reactions
3
5
13 15
in most cases ). Overall, these data suggest that the C, N ACES
1
3
15
and C, N urea copolarization method could be used to determine
the pH in an imaging experiment.
13
15
Figure 4. Measurement of pH with C, N ACES. The calculated pH values of 7.16 and
.86 correlated with the values of 7.22 and 6.91 measured on a pH meter following the
6
13
15
experiment. The carboxylate of C,
adjacent N.
N ACES is a doublet owing to coupling with the
15
In order to assess the properties of this system under imaging
conditions, a five compartment phantom was imaged on a clinical
3
T scanner (Figure 5). Again, the dramatic chemical shift change of
ACES in response to pH allowed for rapid determination of pH.
There was a slight difference in pH calculated from the chemical
shift and that measured on a pH meter following the experiment.
These differences, averaging 0.18 pH units, may be due to a
combination of factors, including temperature (supplemental figure
8
), concentration (3 mM in the phantom, supplemental figure 9),
the presence of phosphate buffer in the solution (supplemental
table 3), or the early time point used for imaging (supplemental
table 10), and are similar to previously described phantom
1
3
36,
experiments for imaging pH using hyperpolarized C bicarbonate.
3
7
At present, we propose that the accuracy of measured vs.
calculated pH is within 0.1 – 0.2 pH units, although a much larger
series of experiments would be necessary to truly estimate the
accuracy (Supplemental figure 11). In vivo pH imaging is a key
future goal for this probe. Based on the phantom experiments, we
predict that injection of 0.5 mg ACES should be sufficient to
generate appropriate signal to noise in a mouse, for a total dose of
2
5 mg/kg. Since the administered probe could potentially buffer pH,
one important goal is the optimization of signal to noise ratio, as
well as accuracy of the method. In order to achieve this goal,
synthesis of other isotopically labelled variants is currently
1
4
underway in our laboratory, including N (to minimize signal loss
13
15
13 15
Figure 5. Five compartment phantom for imaging pH using C, N ACES and C,
N
1
5
2
due to the splitting by N), and H labelled probes (to improve the
of the molecule).
Overall, these data demonstrate that hyperpolarized C, N ACES Summary of calculated and measured pH values.
urea A) Phantom design incorporated 5 compartments with varying pH. B) 2D chemical
shift imaging through the phantom. C) Representative voxel from position 2. D)
T
1
1
3
15
1
3
can be used to determine pH using C magnetic resonance
spectroscopy. Initially, a series of compounds was screened for a
large chemical shift change over the physiologically relevant pH Acknowledgements
range. Of these compounds, Good’s buffers were the most
R.R.F. acknowledges support from NIH 5T32EB0011631-10, a
promising, owing to properties considered in their initial design,
1
3
15
University of California, San Francisco Department of Radiology
Seed Grant, a Radiology Society of North America research fellow
grant, and an SNMMI-ERF William and Mitzi Blahd, MD Pilot Grant.
D.M.W. acknowledges support from NIH R01CA166766. C.V.M.
acknowledges support from NIH K01DK099451. This project was
also supported by NIH center grant P41EB013598.
coupled with the vast experience of their use. We selected C, N
ACES owing to the large chemical shift change over the physiologic
range and lack of chelation properties. This compound was applied
to pH measurement in an NMR spectrometer and in a chemical shift
1
3
15
imaging experiment on a clinical 3T MRI scanner. C, N ACES is a
1
3
promising tool for imaging pH using hyperpolarized C MRS, and
we anticipate application of this probe to imaging in animal models
of malignancy.
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