Letter

Cite This: ACS Sens. XXXX, XXX, XXX−XXX pubs.acs.org/acssensors

13
Esterase-Catalyzed Production of Hyperpolarized C‑Enriched
Carbon Dioxide in Tissues for Measuring pH
Nesmine Maptue,† Weina Jiang,† Crystal Harrison,† Alexander M. Funk,† Gaurav Sharma,†
Craig R. Malloy,†,‡,§,∥ Dean Sherry,†,‡,¶ and Chalermchai Khemtong*,†,‡
†
Advanced Imaging Research Center, ‡Department of Radiology, and §Department of Internal Medicine, University of Texas
Southwestern Medical Center, Dallas, Texas 75390, United States
∥
Veteran Affairs North Texas Health Care System, Dallas, Texas 75216, United States
¶
Department of Chemistry, University of Texas at Dallas, Richardson, Texas 75080, United States
*
S Supporting Information
Downloaded from pubs.acs.org by UNIV PARIS-SUD on 11/11/18. For personal use only.

ABSTRACT: 13C Magnetic resonance imaging of hyper-

polarized (HP) 13C-enriched bicarbonate (H13CO3−) and
carbon dioxide (13CO2) is a novel and sensitive technique for
tissue pH mapping in vivo. Administration of the HP
physiological buffer pair is attractive, but poor polarization and
the short T1 of 13C-enriched inorganic bicarbonate salts are
major drawbacks for this approach. Here, we report a new class
of mixed anhydrides for esterase-catalyzed production of highly
polarized 13CO2 and H13CO3− in tissue. A series of precursors
with different alkoxy and acyl groups were synthesized and
tested for chemical stability and T1. 13C-enriched ethyl acetyl
ACS Sens.

carbonate (13C-EAC) was found to be the most suitable

candidate due to the relatively long T1 and good chemical stability. Our results showed that 13C-EAC can be efficiently and
rapidly polarized using BDPA. HP 13C-EAC was rapidly hydrolyzed by esterase to 13C-enriched monoacetyl carbonate (13C-
MAC), which then decomposed to HP 13CO2. Equilibrium between the newly produced 13CO2 and H13CO3− was quickly
established by carbonic anhydrase, producing a physiological buffer pair with 13C NMR signals that can be quantified for pH
measurements. Finally, in vivo tissue pH measurements using HP 13C-EAC was successfully demonstrated in the liver of healthy
rats. These results suggest that HP 13C-EAC is a novel imaging probe for in vivo pH measurements.
KEYWORDS: pH sensor, hyperpolarization, 13C MRI, ethyl acetyl carbonate, esterase, carbon dioxide, bicarbonate

C arbon dioxide (CO 2 ), the metabolic product of

catabolism in all tissues, is rapidly converted to carbonic
acid (H2CO3) by the ubiquitous enzyme, carbonic anhy-
attractive because it involves administration of a physiological
base, bicarbonate, with excellent MR imaging sensitivity.
However, multiple challenges remain before this method can
drase.1,2 Even though carbonic acid is considered a weak acid be applied for routine in vivo pH measurements. First, the HP
(pKa = 6.35), the amount of bicarbonate (HCO3−) present in
13
C MR signal of H13CO3− is relatively short-lived because of
blood near physiological pH values (∼25 mM) contributes fast T1 relaxation (T1 = ∼10 s). DNP of inorganic bicarbonate
significantly to the total buffering capacity of blood. The salts is also challenging, generally resulting in low polarization
equilibrium between CO2 and HCO3− is altered most levels (∼16%).5 Gallagher et al. achieved an improved 13C
polarization level using cesium [13C]bicarbonate and demon-
dramatically in tissues that produce excess acid. Consequently,
strated pH imaging of subcutaneous EL4 tumors in mice.6 The
the ratio of CO2 to HCO3− has been used as an index of tissue alkaline metal ions were removed by ion exchange prior to
pH using hyperpolarized (HP) 13C MRI.3 HP 13C MRI is a administration due to potential Cs toxicity.7 Several studies
novel imaging method that allows for insensitive nuclei such as have evaluated the use of molecular precursors to produce HP
13
C to be imaged, owing to the significantly improved NMR 13
C imaging probes in situ either by chemical or enzymatic
sensitivity afforded by dynamic nuclear polarization (DNP).4 decompositions of the precursors. These approaches involve
The hyperpolarized spin state is not in equilibrium and returns the polarization of chemically stable 13C-enriched compounds
to thermal equilibrium as a function of spin−lattice relaxation with a long T1 and the desired molecular sensing probes are
time, T1. Using DNP, various salts of 13C-enriched bicarbonate
(H13CO3−) have been hyperpolarized, and upon dissolu- Received: September 25, 2018
tion and administration, the resulting HP H13CO3− rapidly Accepted: November 6, 2018
equilibrates in vivo to form HP 13CO2. This approach is Published: November 6, 2018

© XXXX American Chemical Society A DOI: 10.1021/acssensors.8b01097

ACS Sens. XXXX, XXX, XXX−XXX
ACS Sensors Letter

produced after the dissolution of the HP agents.8−13 So far, primarily take place only after the injection of the HP 13C
two molecular precursors for HP 13CO2/H13CO3− have been imaging agent.
evaluated for pH imaging applications. Ghosh et al. produced To test our hypotheses, we first synthesized 13C-enriched
HP 13CO2 by oxidation of HP 13C α-keto acids with H2O2.9 In ethyl acetyl carbonate (13C-EAC) from 13C-enriched ethyl
another study by Korenchan et al., HP H13CO3− and 13CO2 chloroformate and acetic acid in diethyl ether using N-
were produced by chemical hydrolyses of HP 13C organic methylmorpholine as a base (Supporting Information). We
carbonate compounds.12 Although the longer T1 and higher found that 13C-EAC was relatively stable when stored in an
polarization levels of the precursor molecules may be aprotic solvent at low temperature but gradually hydrolyzed in
beneficial, the required decarboxylation delayed the probe water under ambient conditions. During initial trials to polarize
injection, again resulting in significant HP 13C signal losses. 13
C-EAC using the trityl radical OX063, it was found that the
More importantly, these approaches still involve administration radical itself quickly hydrolyzed the compound. However, the
of HP H13CO3− and 13CO2, probes with very short T1 values, hydrophobic and relatively inert radical BDPA22,23 was found
offsetting any advantages the proposed HP 13CO2-precursor to be fully soluble in 13C-EAC without causing any
molecules offer. HP 13CO2 can also be produced in tissues via decomposition. Moreover, the resulting neat-EAC-BDPA (40
the decarboxylation of HP 13 C-pyruvate by pyruvate mM) formed a glassy solid when frozen and nicely polarized
dehydrogenase (PDH).14,15 However, this method requires without a glassing matrix. From a polarization buildup of 13C-
high flux through PDH which may not occur in vivo even in EAC/BDPA (Figure 1a), it is clear that 13C-EAC polarization
highly oxidative tissues because of the presence of competing
substrates such as fatty acids.16 Furthermore, PDH activity is
inherently low in some tissues such as cancers. 17,18
Consequently, administration of HP [1-13C]pyruvate is not a
reliable method for generating HP 13CO2.
Here, we report the development of mixed anhydride
compounds as precursors of HP 13CO2 and H13CO3− for tissue
pH measurement. Typical mixed anhydrides consist of an
alkoxy (R′O-) group and an acyl (R″CO-) moiety (Scheme 1).
Figure 1. (a) DNP polarization buildup of neat 13C-EAC polarized
with BDPA (40 mM) in a HyperSense polarizer. (b) Arrayed 13C
Scheme 1. Proposed Mechanism of Esterase-Catalyzed NMR spectra of HP 13C-EAC (2 mM in ethanol) following ∼1 h of
Production of HP 13CO2 and H13CO3− from 13C-Enriched polarization acquired every 5 s with a 10° flip angle at 400 MHz.
Mixed Anhydridesa

reached the maximum level in a very short time (∼10 min)

confirming that BDPA is an excellent radical for 13C-EAC
polarization. Dissolution of HP 13C-EAC was first done with
ethanol to avoid potential hydrolysis. Arrayed 13C NMR
spectra acquired every 5 s of 2 mM 13C-EAC in ethanol are
shown in Figure 1b. The T1 of the 13C-enriched carbonate at
9.4 T was 30 s. The liquid state signal enhancement calculated
from signal intensities of the first HP spectrum and thermally
polarized signal (Figure S3) was ∼30,000-fold, corresponding
to ∼25% polarization. In addition to EAC, other mixed
a
CA = carbonic anhydrase.
anhydride compounds with different alkoxy and acyl groups
We hypothesized that the ester functional group of a mixed were also synthesized to evaluate the chemical stability and
anhydride would be readily hydrolyzed by carboxyl esterase T1’s of this class of compounds (Table S1, SI). We found that
13
(Step 1). The hydrolyzed product monoacyl carbonate would C-EAC possesses the most favorable properties among those
then undergo a unimolecular decomposition to produce CO2 molecules for in situ production of HP 13CO2. Larger mixed
(Step 2). Since these processes take place in vivo, the newly anhydrides are more stable, but the shorter T1 and lower water
produced CO2 will be rapidly equilibrated to HCO3− by solubility are major drawbacks. Despite the longer T1, methyl
carbonic anhydrase (Step 3) and the resulting HCO3−/CO2 acetyl carbonate is less stable and more importantly produces
ratio will reflect the local tissue pH. Based on the design of toxic methanol as a byproduct after the esterase hydrolysis.
these imaging probes, 13C-enrichment of only the carbonate Biocompatible ethanol and acetate are produced as byproducts
carbon is required for in situ HP 13CO2 production. Given the following the esterase hydrolysis of HP 13C-EAC, thereby
high esterase activity in plasma,19,20 we expect that the mixed minimizing toxicity concerns.
anhydride compounds would be readily hydrolyzed in the We next investigated the applicability of HP 13C-EAC as a
circulation soon after being administered. Mixed anhydride precursor for in situ production of HP 13CO/H13CO3−. The
molecules that survive the hydrolysis by circulating esterase main goals were to (1) evaluate the chemical stability of HP
13
can also be hydrolyzed in esterase-rich organs such as the liver C-EAC in a physiological buffer solution; (2) assess whether
and kidneys.21 We also expect that the sequence of chemical HP 13C-EAC is sensitive to esterase activity; and (3) evaluate
reactions following the hydrolysis would be relatively fast and whether the esterase-hydrolyzed product decomposes within
produce enough H13CO3− and 13CO2 for pH measurements. the HP 13C signal time frame to produce HP 13CO2. First, we
This approach also allows for administration of relatively stable collected an array of 13C NMR spectra of HP 13C-EAC
HP 13C compounds with high polarization levels. The dissolved in PBS buffer with each spectrum acquired every 5 s
production of HP 13CO2 and H13CO3− is also expected to (pH = 7.4, Figure S4). A summed 13C NMR spectrum over the
B DOI: 10.1021/acssensors.8b01097
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ACS Sensors Letter

course of the array (lower panel of Figure 2a) shows some the signal intensity of HP 13CO2 increased much more slowly
degree of HP 13C-EAC hydrolysis in PBS. The hydrolyzed and peaked at ∼50 s. Given that decomposition of 13C-MAC is
not enzymatically catalyzed, one would expect similar kinetics
for the appearance of HP 13CO2 in all three samples. However,
the apparent decomposition rates of HP 13C-MAC follow the
decreasing trend as liver homogenate > plasma > esterase.
Much faster decomposition of HP 13C-MAC was observed in
the liver homogenate sample as evident from the sharp decay
of the HP 13C-MAC signal curve. These results suggest that
the presence of proteins and divalent cations present in the two
biological samples may facilitate the hydrolysis of 13C-MAC,
but the exact mechanism is yet to be delineated. Nevertheless,
the rapid decomposition of HP 13C-MAC is highly beneficial
for the purpose of producing HP 13CO2 for imaging tissue pH.
The HP H13CO3− peaks in Figure 2a and signal-time curves in
Figure 2b demonstrate that much of the newly generated HP
13
CO2 in the liver homogenate sample was rapidly converted to
HP H13CO 3− by carbonic anhydrase. While carbonic
anhydrase is not expected in the plasma sample,25,26 a small
amount of the enzyme may be present due to lysed red blood
cells during the plasma preparation. Consequently, the
H13CO3− signal in the plasma was much lower. The isolated
esterase solution did not contain carbonic anhydrase enzyme.
Therefore, H13CO3− was produced solely by chemical
hydration of 13CO2 in this sample. Despite the presence of
carbonic anhydrase in both plasma and liver homogenate
Figure 2. (a) Representative 13C NMR spectra of HP 13C-EAC (2 samples, the pH values estimated from H13CO3−13CO2 ratios
mM) in PBS (pH = 7.4), isolated esterase solution, rat plasma, and rat were much lower than the values measured by a glass electrode
liver homogenate. Each spectrum is a sum of all spectra acquired over of the same solutions. This is likely due to the high
the course of HP 13C-signal lifetime (∼3 min). A small peak at 156.3
concentrations of newly produced 13CO2 in these static
ppm is the non-13C-enriched acetyl carbon. (b) HP 13C NMR signal-
time curves of 13C-MAC, 13CO2, H13CO3−, and 13C-EAC (divided by systems. The 13CO2/H13CO3− ratio was therefore not in an
5) normalized to the total HP 13C signal from the same samples. equilibrium within the HP 13C signal time frame.5 Our results
demonstrate that HP 13C-EAC is quite stable with slight
hydrolysis in buffer media. The presence of esterase enzyme in
product, 13C-enriched monoacetyl carbonate (13C-MAC), plasma and liver homogenate significantly accelerated HP 13C-
appeared at 160.0 ppm. 13CO2 and H13CO3− resonances are EAC hydrolysis, producing HP 13CO2 that can be readily
also visible at 125.0 and 160.6 ppm, respectively, confirming
13
converted to H13CO3− by carbonic anhydrase for tissue pH
CO2 production from 13C-MAC decomposition. The measurements.
H13CO3− peak is small because carbonic anhydrase was not 13
C chemical shift imaging (CSI) of a phantom containing
present. We then evaluated the esterase-catalyzed hydrolysis of solutions of HP 13C-EAC in PBS and liver homogenate are
HP 13C-EAC in the presence of isolated porcine liver esterase shown in Figure 3. In this phantom, two small vials inside a 20
and in rat plasma and liver homogenate solutions, both of mL beaker filled with water were added a solution of HP 13C-
which contain carboxyl esterase.20,24 Arrayed 13C NMR spectra EAC in PBS or HP 13C-EAC in liver homogenate ([13C-EAC]
of HP 13C-EAC in esterase solution, plasma, and liver = 15 mM). Figure 3a−c show HP 13C signal intensity maps of
homogenate (Figure S4) show the evolution of 13C-MAC, 13
CO2, H13CO3−, and 13C-MAC acquired ∼15 s after mixing
13
CO2, and H13CO3− peaks soon after mixing. Summed 13C overlaid on a 1H reference image. Higher intensity of 13CO2 is
spectra of HP 13C-EAC in these samples are shown in Figure shown in the left vial containing the liver homogenate solution
2a. The spectra confirm higher production of these hydrolyzed than the right vial of HP 13C-EAC in PBS. This confirms the
13
C-species in the samples that contain carboxyl esterase, as rapid hydrolysis of HP 13C-EAC in the presence of esterase
expected. The presence of enzyme esterase resulted in rapid enzyme coupled with faster 13C-MAC decomposition in the
hydrolysis of HP 13C-EAC to HP 13C-MAC. Plots of signal liver homogenate sample, as previously discussed. Intensity
intensities of 13C-MAC, 13CO2, and H13CO3− normalized to maps of 13C-MAC and H13CO3− are not distinguishably
the total HP 13C signal over the course of the NMR acquisition different in these two vials. However, the 13C-EAC maps in
are shown in Figure 2b. The signal-intensity plots confirm that Figure 3d show a noticeably lower signal in the liver
HP 13C-MAC was produced instantly in esterase, plasma, and homogenate vial due to fast 13C-EAC decomposition by
liver homogenate samples following the mixing. esterase hydrolysis. The results show that the production of
The decomposition of HP 13C-MAC to HP 13CO2 in both HP 13CO2 and H13CO3− from esterase-catalyzed hydrolysis of
biological samples was quite fast as confirmed by the HP HP 13C-EAC can be imaged by chemical shift imaging. The
13
CO2 signal-time curves. Interestingly, the HP 13CO2 curve of H13CO3− and 13C-MAC peaks can be resolved by peak-fitting
the esterase sample has a distinctively different kinetics both peaks during postimage processing and signal intensity
compared to those of the two biological samples. In plasma maps of these two hydrolyzed products can be generated. It
and liver homogenate, the signals reached an apex at ∼20 s must be stated that this relatively small chemical shift
before decaying due to T1 relaxation. In the esterase solution, separation (Δδ = 0.6 ppm) could become a challenge in the
C DOI: 10.1021/acssensors.8b01097
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ACS Sensors Letter

Our results showed that HP 13C-MAC, 13CO2, and H13CO3−

peaks appeared soon after the injection of HP 13C-EAC
(Figure 4b). The signal of the parent compound HP 13C-EAC
was very small in every spectrum compared to its downstream
metabolites. This indicates that the hydrolysis of 13C-EAC by
esterase is very rapid in vivo. Although the HP 13C-MAC and
HP H13CO3− peaks in this in vivo result are somewhat
overlapped due to greater field inhomogeneity, the two
resonances are resolved and the peak intensities of the two
HP compounds can be quantified by peak fittings using
Gaussian and Lorentzian functions (Figure S6). The intensities
of the H13CO3− and 13CO2 signals are plotted as a function of
time in Figure 4b. A ratio between HP H13CO3− and 13CO2
signal intensities was calculated over 21 s (7 time points) while
Figure 3. CSI of a phantom containing HP 13C-EAC (15 mM) in PBS HP 13CO2 signal intensity can be reliably measured. A bar
(right tube) and liver homogenate (left tube). (a-d) Signal intensity graph showing an average value of these ratios are shown in
images of HP 13CO2, 13C-MAC, H13CO3−, and 13C-EAC. A total HP Figure 4c. From these values, the average HP H13CO3−/13CO2
13
C intensity map and a 1H reference image are shown in (e) and (f), signal intensity ratio was 11.9:1 and the average pH value was
respectively. (g,h) 13C spectra of the region-of-interest (ROI) 7.24 ± 0.08 (Figure 4d). This value agrees very well with the
depicted in (e). The left and right intensity histograms are for pH of rat livers reported previously.27 It is important to note
images a-c and images d-e, respectively. CSI parameters: matrix = 8 × that in all three rats injected with HP 13C-EAC, there were no
8, FOV = 45 × 45 mm2, slice thickness = 10 mm, flip angle = 10°,
number of points = 512; spectral width = 5 kHz. visible signs of acute toxicity following the injection as
observed from the normal heart and respiratory rates. All
rats also recovered normally after the experiments, suggesting
in vivo settings because of field inhomogeneities. Nonetheless, that 13C-EAC is biocompatible.
this concern could be alleviated with increasing interest in In conclusion, we have demonstrated that tissue pH can be
high-field MRI where larger chemical shift separation can be measured in vivo using HP 13C-EAC. By taking advantage of
achieved. the abundant esterase and carbonic anhydrase activities in
Finally, HP 13C-EAC was also presented to live animals. In mammalian tissues, HP 13CO2 and H13CO3− can be rapidly
this experiment, rats were administered with a solution of HP produced in situ and tissue pH can be calculated from HP 13C
13
C-EAC in PBS (3 mL, ∼80 mM) via a tail vein catheter after signal ratios of the physiological buffer pair. The T1, reasonable
a quick removal of BDPA by filtration. Sequential 13C NMR chemical stability, and high polarization level of HP 13C-EAC
spectra collected form the liver region only (n = 3) were are also advantageous for HP 13C imaging applications,
acquired every 3 s using 20° pulses. A HP 13C spectrum (sum potentially allowing for preinjection quality control procedures
of 7 spectra) over 21 s period after the HP 13C-EAC injection
such as radical removal without a significant signal loss. The
is shown in Figure 4a, showing all expected HP 13C species.
results suggest that HP 13C-EAC is an attractive pH imaging
agent for in vivo assessment of abnormal tissue pH associated
with many diseases.

■
*
ASSOCIATED CONTENT
S Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acssen-
sors.8b01097.
Synthesis, characterization, and hyperpolarization of
mixed anhydrides; detailed experiments for hydrolysis
analyses of HP 13C-EAC; MRI parameters for 13C-CSI
of HP 13C-EAC (PDF)

■ AUTHOR INFORMATION
Corresponding Author
Figure 4. In vivo pH measurements of a rat liver. (a) 13C NMR *E-mail: [email protected].
spectrum of rat liver following HP 13C-EAC (80 mM in PBS)
administration. The spectrum is a sum of 7 spectra acquired every 3 s ORCID
over 21 s with 10° pulses at 4.7T. (b) Signal intensities of HP Gaurav Sharma: 0000-0002-1754-7163
H 13 CO 3 − and 13 CO 2 from the rat liver; (c) Average HP Chalermchai Khemtong: 0000-0003-1976-8098
H13CO3−/13CO2 signal ratio calculated over 21 s. (d) Average liver
pH value estimated from the HP H13CO3−/13CO2 ratio. (e) 1H Notes
reference image showing the liver for HP 13C MRS. The authors declare no competing financial interest.
D DOI: 10.1021/acssensors.8b01097
ACS Sens. XXXX, XXX, XXX−XXX
ACS Sensors Letter

■ ACKNOWLEDGMENTS
We thank the following agencies for financial support: NIH
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E DOI: 10.1021/acssensors.8b01097
ACS Sens. XXXX, XXX, XXX−XXX