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Hyperpolarized 13C-labeled bicarbonate (H13CO3-) for in vivo pH measurement with 13C magnetic resonance spectroscopy

Hyperpolarized H13CO3-
, PhD
National Center for Biotechnology Information, NLM, NIH
Corresponding author.

Created: ; Last Update: April 12, 2010.

Chemical name:Hyperpolarized 13C-labeled bicarbonate (H13CO3-) for in vivo pH measurement with 13C magnetic resonance spectroscopy
Abbreviated name:Hyperpolarized H13CO3-
Synonym:13C-bicarbonate, 13CO2
Agent Category:Compounds
Target:Carbonic anhydrase (tissue pH)
Target Category:Enzymes
Method of detection:Magnetic resonance imaging (MRI)/magnetic resonance spectroscopy (MRS)
Source of signal / contrast:Hyperpolarized 13C
  • Checkbox In vitro
  • Checkbox Rodents
No structure is available.



Magnetic resonance spectroscopy (MRS) is a technique that allows the non-invasive detection of multiple small metabolites within cells or extracellular spaces in vivo (1-4). Although MRS is theoretically applicable to any nucleus possessing spin, the more frequently investigated applications are in proton (1H) and carbon-13 (13C) (5-7). 13C MRS is superior to 1H MRS in many respects (3, 7-9). 13C MRS can provide specific information about the identity and structure of biologically important compounds. The chemical shift range for carbon (~250 ppm) is much larger than that for proton (~15 ppm), allowing for improved resolution of metabolites. However, 13C MRS is limited by the low natural abundance of 13C (1.1%) and its very low nuclear spin polarization (2.5 × 10-6 polarization at 3 T and 37ºC) (2, 3). Several techniques have been used to overcome these limitations, including dynamic nuclear polarization (DNP), which introduces one or more 13C molecules into a metabolic substrate (2, 3, 9). Because the T1 relaxation time of 13C in small molecules is much longer than that of 1H (0.1–2.0 s in a magnetic field of 0.1–3.0 T), hyperpolarized 13C-labeled tracers can be generated outside the subject and the magnetic resonance scanner (7). Nearly 100% nuclear polarization for 1H and 50% for 13C can be achieved in various organic molecules when DNP is performed in a strong magnetic field and at cryogenic temperatures. Replacing the 12C isotope (98.9% natural abundance) with the 13C isotope at a specific carbon or carbons in a metabolic substrate does not affect the substrate’s biochemistry. Hyperpolarized 13C-labeled substrates can provide >10,000-fold enhancement of the 13C MRS signals from the substrate and its subsequent metabolic products, allowing the assessment of changes in metabolic fluxes in vivo and the imaging of blood vessels and tissue perfusion without background signal from surrounding tissues (1, 3, 4, 10-14).

13C MRS with DNP technique has also been investigated for measuring tissue pH in vivo (4, 15). HCO3- is the primary extracellular buffer, and it resists changes in pH through interconversion with CO2 in the reaction catalyzed by carbonic anhydrase. In principal, tissue pH can be determined from 13C MRS measurements of endogenous H13CO3- and 13CO2 because their concentration ratio can be used to calculate pH from the Henderson-Hasselbalch equation with an acid dissociation constant (pKa) of 6.17 in vivo. On the basis of this principal, Schroeder et al. measured the pH in diseased and healthy cardiac myocytes with simultaneous detection of hyperpolarized [1-13C]pyruvate-derived H13CO3- and 13CO2 (15). Their results suggest that hyperpolarized [1-13C]pyruvate with MRS detection of its derived H13CO3- and 13CO2 can be used to measure the intracellular pH (pHi) of cardiomyocytes in vivo. Similarly, Gallagher et al. generated a non-toxic, pH-probe, hyperpolarized H13CO3- and exploited the pH in tumors with measurement of the H13CO3- and 13CO2 concentration ratio after administration of hyperpolarized H13CO3- (4).

The tumor microenvironment is characterized by low extracellular pH (pHe) and neutral-to-alkaline pHi (16, 17). The average pHe could be as low as 6.0. A pH gradient (pHi > pHe) exists across the cell membrane in tumors. This gradient is contrary to that found in normal tissues, in which pHi (7.2–7.4) is lower than pHe. In addition, diffusion of the H+ ions along concentration gradients from tumors into adjacent normal tissues creates a peritumoral acid gradient. Accurate measurement of the pH in tissues is of diagnostic and therapeutic value. Imaging with small-molecule agents has been tested for measuring tumor pH. However, agents based on 1H, 31P, or 19F MRS are limited by the inherent low sensitivity of spectroscopy and small pH-dependent chemical shift of these agents (18, 19). The approach with gadolinium (Gd3+) chelate relaxation agents, which show a pH-dependent hydrogen exchange to the Gd3+-bound water, requires an accurate determination of the agent concentration, which in practice is difficult to achieve in vivo (20). Although positron emission tomography and optical imaging are sensitive, they appear have difficulty obtaining a pH map at high resolution (21-23). Furthermore, most of the published probes predominantly measure the pH within cells, which is more resistant to pH changes than the extracellular space. The data obtained by Gallagher et al. from hyperpolarized H13CO3- indicated that hyperpolarized H13CO3- provided a means to measure the pHe rather than the pHi (4). Given the range of pathological conditions in which the acid–base balance is altered, this technique may prove to be of diagnostic value not only in oncology but also in the imaging of ischemia and inflammation (4).



Gallagher et al. described the generation of hyperpolarized H13CO3- in detail (4). Briefly, CsH13CO3 was first prepared by slowly adding 13CO2 to CsOH hydrate. Up to 98% of the bicarbonate was labeled with 13C at this step. 13C-Labeled CsH13CO3 was then dissolved in water and glycerol. After addition of free radical (OX063) and Gd3+ chelate, the solution was polarized in a polarizer under a 93.982 GHz and 100-mW microwave source for 2 h. The frozen sample was dissolved in phosphate buffer (pH 7.5) containing diaminoethane tetraacetic acid heated to 180°C and pressurized to 10 bar. Cs was rapidly removed with an ion-exchange column to generate the pure hyperpolarized H13CO3-. The final concentration of H13CO3- was ~100 mmol at 37°C. The polarized H13CO3- was 16% in the solution state, which represents a 20,000-fold increase above thermal equilibrium (at 9.4 T and 37°C).

In Vitro Studies: Testing in Cells and Tissues


Gallagher et al. first tested the feasibility to measure the pH in phosphate buffers in vitro with hyperpolarized H13CO3- (4). The pH in each voxel was calculated from the relative concentrations of H13CO3- and 13CO2 with the Henderson-Hasselbalch equation. In the presence of carbonic anhydrase in buffers, the estimated pH was closely correlated with that measured using a pH meter (average pH difference ± SD, 0.07 ± 0.03; n = 4) but was slightly overestimated in the absence of the enzyme (0.32 ± 0.13; n = 6) (P = 0.01). This close correlation reflects the rapid establishment of chemical equilibrium and equilibration of polarization between H13CO3- and 13CO2 catalyzed by carbonic anhydrase.

Animal Studies



Gallagher et al. performed 13C spectra and spectroscopic imaging (at 9.4 T) in mice bearing murine lymphoma tumors (4). One group of mice was pretreated with ammonium chloride to decrease the tissue pH, and another group of mice was pretreated with sodium bicarbonate solution to increase the tissue pH. 31P spectra were also acquired after the intraperitoneal administration of 3-aminopropylphosphonate (3-APP) for comparative study. Spectra of H13CO3- and 13CO2 from tumor slices (5 mm thick) gave a calculated pH of 6.71 ± 0.14 (n = 12) compared with a non–slice-selective pH, which included a weighted mean of the tumor pH and that of surrounding tissue, of 7.09 ± 0.10 (n = 8) (P < 0.01). Administration of sodium bicarbonate in the drinking water increased the measured tumor pH to 7.02 ± 0.04 (n = 5) (P < 0.01). Gavage with ammonium chloride decreased the pH to 6.47 ± 0.11 (n = 6) (P < 0.01). The pHi, estimated from the chemical shift of the intracellular inorganic phosphate resonance, was 7.44 ± 0.16 in untreated animals (n = 7) and was similar in mice treated with bicarbonate (7.47 ± 0.15 (n = 4) (P = 0.75)) or ammonium chloride (7.36 ± 0.08 (n = 6) (P = 0.30)). Correlation between the tumor pH determined with hyperpolarized H13CO3- and the pH determined with the 3-APP probe suggested that hyperpolarized H13CO3- predominantly measured the pHe. This probably reflects the higher concentration of extracellular versus intracellular bicarbonate, as well as the rapid acquisition of spectra immediately after injection of hyperpolarized bicarbonate. Bicarbonate injection did not significantly change the tumor pHe as shown with 3-APP–based 31P MRS measurement (pH change = 0.02 ± 0.03; n = 4). Superimposition of the pH maps over the corresponding proton image of tissue water showed that the highest concentration of 13CO2 was in the tumor. The H13CO3- signal was highest in the aorta, and there was little difference in signal intensities between muscle and tumor.

Signal from the hyperpolarized nuclear spin decreases rapidly because of the decay of polarization; thus it is possible that small differences in the rate at which H13CO3- and 13CO2 lose polarization will lead to variations in the calculated pH with time. In the study by Gallagher et al., this was not observed over a period of 20 s measurement in vivo (data not shown) and could be explained by the fact that H13CO3- and 13CO2 are interconverted so rapidly that their apparent T1 values become equal (4). The measured T1 was 10.1 ± 2.9 s (n = 9) for H13CO3- and 9.8 ± 2.5 s for 13CO2 (n = 7) (P = 0.83). Theoretically, in the absence of 13CO2 resonance saturation, decay of the H13CO3- resonance will be dominated by its T1. In the presence of 13CO2 resonance saturation, the H13CO3- signal will decay with a time constant given by 1/(1/T1 + k), where k is the rate constant for the conversion of H13CO3- to 13CO2. Gallagher et al. showed that inhibition of the carbonic anhydrase with acetazolamide abolished this decrease in the H13CO3- signal, demonstrating the importance of the enzyme for the exchange of polarization between the two molecules (4). The apparent exchange rate constant was approximately eight times greater than the polarization loss rate constant (1/T1).

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


No references are currently available.

Human Studies


No references are currently available.


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