Determining the Rate of Carbonic Anhydrase Reaction in the Human Brain

Carbonic anhydrase plays important role in life. This study sought to demonstrate the feasibility of detecting carbonic anhydrase activity in the human brain in vivo. After oral administration of [U-13C6]glucose, 13C saturation transfer experiments were performed with interleaved control spectra and carbon dioxide saturation spectra. Proton nuclear Overhauser effect pulses were used to increase signal to noise ratio; no proton decoupling was applied. Results showed that the 13C signal of bicarbonate was reduced by 72% ± 0.03 upon saturating carbon dioxide. The unidirectional dehydration rate constant of the carbonic anhydrase reaction was found to be 0.28 ± 0.02 sec−1 in the human brain. These findings demonstrate the feasibility of measuring carbonic anhydrase activity in vivo in the human brain, which makes it possible to characterize this important enzyme in patients with brain disorders.

signal, as expected from the Henderson-Hasselbalch equation [1]. When the two spectra were compared, signals of bicarbonate and carbon dioxide were increased by 101% and 60.5%, respectively.
The 13 C saturation transfer effect of the uncatalyzed exchange between carbon dioxide and bicarbonate was investigated using the same phantom at ambient temperature. Figure 2 shows the observed 13 C saturation transfer effect due to the exchange between carbon dioxide and bicarbonate in the absence of catalysis by CA. The control spectrum (Fig. 2a) was the same as the NOE-enhanced spectrum in Fig. 1. We found that the bicarbonate signal was appreciably reduced upon radio-frequency (RF) saturation of carbon dioxide (Fig. 2b), and that the signal of carbon dioxide disappeared (Fig. 2b). The difference spectrum revealed an 8.2% decrease at ambient temperature, and pH = 7.2 caused by the uncatalyzed exchange between carbon dioxide and bicarbonate in the phantom (Fig. 2c). The T 1 of bicarbonate in the phantom was found to be 35 s, as measured using the equation for T 1 calculation given by Xu and colleagues 22 . The same T 1 was found with and without NOE enhancement. Using Eq. (1) (see Methods), the uncatalyzed unidirectional dehydration rate constant was 0.0058 sec −1 .
During in vivo scans, blood samples were collected every 10 minutes to measure blood glucose concentrations. Although the total glucose reading fluctuated during the scan, signals of bicarbonate and other metabolites increased steadily and reached their approximate maxima at 100-120 minutes after oral administration (see Figs 3 and 4).
Saturation transfer effect was measured from three healthy volunteers. A typical time-course of control spectra from human brain after oral administration of [U- 13 C 6 ]glucose without proton decoupling is shown in Fig. 3. The spectra of carboxylic/amide carbons with oral administration of [U- 13 C 6 ]glucose were comparable to those   13 C saturation transfer effect in the phantom due to exchange between carbon dioxide and bicarbonate in the absence of catalysis by carbonic anhydrase (CA). The control spectrum (a) was the same as the NOE-enhanced spectrum in Fig. 1. The bicarbonate signal was appreciably reduced upon radio frequency (RF) saturation of carbon dioxide (b). Note that the signal of carbon dioxide disappeared in (b). The difference spectrum (c) revealed an 8.2% decrease at ambient temperature and pH = 7.2 caused by the uncatalyzed exchange between carbon dioxide and bicarbonate in the phantom.  (Fig. 3). Because of the large homonuclear 13 C-13 C coupling (51 Hz) due to the administration of uniformly 13 C-labeled glucose, the signal from glutamate C5 appeared as a doublet. Signals from other carboxylic/ amide carbons also experienced homonuclear splitting as well as line-broadening due to long-range heteronuclear couplings, and their resonances overlapped each other in the spectrum. No signal from carbon dioxide was detected in vivo 25 . Because all six 13 C labels of [U- 13 C 6 ]glucose contribute to the final bicarbonate 13 C signal-due to the actions of pyruvate dehydrogenase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase-the 13 C signal of bicarbonate became much stronger than that acquired in the previous study with intravenous infusion of [2-13 C]glucose 26 .
The signal intensities of bicarbonate from the three subjects as a function of time is given in Fig. 4 showing that the bicarbonate signals increased approximately monotonically. For the three subjects, the same time dependencies were also consistently observed for signals from the other metabolites (glutamate C1 and C5, glutamine C1 and C5, and aspartate C1 and C4).
The spectra of 13 C saturation transfer experiments obtained from a single subject, measured between 110 and 122 minutes after the oral administration of [U-13 C 6 ]glucose, is shown in Fig. 5. The top spectrum (Fig. 5a) is the control spectrum with the 13 C irradiation at 228 ppm, the middle one (Fig. 5b) is the spectrum obtained   (Fig. 5c) is the difference spectrum. Based on the results of the three subjects, the signal intensity of bicarbonate was found to be reduced by 72% ± 0.03 (mean ± SD, n = 3) due to saturating carbon dioxide. T 1 of bicarbonate resonance was measured from three different subjects using the inversion recovery method. The results of in vivo T 1 measurements of 13 C bicarbonate using the 13 C metabolite null method were plotted in Fig. 6. Trace (a) is the non-inverted spectrum and trace (b) is the inverted spectrum with null signal at a recovery time of 7.0 s. The measured T 1 was 9.6 ± 0.3 s (mean ± SD, n = 3). Using Eq. 1 (see Methods), with the measured values of M sat /M n°sat and the mean of T 1 (9.6 s), the pseudo first-order rate constant for the unidirectional dehydration (k) in the human brain was determined to be 0.28 ± 0.02 sec −1 (n = 3).

Discussion
This 7 Tesla MRS study is the first to demonstrate that CA activity can be detected in vivo in the human brain. Notably, although all of our experiments were performed using a 13 C saturation transfer technique at 7 Tesla, the intense labeled bicarbonate 13 C signal observed after administration of uniformly 13 C-labeled glucose, and the dramatic change in the bicarbonate signal (72% reduction) observed in conjunction with carbon dioxide saturation, suggests that this technique could be readily extended to lower and more clinically relevant field strengths such as 3 Tesla.
The 13 C signal of bicarbonate at 160.7 ppm has been observed in the human brain when detecting carboxylic/amide carbons with infusion of [1-13 C]glucose 27 , [1-13 C]acetate 28 , and [2-13 C]glucose 26 infusions.  Although the concentration of bicarbonate in the brain under normal physiological conditions is relatively high (>20 mM) 29 , previous reports noted that the observed 13 C signal intensity of bicarbonate was relatively weak compared with the signal intensity of many other 13 C-labeled metabolites. There may be several reasons for this. First, to acquire carboxylic/amide carbons from amino acids, the 13 C excitation pulse may be optimally placed on the carboxylic/amide carbons near 180.0 ppm. If the bandwidth of the 13 C excitation pulse is not wide enough, particularly for high field experiments, 13 C-labeled bicarbonates may not be appropriately excited. Second, when a single 13 C-labeled substrate is administered in 13 C MRS studies, only one 13 C label per substrate molecule can be incorporated into bicarbonate. Therefore, the fractional enrichment of 13 C-labeled bicarbonate is relatively low. To increase the bicarbonate signal for the present saturation transfer experiments, we: (a) placed the center frequency of the 13 C excitation pulse at the bicarbonate frequency of 160.7 ppm, (b) used a TR of 30 s for the saturation transfer measurements and 55 s for the T 1 measurements, and (c) administered [U-13 C 6 ]glucose to the subjects so that all six 13 C labels of the substrate would contribute to the final bicarbonate 13 C signal via the actions of pyruvate dehydrogenase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase. As a result of these approaches, the bicarbonate signal was increased significantly. Coupled with the very large saturation transfer effect catalyzed by CA, the overall sensitivity of this experiment was uniquely high for 13 C MRS.
Due to the rapid, CA-catalyzed exchange between carbon dioxide and bicarbonate, as well as the rapid exchange between the proton of bicarbonate and water, the small two-bond 1 H-13 C scalar coupling in bicarbonate is not observable even in the absence of heteronuclear decoupling in both phantom and in vivo scans. Therefore, proton decoupling was not applied in this study, which also helped to largely reduce RF deposition and the associated specific absorption rate. Results from the 13 C-labeled bicarbonate phantom experiment (Fig. 1) demonstrated that the bicarbonate signal was increased by a factor of 2.0 due to the NOE via dipole-dipole interactions between bicarbonate 13 C labels and surrounding protons. Interestingly, although carbon dioxide was not protonated at all, a highly significant (~60%) NOE enhancement of the carbon dioxide signal was also observed. This is likely due to a carry-over of the NOE effect from bicarbonate via interconversion between the two molecules. The magnitude of the NOE on bicarbonate found in this study is very similar to that observed in our previous 7 Tesla phantom study, where signals from glutamine and aspartate were increased by a factor of 2.3 24 . In short, we have successfully measured a large and quantifiable carbon saturation transfer effect in the human brain by combining multiple mechanisms to enhance the bicarbonate 13 C signal with the relatively long 13 C T 1 of bicarbonate and the fast exchange between carbon dioxide and bicarbonate catalyzed by CA.
The present study further demonstrated that the 13 C signal of carbon dioxide was detected in the phantom of 13 C enriched bicarbonate phantom (Fig. 2), but not in human brain. There are several possible reasons for this. First, the concentration of dissolved free carbon dioxide gas in brain tissue is ~1 mM at normal physiological condition 29 , which makes it difficult to detect in vivo in the brain. Second, the intrinsic T 1 of the unprotonated carbon dioxide is expected to be longer than that of the protonated bicarbonate. Third, detection of the carbon dioxide resonance in vivo could also be hampered by the off-resonance effect of the excitation pulse placed at the resonance frequency of bicarbonate. Because the pseudo first-order dehydration rate constant depends on M sat /M nosat of bicarbonate and its T 1 only, the concentration of carbon dioxide has no effect on its quantification. It should be noted that the concentrations of carbonic acid and carbonate were negligibly low at physiological (neutral) pH. They are also in rapid exchange with bicarbonate. Consequently, their in vivo 13 C MRS signals were not observed either.
Following traditional saturation transfer techniques, the frequency of the control irradiation in our scans was initially set at 196.4 ppm, the same frequency difference from bicarbonate (35.7 ppm) but on the downfield side of the bicarbonate resonance. However, at this frequency setting, a small amount of residual signal was observed in the difference spectra in the region from 174.0 ppm to 176.0 ppm, which is dominated by glutamate C1 resonance. The residual signal can be explained by partial saturation of the C1 resonance of α-ketoglutarate hydrate, possibly bound to larger molecules. However, the exact source of this residual signal is unknown. After the control pulse was slightly moved downfield to 228.0 ppm, the residual signal in the difference spectra disappeared, and this shifted frequency was used for all control scans. Nevertheless, this small frequency shift should not have affected the bicarbonate signal because: (1) previous in vivo experiments repeatedly showed the nonexistence of nonspecific background magnetization transfer among the very diluted 13 C spins 22,23 and (2) the large frequency separation between the control frequency (228.0 ppm) and the resonance frequency of bicarbonate (160.7 ppm) ruled out any RF spillover effects commonly seen in 31 P saturation transfer experiments. 13 C-labeled substrates are commonly infused into human subjects via two intravenous (IV) insertions on a subject's forearms; one is used to monitor blood glucose levels and the other is used to infuse the 13 C-labeled substrates. There are many advantages of using intravenous glucose infusion including increased delivery of glucose into the brain. However, placing two IV catheters may increase the chance of causing pain to the subject and other adverse effects, including possible clogging of the infusion line that may interrupt the infusion process and cause swelling. In this study, glucose solution was administered orally [30][31][32] so that only one IV line was needed to monitor blood glucose levels. With the dosage of 0.75 grams 99% 13 C enriched glucose per kg of body weight, the amount of the glucose drunk by the subjects was between 40-50 grams, depending on body weight. The amount of glucose orally administrated into the subjects is very similar to that administrated via IV infusion in our previous infusion protocol 24,26 . The metabolism of 13 C-labeled metabolites is slower with oral administration due to delayed glucose uptake by brain, but the peak fractional enrichment of 13 C-labeled metabolites is not smaller than that associated with the intravenous infusion method if sufficient time is allowed 32 . In our study, larger fluctuations of blood glucose levels were observed (data not shown). The use of a longer catheter line in the 7 T magnet and the increased amount of "dead" blood volume (therefore dilution of blood by saline) may have contributed to this observation. Nonetheless, 13 C enrichment of the brain metabolites increased steadily over the time course (Fig. 3). During the interleaved data acquisition (TR = 30 s), each pair of control spectrum and carbon dioxide-saturated spectrum was taken in one minute. Within one minute, the fractional enrichment of SCIeNtIfIC REPORTS | (2018) 8:2328 | DOI:10.1038/s41598-018-20746-x bicarbonate could be considered stable because of the large dampening effect of various decarboxylation reactions, including the tricarboxylic acid cycle with a time constant of approximately one hour. Therefore, the spectroscopically observed saturation transfer observed here is considered very accurate.
Previous studies reported that the T 1 of bicarbonate was 11.8 s in the anesthetized rat brain at 11.7 Tesla 25 . To accurately measure T 1 of bicarbonate in human brain at 7 Tesla, a TR of 55 s was applied to satisfy the condition of TR ≥ 5 T 1 . Our measurement (n = 3) showed that T 1 of bicarbonate in human brain at 7 Tesla was 9.6 ± 0.3 (n = 3). The shorter T 1 found in this study may be attributable to generally faster relaxation in the more iron-rich human brain.
Finally, although only the cerebral carbon dioxide-bicarbonate exchange catalyzed by CA was investigated in this study, it is expected that the in vivo 13 C saturation transfer spectroscopy method can also be used to study the CA-catalyzed carbon dioxide-bicarbonate exchange in other organs/tissues because of the wide distribution of CA, carbon dioxide, and bicarbonate.
In summary, this study demonstrated that bicarbonate and carbon dioxide are in rapid exchange in the human brain under catalysis by CA. The exchange is sufficiently rapid in vivo to lead to a large and quantifiable 13 C saturation transfer effect. The signal intensity of bicarbonate was significantly boosted by administering [U-13 C 6 ] glucose. Because the 13 C bicarbonate signal was not overlapped by that of scalp lipids, spatial localization methods such as SPLASH 33 may be used to extract CA activity from a predefined brain region with natural anatomical boundaries.

Methods
In vivo 13 C MRS experiments were performed on a Siemens Magnetom 7 Tesla scanner (Siemens Healthcare, Erlangen, Germany) with VB17 software. The in-house built RF coil assembly comprised a circular 13 C coil (diameter = 7 cm) and a quadrature half-volume proton coil. They were mounted on three vertically stacked semi-cylindrical plastic tubes. Each proton loop had a single-tuned 1 H cable trap constructed using the RG-316 cable. A 13 C/ 1 H dual-tuned cable trap, built inside a shielded box, was connected to the 13 C coil. At 75 MHz, the isolation between the 13 C coil and both proton loops was −38 dB. At 300 MHz, the isolation between the two proton loops and between the 13 C coil and both proton loops was −20 and −40 dB, respectively. The coil assembly was connected to the 7 Tesla scanner via an interface box (Quality ElectroDynamics, Mayfield Village, Ohio, USA) that included transmit-receive switches, pre-amplifiers, and RF filters for both channels, as well as a quadrature combiner for the proton channel. Details of the coil design, fabrication, its RF safety evaluation and positioning of human subjects were described in a previous publication from our laboratory 24 .
Healthy human subjects (n = 6) were recruited and consented using the procedures approved by the Institutional Review Board (IRB) of the National Institute of Mental Health (NIMH). The administration of 13 C enriched glucose solution has been approved by IRB-NIMH and the National Institutes of Health (NIH) Clinical Center Pharmacy Department. All experimental protocols and methods were performed in accordance with the guidelines and regulations of NIH MRI Research Facility. Subjects fasted at least 12 hours before MRS scans. A solution of 20% w/w 99% enriched [U-13 C 6 ]glucose was orally administrated to subjects at a dose of 0.75 g [U-13 C 6 ]glucose per kg of body weight 32 before initiation of 13 C MRS scans. One antecubital vein was cannulated for withdrawing blood every 10 minutes to monitor blood glucose levels during the entire study.
A gradient-echo based 3-plane localizer was first used to properly position each subject. Static magnetic (B 0 ) field shimming was performed using Siemens 3D Shim tool that included full first-and second-order shims, and axial terms of third-order shims (Z 3 , Z 2 X, Z 2 Y, Z(X 2 − Y 2 )). A voxel of 5 × 5 × 5 cm 3 cube was selected to perform B 0 shimming in the occipital lobe directly above the 13 C surface coil. A point resolved spectroscopy (PRESS) sequence 34 was used to acquire water spectrum from the shimmed voxel in order to evaluate the convergence of shim adjustments. Typical water linewidth (full width at half maximum) from the 125 cm 3 cubic voxel was 14.8 ± 2.1 Hz. The procedures used to calibrate RF transmit power for the proton and 13 C coils have been previously described 24 . Briefly, RF power in the proton channel was calibrated in-situ using a two-dimensional stimulated echo method that generates a one-dimensional profile along the y-column. The transmit voltage of a proton excitation pulse was adjusted to generate a 180 o null along the y-direction. This voltage was used as a reference for NOE pulses. The RF power of the 13 C coil was calibrated using a phantom of a three-liter cylindrical bottle filled with distilled water and 6 grams NaCl. 13 C transmit voltage for a 500 μs 90° hard pulse was determined using the maximum signal of [1-13 C]glucose in a small sphere inside the cylinder.
The following parameters were used to acquire phantom 13  In vivo 13 C saturation transfer spectra were acquired from three healthy subjects using a modified Siemens free induction decay sequence. The sequence had an interleaved acquisition scheme: {control irradiationbicarbonate excitation -acquisition} -{carbon dioxide saturation -bicarbonate excitation -acquisition}. The excitation hard pulse (0.25 ms) was placed at the resonance frequency of bicarbonate (160.7 ppm). Acquisition parameters were SW = 8 kHz, data points = 2048, acquisition time = 256 ms, and TR = 30 s. During the entire recycle delay of 30 s, a 50 ms pulse block was repeatedly executed. In each pulse block, a 1.0 ms proton hard pulse (120°) was applied first at water frequency to generate heteronuclear NOE, then a 48.0 ms continuous wave pulse (γB 1 = 50 Hz) was used to saturate carbon dioxide at 125.0 ppm or for control irradiation at 228.0 ppm. No proton decoupling was applied. The average RF power was less than 2.8 W. Each interleaved dataset contained 24 spectra (NA = 24). Twelve odd-number spectra had control irradiation at 228.0 ppm, and 12 even-number spectra had a saturation pulse placed on carbon dioxide at 125.0 ppm.
Prior to performing in vivo studies, phantom experiments were conducted to evaluate NOE and the saturation transfer effects of the uncatalyzed exchange between carbon dioxide and bicarbonate. The phantom was a three-liter cylindrical bottle filled with distilled water and 6 g NaCl. The loading effect of this phantom is approximately the same as that of an adult human head based on the measurement of loaded Q-value of the coils. Inside SCIeNtIfIC REPORTS | (2018) 8:2328 | DOI:10.1038/s41598-018-20746-x the bottle, a 7-cm sphere containing 20 mM NaH 13 CO 3 (pH = 7.2) was attached to the cylindrical surface of the bottle directly above the 13 C coil. The interleaved 13 C pulse sequence described above was used in the phantom experiments with the following changes: (a) the 13 C excitation pulse was placed at the frequency of carbon dioxide (125.0 ppm), (b) TR = 35 s, NA = 16, and (c) control spectra were acquired without any 13 C pre-irradiation.
The T 1 of in vivo 13 C bicarbonate signal is relatively long 25 . When bicarbonate is partially saturated due to insufficient TR, the pseudo first-order rate constant for unidirectional dehydration (H + + HCO 3 − → CO 2 + H 2 O) can be calculated using the following equation [ where M sat is the bicarbonate 13 C magnetization measured while saturating carbon dioxide, M nosat is the bicarbonate 13 C magnetization without saturation of carbon dioxide, TR is repetition time, T 1 is the spin-lattice relaxation time of bicarbonate without saturation of carbon dioxide, and k is the pseudo first-order rate constant for the unidirectional dehydration reaction. In vivo T 1 of bicarbonate was measured using the inversion-recovery null method without saturating carbon dioxide. A hyperbolic secant pulse (30 ms) was used to invert the bicarbonate 13 C signal. For T 1 measurements, TR was set to 55 s.
Data availability statement. The data of this study are available from the corresponding author upon request.