In vivo detection of γ-glutamyl-transferase up-regulation in glioma using hyperpolarized γ-glutamyl-[1-13C]glycine

Glutathione (GSH) is often upregulated in cancer, where it serves to mitigate oxidative stress. γ-glutamyl-transferase (GGT) is a key enzyme in GSH homeostasis, and compared to normal brain its expression is elevated in tumors, including in primary glioblastoma. GGT is therefore an attractive imaging target for detection of glioblastoma. The goal of our study was to assess the value of hyperpolarized (HP) γ-glutamyl-[1-13C]glycine for non-invasive imaging of glioblastoma. Nude rats bearing orthotopic U87 glioblastoma and healthy controls were investigated. Imaging was performed by injecting HP γ-glutamyl-[1-13C]glycine and acquiring dynamic 13C data on a preclinical 3T MR scanner. The signal-to-noise (SNR) ratios of γ-glutamyl-[1-13C]glycine and its product [1-13C]glycine were evaluated. Comparison of control and tumor-bearing rats showed no difference in γ-glutamyl-[1-13C]glycine SNR, pointing to similar delivery to tumor and normal brain. In contrast, [1-13C]glycine SNR was significantly higher in tumor-bearing rats compared to controls, and in tumor regions compared to normal-appearing brain. Importantly, higher [1-13C]glycine was associated with higher GGT expression and higher GSH levels in tumor tissue compared to normal brain. Collectively, this study demonstrates, to our knowledge for the first time, the feasibility of using HP γ-glutamyl-[1-13C]glycine to monitor GGT expression in the brain and thus to detect glioblastoma.


Results
Characterization of HP γ-glutamyl-[1-13 C]glycine. HP γ-glutamyl-[1-13 C]glycine was synthesized as previously described 31 , dissolved in NaOH solution and mixed with OX063 and glycerol. Following polarization for 1.5 h, the resonance of γ-glutamyl- [1-13 C]glycine was detected at 177.5 ppm with a polarization level (back calculated to time of dissolution) of 22.9 ± 6.7% (n = 3) when compared to the thermal spectrum (Fig. 1A). T 1 values of HP γ-glutamyl-[1-13 C]glycine were measured in solution at 3T and 11.7T by fitting the dynamically acquired HP 13 C spectra (Fig. 1B) with a monoexponential curve after correcting for flip angle. The T 1 of HP γ-glutamyl-[1-13 C]glycine was 33 ± 3.5 s at 3T (n = 3) and 18 ± 2.5 s (n = 3) at 11.7T, consistent with an expected reduction in the T 1 of 13 C carbonyl groups at higher field and were comparable with published values 31 . In vivo HP studies in healthy and glioblastoma-bearing rats at 3T. Dynamic HP 13 C slab acquisitions using a spectral spatial slice selective scheme were performed on glioblastoma-bearing rats once the tumor reached a volume of 0.27 ± 0.06 cm3 ( Fig. 2A,B). Tumor-free healthy rats were used as controls. The signal from the substrate HP γ-glutamyl-[1-13 C]glycine could be detected in both healthy and tumor-bearing animals (Fig. 2C). Comparison of HP γ-glutamyl- [1-13 C]glycine SNR showed no statistically significant difference between tumor-bearing and healthy rats (134.8 ± 16.5 a.u. vs 155.7 ± 31.8 a.u. for control and tumor animals respectively; p = 0.6; n = 7; Fig. 2D), indicating no significant difference in substrate delivery. The SNR of the product [1-13 C]glycine (173.7 ppm) was below detection in individual spectra of the slab dynamic acquisition (Fig. 2B), but the sum of the dynamic 13 C spectra showed improved SNR and [1-13 C]glycine could be readily detected in all tumor-bearing animals and in some tumor-free healthy animals (Fig. 2C). Importantly however, the SNR of HP [1-13 C]glycine in tumor-bearing rats was significantly higher relative to healthy rats (2.58 ± 1.00 a.u. vs 6.96 ± 1.49 a.u. for control and tumor animals respectively; p = 0.046; n = 7; Fig. 2E) as was the ratio of HP [1-13 C]glycine to γ-glutamyl-[1-13 C]glycine (0.021 ± 0.008 a.u. vs 0.046 ± 0.004 a.u. for control and tumor animals respectively; p = 0.027; n = 7; Fig. 2F). Additionally, the ratio of [1-13 C]glycine to γ-glutamyl-[1-13 C]glycine was positively correlated with the tumor fraction in the slab (R² = 0.8188; Supplementary Fig. 1).

Ex vivo evaluation of GGT enzyme expression and GSH levels in glioblastoma and normal brain.
GGT expression was previously shown to be higher in glioblastoma relative to normal brain 17 . In order to confirm that the higher production of HP [1-13 C]glycine from HP γ-glutamyl-[1-13 C]glycine in tumor relative to normal brain was linked to higher GGT expression, we examined GGT levels by western blotting in tumor and contralateral normal-appearing brain tissue from tumor-bearing rats. Brain tissue isolated from tumor-free healthy controls was also examined. As shown in Fig. 4A, glioblastoma tumor tissues showed higher expression of the GGT isoforms GGT1 and GGT2 compared to contralateral normal-appearing or tumor-free healthy brain tissues (3.06 ± 0.31 a.u., 1.00 ± 0.08 a.u. and 1.14 ± 0.14 a.u. for tumor, contralateral normal-appearing brain and healthy brain respectively; p = 0.03 tumor versus contralateral normal-appearing brain and p = 0.02 tumor versus healthy brain; Fig. 4A,B, Supplementary Fig. 2).
In terms of function, GGT plays a key role in maintaining cellular GSH levels. To assess whether higher tumor GGT activity was linked to higher GSH levels, we measured GSH levels by 1 H-MRS in extracts from U87 tumors, contralateral normal-appearing brain and healthy normal brain isolated from tumor-free animals. Representative 1 H-MRS spectra are shown in Fig. 5A. Quantification of the data indicated that GSH levels (126.6 ± 16.3pmol, 42.8 ± 9.1pmol and 28.0 ± 8.0pmol per mg of wet tissue of tumor, contralateral normal-appearing brain and healthy brain respectively) were significantly higher in U87 tumors compared to contralateral normal brain (p = 0.006; n = 5) and compared to healthy brain tissue (p = 0.004; n = 3, Fig. 5B).

Discussion
HP γ-glutamyl-[1-13 C]glycine has previously been used to assess GGT activity in kidneys and in an ovarian carcinoma model [31][32][33] . The goal of this study was to evaluate the feasibility of using HP γ-glutamyl-[1-13 C]glycine to non-invasively probe GGT expression in glioblastoma in the rat brain in vivo. We have demonstrated, to our knowledge for the first time, that the dynamic, real-time conversion of HP γ-glutamyl-[1-13 C]glycine to [1-13 C]glycine can be monitored in vivo in normal rat brain and in tumor-bearing animals. Furthermore, in tumor-bearing animals we were able to show, using 2D EPSI imaging, that elevated [1-13 C]glycine production was localized to the tumor region. Importantly, our metabolic imaging data are linked to elevated GGT expression and elevated GSH levels in tumor tissue relative to contralateral normal-appearing brain and healthy normal  in glioma tumor compared to contralateral normalappearing brain tissue and healthy brain tissue. β-actin was used as loading control. Complete blots can be seen in Supplementary Fig. 2. (B) Quantification of GGT levels for the three groups. Protein expression normalized to β-actin values: 3.06 ± 0.31 a.u., 1.00 ± 0.08 a.u. and 1.14 ± 0.14 a.u. for tumor, contralateral normal-appearing brain and healthy brain respectively. Black: tumor; Striped black bar: Contralateral normal-appearing brain; Red: Healthy brain. *p < 0.05. (2020) 10:6244 | https://doi.org/10.1038/s41598-020-63160-y www.nature.com/scientificreports www.nature.com/scientificreports/ brain tissues. Collectively, these results identify HP γ-glutamyl-[1-13 C]glycine as a novel, non-invasive probe that is associated with cellular redox in brain tumors.
Previous studies have evaluated the expression of GGT in normal human brain, biopsies from brain tumors of different grades including GBM, and GBM cell lines including U87 18,19 . GGT expression was over 4-fold higher in GBM when compared to normal brain. Furthermore, GGT expression in U87 cells was comparable to its expression in GBM patient biopsies 18 . These results point to the validity of our studies with the U87 model, and the significance of our findings for imaging GBM tumors in patients.
γ-glutamyl-[1-13 C]glycine fits the essential technical requirements of a useful HP probe 24 . Thanks to localization of the GGT enzyme on the outer surface of the cell membrane, γ-glutamyl-[1-13 C]glycine metabolism occurs in the extracellular space and not in the cytosol or mitochondria as is the case for most HP probes including HP [1-13 C]pyruvate 24 and [1-13 C]dehydroascorbic acid (DHA) 34,35 , or [1,3-13 C]acetoacetate [36][37][38] , respectively. This is a significant advantage as additional transport though the cell membrane would increase the time between HP injection and enzymatic reaction and therefore decrease SNR. Compared to other probes 24,39 , the liquid state polarization level (~23%) of γ-glutamyl-[1-13 C]glycine was relatively high, and the T 1 relaxation time at clinical field strength (3T) was reasonably long (~33 s), such that it was possible to probe the metabolism of γ-glutamyl-[1-13 C]glycine in vivo in an orthotopic glioma model. The chemical shift separation of 4 ppm between substrate (γ-glutamyl-[1-13 C]glycine; 177.7 ppm) and product ([1-13 C]glycine; 173.7 ppm) was also sufficient to allow visualization of product formation at the spectral resolution of our in vivo studies at 3T. Finally, when considering suitability for clinical translation, our study observed no noticeable changes in breathing during and after infusion of HP γ-glutamyl-[1-13 C]glycine (48.6 mM). This observation was also consistent with a previous and more detailed study conducted by some of the authors of this manuscript 31 : no physiological changes associated with γ-glutamyl-[1-13 C]glycine infusion were observed when monitoring breathing, blood pressure and heart rate using a pneumatic pillow, an arterial catheter and an intra-arterial blood pressure sensor.
GGT is a membrane-bound enzyme facing the extracellular compartment. It can be detected in normal brain, but is significantly higher in tumor 19 . There was no significant difference in the SNR values of HP γ-glutamyl-[1-13 C]glycine in orthotopic U87 tumor-bearing rats and tumor-free healthy controls, consistent with a similar delivery to both tissues. Also the EPSI imaging results showed no difference in the uptake of HP γ-glutamyl-[1-13 C]glycine between tumor and healthy brain area. Low [1-13 C]glycine production in the normal brain and a significantly higher production in the tumor region are therefore most likely a reflection of the elevated tumor GGT expression. However, we cannot exclude higher delivery of HP γ-glutamyl-[1-13 C]glycine to the more permeable tumor region also contributing to higher glycine production. After GGT removes the glutamyl group from GSH, cysteinylglycine is cleaved by an extracellular dipeptidase to the constituent amino acids cysteine and glycine, and the released amino acids are transported into the cell via dedicated transporters. We did not observe any consistent asymmetry in the lineshape of glycine peak, however, considering our line width (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) and the relatively long T 1 of glycine (45 s at 9.4T 31 ) we cannot rule out that some of the HP γ-glutamyl-[1-13 C]glycine is intracellular. Importantly however, the precise compartmental localization of glycine would not be expected to affect our conclusions regarding its association with tumor.
Our slab results indicating that HP [1-13 C]glycine production is significantly higher in tumor-bearing animals point to the utility of HP γ-glutamyl-[1-13 C]glycine for non-invasively monitoring GGT activity in orthotopic glioblastoma in vivo. However, the SNR of our dynamic slab spectra was limited. Further improvements should therefore be considered to improve the slab data acquisition scheme. For example, one approach could be to combine a variable flip angle pulse sequence with the multiband method used here. As previously described, this approach would utilize progressively increasing flip angles between excitations and for each metabolite. The flip angles would be optimized to account for T 1 relaxation, prior RF excitations and metabolic conversion in order to improve the SNR of the metabolites 40 . Additionally, further improvements could be obtained through changes in hardware. The use of a 13 C circular polarized coil can provide a square root of 2 SNR improvement Figure 5. GSH levels in tumor, contralateral normal-appearing brain tissue and healthy brain tissues evaluated by MRS of extracts. (A) Typical 500 MHz 1 H MRS spectrum of the aqueous fraction of tumor, contralateral normal-appearing brain and healthy brain where the GSH regions are highlighted. (B) Quantification of GSH levels. 126.6 ± 16.3pmol, 42.8 ± 9.1pmol and 28.0 ± 8.0pmol per mg of wet tissue of tumor, contralateral normal-appearing brain and healthy brain respectively. Black: tumor; Striped black bar: Contralateral normalappearing brain; Red: Healthy brain. **p < 0.01. (2020) 10:6244 | https://doi.org/10.1038/s41598-020-63160-y www.nature.com/scientificreports www.nature.com/scientificreports/ over the linear polarized 13 C volume coil used in this study 41,42 . In the case of the EPSI studies, the voxel size used (346.7 μl) is larger than reports in literature for in vivo preclinical studies utilizing, for example, HP pyruvate [43][44][45][46][47][48] . Further improvements of the SNR and therefore the spatial resolution of the 2D EPSI acquisition scheme could be achieved through the above-mentioned variable flip angle scheme or the use of a circular polarized volume coil. An alternative approach could be to apply a high flip angle pulse on [1-13 C]glycine and utilize all its magnetization at the time point when the [1-13 C]glycine signal is maximal based on the dynamic slab acquisition. This approach has been previously reported for other probes by us and others 44,[49][50][51] .
GGT is a membrane-bound enzyme that plays a key role in the metabolism of GSH. GGT catalyzes the degradation of extracellular GSH, thereby allowing recovery of constituent amino acids, including cysteine, which is often rate-limiting, for subsequent intracellular GSH re-synthesis. Due to the role of GSH as the principal water-soluble antioxidant within the cell, GGT has traditionally been regarded as crucial to cellular protection against oxidative stress. Cancer cells, in particular, suffer from higher levels of oxidative stress relative to normal cells. GGT expression is accordingly higher in cancer, including in high-grade primary glioblastoma, and non-invasive analysis of GGT activity, therefore, is associated with tumor antioxidant levels. Here we quantified GSH ex vivo, however prior work has used optimized 1 H MRS sequences to separate GSH from the overlapping glutamate and glutamine peaks and assess steady-state GSH levels in patients in vivo [52][53][54] . Consistent with our preclinical findings, these studies showed that in brain tumors GSH levels were higher than in normal-appearing brain 55,56 . In complementary HP studies probing metabolic fluxes in animal models, previous investigations have used HP DHA 34,35 and HP [1,3-13 C]acetoacetate [36][37][38] , to non-invasively assess cellular redox status. However, the clinical translation of HP DHA is limited by potential toxicity issues 35 while the conversion of HP acetoacetate to HP β-hydroxybutyrate is a measure of mitochondrial redox status. Our study identifies HP γ-glutamyl-[1-13 C] glycine as a promising probe that can report on GGT and associated cellular redox to provide complementary information to GSH.
Comparison of orthotopic U87 tumor-bearing rats to tumor-free healthy controls showed a significantly higher level of HP [1-13 C]glycine in tumor-bearing rats that positively correlated with tumor fraction. Spatial localization demonstrated that HP [1-13 C]glycine was elevated in tumor tissue compared to normal brain. Importantly, the lower level of HP [1-13 C]glycine production in healthy rats precludes the possibility that the HP [1-13 C]glycine detected in tumor animals was exclusively produced in the kidney 16 and then delivered to the brain. This is an important consideration given that, in many cancers, GGT can cleave GSH in interstitial fluid and blood 4,57 . Considering previous work showing that the ratio of [1-13 C]glycine to total HP signal (γ-glutamyl-[1-13 C]glycine plus [1-13 C]glycine) is positively correlated with GGT activity 31 , previous studies showing higher levels of GGT in U87 glioma cells compare to normal brain 19 , and our observation that higher HP [1-13 C]glycine production in tumor was associated with higher GGT expression in tumor tissue, our results collectively point to the potential of γ-glutamyl-[1-13 C]glycine as a HP probe to detect brain tumor in vivo in real time. Furthermore, tumor therapy, and in particular radiation, can lead to "pseudoprogression" -MRI changes that cannot easily distinguish between tumor recurrence and treatment effects [58][59][60] . Because HP γ-glutamyl-[1-13 C]glycine detects a molecular event specific to tumor, it could also help in evaluating treatment outcome. Indeed, the potential of a GGT-specific probe for distinguishing tumor recurrence from post-radiation effects has already been demonstrated in an ex vivo study using a GGT specific fluorescent probe 18 .
In summary, our study identifies HP γ-glutamyl-[1-13 C]glycine as a probe for monitoring GGT activity in orthotopic glioblastoma in vivo. Higher [1-13 C]glycine production in the tumor relative to normal brain in vivo was associated with higher GGT expression and higher steady-state GSH in the tumor. Given the critical role of GGT in redox homeostasis, our findings add to the repertoire of methods that can help to non-invasively assess redox in the brain and in brain tumors, and to more clearly distinguish tumor from normal brain.
Relaxation and polarization levels. Following dissolution, HP γ-glutamyl-[1-13 C]glycine was rapidly transferred to a horizontal 3T (Bruker BioSpec 105 mm bore diameter, n = 3, TR = 3 s/FA = 10°) or a vertical 11.7T (INOVA, Agilent Technologies, n = 3, TR = 3 s/FA = 13°,) system to evaluate T 1 . Percent polarization was quantified at 11.7T (n = 3, TR = 300 s/FA = 90°/NR = 5). Spectra were processed and peaks quantified by integration using MestReNova (Mestrelab). For T 1 determination peak integrals were corrected for flip angle and fit with a monoexponential curve. The polarization level in solution was evaluated by comparing the first hyperpolarized spectrum of the dynamic set to the corresponding thermal equilibrium spectrum after correction for flip angle and number of averages and then back calculating the value to the time of dissolution (20 to 25 s prior to first spectral acquisition).
Animal studies. Orthotopic glioma model. U87 cells were received from the UCSF Brain Tumor SPORE Biorepository and were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 2 mM glutamine, and 100 U/ml penicillin and streptomycin under normoxic conditions for no more than 30 passages before inoculation. The cell line was authenticated by short tandem repeat fingerprinting (Cell Line Genetics) within 6 months of the study. All animal studies were performed in accordance (2020) 10:6244 | https://doi.org/10.1038/s41598-020-63160-y www.nature.com/scientificreports www.nature.com/scientificreports/ with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of California San Francisco Institutional Animal Care and Use Committee (IACUC Protocol No: AN170079). 17 male athymic nu/nu rats (5 weeks old) were investigated. 7 animals were used as age-matched tumor-free controls. In 10 animals (6 used for slab acquisition, 3 for imaging and 1 for both), tumors were generated by implanting U87 glioblastoma cells (3 × 10 5 ) by intracranial injection as previously described 49 .
MRI and HP 13 C MRS in vivo studies. All measurements were performed on a horizontal 3T scanner (BioSpec 105 mm bore diameter, Bruker) equipped with a dual-tuned 1 H-13 C volume coil (42 mm inner diameter, Bruker). Animals were anesthetized and maintained using isoflurane (1-2% in O 2 ) and placed head first in the prone position. Animal breathing was monitored using a small animal breathing monitoring system (MR-compatible Small Animal Monitoring, SA Instruments, USA) during all acquisitions. Axial and sagittal anatomical T 2 -weighted images were recorded using a spin echo (TurboRARE) sequence (TE/TR = 64/3484 ms, FOV = 35 × 35 mm 2 , 256 × 256, slice thickness=1 mm, NA = 10) and used to evaluate tumor location and size. HP studies were performed following injection of 2.2 ml HP γ-glutamyl-[1-13 C]glycine (prepared as described above) via a tail-vein catheter over 15 s. Dynamic 13 C MR spectra were acquired from a 15 mm slab through the brain every 3 s using a flyback spectral-spatial pulse with 30° excitation on product ([1-13 C]glycine) and 4° on substrate (γ-glutamyl-[1-13 C]glycine) ( Supplementary Fig. 3) 61 . In the case of 13 C MRSI, a 2D flyback spectral-spatial echo-planar spectroscopic imaging (EPSI) pulse was used with the same frequency profile as for the slab to provide a spatial resolution of 5.375 × 5.375 × 12 mm 3 , a temporal resolution of 3 s, spectral resolution of 128 points over a spectral bandwidth of 25 ppm. In all cases the scans started 12 s after the start of the HP γ-glutamyl-[1-13 C] glycine injection.
Data processing. Tumor size was measured as the sum of manually contoured tumor areas in each slice multiplied by slice thickness using in-house software 49 . The HP experiments were performed when the tumor volume reached a value of ~0.27 cm 3 . The HP 13 C spectra were analyzed using MestreNova (Mestrelab, Spain). Each spectrum was individually apodized (line broadening = 5 Hz) and phased. Then all the spectra were summed. Resonances for product ([1-13 C]glycine) and substrate (γ-glutamyl-[1-13 C]glycine) were fit with a Lorentzian-Gaussian line shape and their integral was normalized to the standard deviation of the noise. In addition, ratios of [1-13 C]glycine to γ-glutamyl-[1-13 C]glycine were quantified for each animal. The imaging data were processed using in-house Matlab code. For each voxel at every time point, spectra were analyzed after a 3 Hz line broadening by determining the area under each peak by integration. Intensity heat maps were produced by interpolating the data using a Lanczos-2 kernel and normalized to noise, which was evaluated as the standard deviation of the real part of the signal in a voxel outside of the brain. These maps were used to generate the ratio of substrate to product metabolic map. The SNR of γ-glutamyl-[1-13 C]glycine and the ratio of [1-13 C]glycine to γ-glutamyl-[1-13 C]glycine were evaluated in a 76.2 mm 3 volume region of interest comprising of tumor or healthy brain tissue.
Immunoblotting. Tumors and contralateral normal-appearing brain tissues from tumor-bearing animals and healthy brain tissue from tumor-free control animals were excised after the HP MR scan, snap-frozen in liquid nitrogen and stored at −80 °C until further investigation. γ-glutamyl-transferase 1 and 2 (GGT1/2) levels in all samples were quantified using western blotting with β-actin as a loading control as follows. Tissues were lysed using RIPA Buffer (ThermoFisher Scientific) supplemented with 1 μl/ml protease inhibitor cocktail set III (Calbiochem). Lysates normalized to wet tissue weight were then run on 4-20% gels (Bio-Rad) using the SDS-PAGE method and electrotransferred onto nitrocellulose membranes. Membranes were blocked with 5% milk in Tris-Buffered Saline Tween-20 (TBST) and incubated with the primary antibodies anti-GGT1/2 (Santa Cruz Biotechnology sc-393706) at 1:100 dilution and anti-β-actin (Cell Signaling #4970) at 1:5000 dilution overnight at 4°C. HRP-conjugated secondary antibodies (Cell Signaling #7074) at 1:3000 dilution were incubated for 60 min in TBST at room temperature. Immunocomplexes were visualized using ProSignal Pico (Genesee Scientific). Densitometry of the bands was performed using ImageJ software (NIH) to quantify protein expression.
Analysis of tumor tissue. Tissue extraction. The levels of GSH were evaluated by 1 H MRS using snap frozen tissue from tumor, normal-appearing brain tissue or healthy brain tissue (11 to 18 mg wet-weight). The tissues were homogenized in 400 μl ice cold phosphate buffer (PBS) with 1 μl/ml protease inhibitor cocktail set III (Calbiochem) in the presence of TissueLyser beads (TissueLyser LT, QIAGEN). Afterwards a dual-phase extraction method was followed 62 . Briefly, 10 ml ice cold methanol (Sigma-Aldrich) was added to the homogenized tissue. The solution was then vortexed and 10 ml of ice-cold chloroform (Acros Organics) added. After another vortexing, 10 ml of ice cold Milli-Q water was added and a final vortexing performed. Phase separation was achieved by centrifugation for 10 min at 3000 rpm at 4 °C, the phases were separated, and solvents removed by lyophylization. The aqueous phase was then reconstituted for MRS studies in 400 μl phosphate buffer (pH = 7.4) in deuterium oxide (Acros Organics).
MRS acquisition and data analysis. 1 H spectra of the aqueous phase of tissue extracts were recorded using a 500 MHz spectrometer (Bruker BioSpin) equipped with a triple resonance cryoprobe. The 1 H spectra were acquired using a 90° flip angle, 3 s repetition time (TR) with 384 averages. In addition, fully relaxed 1 H spectra were recorded and served to determine correction factors for saturation. The concentration of GSH was quantified by peak integration using MestReNova. The integrals were corrected for saturation, and normalized to mg wet tissue and to an external sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP; Sigma-Aldrich) reference of known concentration.