Introduction

³¹P MRS allows evaluating energy metabolism and dynamic changes of pH in the muscle during physical effort. ¹H MRS can provide cytoplasmic pH information by measuring carnosine, a dipeptide playing a role in pH buffering¹. Carnosine-based pH(pH_1H) determination is an interesting and complementary method to the ³¹P MRS evaluation of pH using Pi(pH_31P), for instance, in dystrophic myopathies presenting “leaky” membranes² or during exercise³. Here, we have extended a previous interleaved ¹H/³¹P multi-voxel pulse sequence for simultaneous pH_1H and pH_31P measurements in muscle4, by adding the acquisition of a dynamic water reference(water_ref) co-localized to the water-suppressed(WS) carnosine MRS voxel. We evaluated the impact the that dynamic water_ref has on pH_1H calculation during an exercise paradigm and compared the pH_1H values with their corresponding pH_31P pairs.

Methods

Experimental Setup.
6 men (42±14 y.o.) participated in this study. Experiments were done at 3 T (Siemens Prisma). The RF coil (RAPID Biomedical) combined a ¹H birdcage transmitter (20-cm inner diameter), a ¹H 18Rx phased-array receiver and a ³¹P 1Tx/3Rx semi-cylindrical transceiver (11-cm resonator length). The subject’s calf was placed facing the ³¹P coil and as close as possible to the center of the ¹H transmit coil.
Exercise paradigm.
Straight-leg plantar flexions were performed every 3 seconds for 13 min on a pneumatic ergometer, gradually increasing the resistance from ~15% to ~30% of maximum voluntary contraction. MRS lasted 25 min, starting 2 before exercise onset.
Interleaved NMR.
An interleaved multi-nuclear sequence was implemented, performing a WET⁵ water suppression (80 Hz saturation bandwidth) and acquiring sequentially a WS ¹H MR spectrum (PRESS, TE=17.9 ms, 2048 points, 8 kHz bandwidth, carrier frequency at 8 ppm), a ³¹P MR spectrum (semi-LASER, TE=21.54 ms, 2048 points, 8 kHz bandwidth, centered at the PCr resonance frequency) and a Water_ref ¹H MR spectrum (PRESS, TE=17.9 ms, 2048 points, 8 kHz bandwidth, carrier frequency at 4.7 ppm) in the same voxel every 3 s (figure 1A). The voxel localization alternated every 3 s between the gastrocnemius (GAS) and the soleus (SOL) muscles (figure 1B).
Data Analysis.
In-house Matlab routines were used for automatic phasing. Individual spectra were then inspected and manual zero and first-order phase correction was performed if needed. Afterwards, spectra were averaged over 1-minute intervals and exported to jMRUI. Spectra were zero-filled (4096 points) and ³¹P MR spectra were apodized (8 Hz, Gaussian filter) before AMARES quantification. For each voxel, the carnosine and water_ref ¹H MR spectra were fitted simultaneously. pH_1H was calculated from the chemical shift of the carnosine C2-H (δ_Carn-C2) resonance and corrected for frequency offsets using the water resonance of the respective water_ref spectra according to⁶: $$pH_{1H}=6.81+log_{10}(\frac{8.57-δ_{Carn-C2}}{δ_{Carn-C2}-7.65})$$. For comparison, pH_1H was also calculated using the residual water from the WS spectra. The chemical shift difference between Pi and PCr (δ_Pi) was used to calculate pH_31P according to⁷: $$pH_{31P}=6.75+log_{10}(\frac{δ_{Pi}-3.27}{5.69-δ_{Pi}})$$.

Results & Discussion

Figure 2 shows the time series of WS ¹H and ³¹P spectra from both voxels from a single subject. In 3 subjects, a broadening of the carnosine C2-H resonance was observed during exercise in GAS. As shown in figure 3, PCr signal amplitude showed a clear drop of at least 50% in GAS accompanied by a mirroring increase in Pi. In contrast, mild to no changes were observed in SOL, as expected during straight-leg plantar flexions from a study exploring 4 knee angles⁸. A high variability was observed for carnosine although the average signal amplitude remained relatively stable in both muscles during the paradigm. pH_1H values presented a systematic increase of ~0.05 units when using the water residue of the WS spectra instead of water_ref (figure 4), demonstrating the interest of a dynamic water reference. The pH_1H and pH_31P time courses presented similar trends (figure 5, top). In GAS, a pH increase in the first minute of exercise was followed by a steady pH decrease during the following 6 minutes of exercise. pH recovery began in the second or third minute of recovery. In SOL, a pH_1H increase was observed only during the second half of the exercise period. Furthermore, when pooling the data of all subjects, a stronger linear correlation between the two pH measuring methods was found (figure 5C-D) in GAS(R²=0.55) than in SOL(R²=0.08). Bland-Altman analysis(figure 5E-F) revealed similar limits of agreement in both muscles but a larger bias in GAS than SOL and the negative mean bias found indicate that in this study pH^31P values were lower than their pH^1H pairs. Splitting or broadening of the carnosine(C2-H) resonance has been reported after performing a 1-hour street run followed by a toe-hopping exercise³. Here, we found both a widening of the carnosine(C2-H) and Pi resonance in GAS during exercise. Nevertheless, care should be taken when interpreting these results as our spectra were averaged a minute. Future studies could include acquisitions performed in GAS only to double the current temporal resolution.

Conclusion

This study showed that adding a dynamic water reference improved pH estimation by ¹H MRS and the results were comparable to pH values measured simultaneously on the same voxel with ³¹P MRS. This method opens the way for new applications of multi-nuclear interleaving in functional studies targeting individual muscles at 3 T.

Acknowledgements

No acknowledgement found.