¹H NMRS of carnosine combined with ³¹P NMRS to better characterize skeletal muscle pH dysregulation in Duchenne muscular dystrophy

Abstract

In recent years, quantitative nuclear magnetic resonance imaging and spectroscopy (NMRI and NMRS) have been used more systematically as outcome measures in natural history and clinical trial studies for Duchenne muscular dystrophy (DMD). Whereas most of these studies have emphasized the evaluation of the fat fraction as an assessment for disease severity, less focus has been placed on metabolic indices measured by NMRS. P NMRS in DMD reveals an alkaline inorganic phosphate (P ) pool, originating from either leaky dystrophic myocytes or an increased interstitial space. H NMRS, exploiting the pH-sensitive proton resonances of carnosine, an intracellular dipeptide, was used to distinguish between these two hypotheses. NMR data were obtained in 23 patients with DMD and 14 healthy subjects on a 3-T clinical NMR system. Both P and H NMRS data were acquired at the level of the gastrocnemius medialis muscle. A multi-slice multi-echo imaging acquisition was performed for the determination of water T and fat fraction in the same region of interest. Whereas nearly all patients with DMD showed an elevated pH compared with healthy controls when using P NMRS, H NMRS-determined pH was not systematically increased. As expected, the carnosine-based intracellular pH was never found to be alkaline in the absence of a concurrent P -based pH elevation. In addition, abnormal intracellular pH, based on carnosine, was never associated with normal water T values. We conclude that, in one group of patients, both H and P NMRS showed an alkaline pH, originating from the intracellular compartment and reflecting ionic dysregulation in dystrophic myocytes. In the other patients with DMD, intracellular pH was normal, but an alkaline P pool was still present, suggesting an extracellular origin, probably revealing an expanded interstitial volume fraction, often associated with fibrotic changes. The data demonstrate that H NMRS could serve as a biomarker to assess the normalization of intramyocytic pH and sarcolemmal permeability following therapy inducing dystrophin expression in patients with DMD.

Free intramuscular Mg2+ concentration calculated using both 31P and 1H NMRS‐based pH in the skeletal muscle of Duchenne muscular dystrophy patients

“…We mention here TRPM7, which is a channel that conducts mainly Ca 2+ and Mg 2+ and is inhibited by low intracellular Mg 2+ concentrations, potentially blocking the channel and leading to intracellular Ca 2+ accumulation. Excess Ca 2+ activates membrane‐bound phospholipases, leading to a breakdown of membrane phospholipids, reflected by an increase in PDE levels, as observed earlier and confirmed in the current study using 31 P NMRS.…”

“…The normal intracellular pH, as found in one of the DMD patient subgroups (ie DMD2), still demonstrated a significantly decreased [Mg 2+ ] compared with healthy controls, illustrating that the method is sensitive to [Mg 2+ ] changes even in the absence of pH differences. Moreover, as well as a significantly different intracellular 1 H NMRS‐based pH between the two DMD subgroups (ie DMD2 and DMD3), with one group not different to the control group (ie DMD2), for Mg 2+ utilizing the 1 H NMRS‐based pH, we have now demonstrated a significant difference both between the two DMD subgroups and between the two subgroups and controls.…”

“…The value of Mg 2+ is calculated using both the pH determined with 31 P NMRS and the 1 H NMRS‐based pH. The naming of DMD groups 2 and 3 stems from our earlier published work. DMD, Duchenne muscular dystrophy; [Mg 2+ ], free intramuscular magnesium concentration; pH w , weighted pH; * P < 0.05; ** P < 0.005; *** P < 0.001; ns, not significant…”

“…We described the processing steps of skeletal muscle 1 H NMR spectra using AMARES (jMRUI) previously. 1 H NMRS‐based pH was determined by the chemical shift difference (σ 2 ) between the imidazole C2‐H resonance of carnosine and the water resonance: For 31 P NMRS, the combined procedure of employing the jMRUI‐based AMARES algorithm (advanced method for accurate, robust and efficient spectral editing) and the Topspin software (Bruker Medical GmbG, Ettlingen, Germany) has been described in detail in two earlier studies. With jMRUI, two components of identical linewidth were determined for the deconvolution of the more neutral cytosolic (P i,a ) and the more alkaline (P i,b ) inorganic phosphate (P i ) resonances (Figure A).…”

23Na MRI depicts early changes in ion homeostasis in skeletal muscle tissue of patients with duchenne muscular dystrophy

“…regions of interest (ROIs) were manually traced on the MESE images within tibialis anterior, tibialis posterior, soleus, and gastrocnemius medialis muscles. 1 H spectroscopy data were processed using jMRUI as described previously . The pH was determined from the chemical shift between carnosine (C 2 ‐H) and residual water resonance according to the modified Henderson–Hasselbalch equation.…”