NEW GENES, FUNCTIONS AND BIOMARKERS: O.6Free Mg2+ intramuscular concentration determined by combined 31P and 1H NMR spectroscopy as a potential outcome measure in Duchenne muscular dystrophy

Abstract

Free intracellular [Mg2+] has been reported to be lower in skeletal muscle of Duchenne muscular dystrophy (DMD) patients. This reduction in [Mg2+] is likely reflecting membrane leakiness and has potentially major consequences due to Mg2+ regulatory role in many cellular processes. Free [Mg2+] determination with 31P NMRS, based on the chemical shift between α- and β-ATP resonances, is highly dependent on a precise knowledge of intracellular pH because of a competition between H+ and Mg2+ ions for binding with ATP. The pH of DMD patients as determined by the 31P NMRS chemical shift of inorganic phosphate (Pi) is abnormally alkaline, resulting either from compromised dystrophic myocytes or from an expanded interstitial space. In the second scenario, free [Mg2+] might be underestimated. Intracellular pH can be determined using 1H NMRS of carnosine. We took advantage of this 1H NMRS-based intracellular determination to determine whether free intramuscular [Mg2+] is in fact abnormally low in DMD patients. Non-localized 31P NMRS data of 64 DMD (9.9±3.1 yrs) and 67 normal boys (12.7±4.1 yrs) were analyzed from the forearm flexors, the leg extensors or the triceps surae and [Mg2+] and pH were calculated from the αATP-βATP and Pi-PCr chemical shifts, respectively. In a subset of DMD patients and controls, [Mg2+] was also calculated using the pH measured by 1H NMRS. 31P NMRS based pH was significantly increased and free [Mg2+] was significantly lower in all DMD muscles. [Mg2+] was then recalculated with pH values based on 1H NMRS as this gives the real intracellular pH. The 1H NMRS-based pH was normal in a subgroup of DMD patients in contrast to the 31P NMRS-based pH. The corresponding [Mg2+] values were significantly different with the two methods. However, the significant difference in [Mg2+] between DMD patients and controls was preserved even when the intracellular pH was similar in both groups. Low free [Mg2+] was confirmed and a likely consequence of membrane leakiness in DMD patients. This makes [Mg2+] a potential functional biomarker of sarcolemma properties restoration by a therapeutic dystrophin expression.