Résumé :
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Altered ratios of phosphate metabolites, observed in 31P NMR spectroscopy (NMRS) of Duchenne patients, were recognized as potential markers of disease years ago1. The increase of therapeutic trials in DMD and in its closest model, the GRMD dog, spurs the need for outcome measures. Availability of high field magnets for large animals prompted us to revisit 31P NMRS in GRMD.Fourteen GRMD (218 mths) and 9 controls (CONT, 2-37 mths) dogs were investigated under anesthesia in a 4T magnet. Resting 31P NMRS was acquired with a 2-cm coil in tibial cranial (TC) or biceps femoris (BF) muscles for 6-12min. The spectrum of TC in a GRMD compared to a CONT dog (Fig) displays low phosphocreatine (PCr), high phospho-monoesters (PME) and -diesters (PDE) relative to ATP, and an enhanced resonance at 0.3ppm downfield of inorganic phophate (Pi), most likely a second Pi pool2. All ratios to ATP were altered in GRMD vs CONT in TC and only PDE was less modified in BF compared to TC. The pH, calculated from the Pi-PCr shift, was unchanged for Pia in GRMD corresponding to a physiological intracellular pHa (6.97±0.05), whilst pHb approached the extracellular range (7.27±0.10) and correlated with pHa in GRMD (R2=0.51). Both Pia and Pib were elevated compared to CONT, and Pia increased with age for GRMD (R2=0.48, p<0.001).Caracterising oxidative ATP synthesis from Pia and Pib by magnetization transfer NMR3 in 5GRMD/4CONT showed equal T1 relaxation times for all pools -unlike shorter T1 of mitochondrial Pi4- and low Pib chemical exchange in all dogs. A Pi from an interstitial compartment would imply improbable equivalent extra- and intracellular volumes. It may originate from suffering cells with a leaky membrane, inadequate cell homeostasis and less efficient pH regulation. Exchange rate of Pia was also reduced in GRMD, but could extraneously result from low glycolytic activity in DMD5.Unlike at low field6, the present GRMD data showed all alterations previously described in DMD children, except for increased pH. Rather, we observed a likely doubling of Pi, with a second more alkaline, less metabolically active pool. The ensemble of these 31P NMRS anomalies, though individually unspecific to dystrophinopathy, have potential value as biomarkers of therapeutic efficacy in DMD.1. Younkin, Neurol., 1987. 37 - 2. Thibaud, Acta Myologica, 2010. 29 - 3. Forsen, J. Chem. Phys, 1963. 39 - 4. Kan, NMR Biomed, 2010. 23 - 5. Lodi, et al., Brain, 1999. 122 - 6. McCully, Muscle Nerve, 1991. 14
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