In zyxwv Vivo zyxw 23Na NMR Studies of Myotonic Dystrophy zy Tammar Kushnir, Tatyana Knubovets, Yacov Itzchak, Uzi Eliav, Menachem Sadeh, Lubov Rapoport, Edna Kott, Gil Navon zyxw Myotonic dystrophy is an inherited multi-system disease. Its pathophysiology leading to muscle malfunction and damage is not well understood. =Na NMR spectroscopy was applied here for an zyxwvutsrqp in vivo comparative study of the calf muscles of 7 myotonic dystrophy patients at various stages of the disease and 11 healthy volunteers. Both the total sodium content, expressed as the ratio of the =Na and 'H water signals, and the fast transverse relaxation time, T,, determined from the triple quantum-filtered spectra, increased in correlation with the severity of the disease. The results demonstrate that =Na NMR enables the quantitation of myotonic dystrophy progression. Key words: myotonic dystrophy; =Na NMR; triple quantum- filtered spectroscopy. INTRODUCTION Myotonic dystrophy (MyD) is the most frequently inher- ited muscle disease in adults. The genetic defect has been identified as an expansion of a CTG triplet repeat se- quence in the 3' noncoding region of a protein kinase gene (1-3). The mechanism by which the genetic defect causes disease is not well understood and currently no adequate treatment exists (4-6). Based on studies of muscle biopsies, it was suggested that MyD is associated with alterations in the regulation of sodium conductance (7). Thus, it is reasonable to assume that a study directed at determining sodium NMR parameters will be valuable in characterizing the pathogenesis of the disease. Paramagnetic shift reagents have been used for in vivo '"Na NMR studies of animals to distinguish between intracellular and extracellular sodium signals (8-10). However, these compounds cannot be used in human studies due to their toxicity. This is why we decided to use triple quantum-filtered (TQF) NMR spectroscopy in order to get information on the relative contribution of the intracellular and extracellular sodium compartments (11, 12). In this technique, only contribution from so- dium ions that have biexponential zyxwvutsr T, relaxation (bound to macromolecules or in fast exchange with them) is MRM 37:192-196 (1997) zyxwvutsrqpo From the Diagnostic Imaging Department MRI Institute (T.K., Y.I.) and Neurologic Department (M.S.), The Chaim Sheba Medical Center, Tel Hashomer; the School of Chemistry (T.K.. U.E.. G.N.). Tel Aviv University, Tel Aviv; and the Neurologic Department (L.R., E.K.), Sapir Medical Center, Kfar Saba, Israel. Address correspondence to: Tammar Kushnir, Ph.D., Diagnostic Imaging Department, MRI Institute. The Chaim Sheba Medical Center, Tel Hashomer 52621. Israel. Received July 10, 1996; revised October 15. 1996: accepted October 15, 1996. This study was partially supported by the grants from the Recanati Foun- dation for Medical Research and I'Association Francaise contre les Myop- athies (AFM). Copyright zyxwvutsrqponm 0 1997 by Williams 8 Wilkins All rights of reproduction in any form reserved. 0740-3194/97 $3.00 detected. Since the fraction of extracellular sodium ions that are bound to macromolecules is smaller than that of the intracellular ions, their Z3Na TQF NMR signal is largely suppressed. While complete elimination can hardly be achieved, as a rule the resultant 23NaTQF NMR spectrum is mostly from intracellular sodium ions. Previous research in the field of 23Na TQF NMR tech- nique enabled both quantification of intracellular so- dium levels and the study of sodium ion dynamics in various animal tissues (13-15). However, 23Na TQF NMR has not been applied to clinical research so far. In this study we used TQF NMR for the investigation of sodium ions in the calf muscles of MyD patients in vivo and compared the results to healthy human muscle. We dem- onstrate that the new method has potential as a diagnos- tic tool for monitoring MyD patients. EXPERIMENTAL Subjects The study group included 11 healthy volunteers and 7 MyD patients. Informed consent was obtained from all the subjects and the study was approved by the autho- rized Helsinki committee. None of the healthy controls had a family history of MyD. Five males and six females aged 17 to 58 were investigated. Clinical evaluation of the patients was done by an experienced neurologist. The muscular disability rating scale (MDRS) was a modified Mathieu et 01. (16) scale. Our modified scale extended the grade 4 of Mathieu et al. into three levels of disability which are now denoted as grades 4-6. None of our patients were in Mathieu's 5th grade (wheel chair). 23NaNMR Measurements NMR spectra were measured using a commercial whole body MRI spectrometer (Gyrex 2T V-Dlx, Elscint, Ltd.) operating at a field strength of 1.91 T (21.5 MHz for '"Na and 81.27 MHz for 'H). Z3Na TQF Nh4R spectra were measured by using a 23Na head coil and the standard pulse sequence for TQF (11, 17): 90" - d2 - 180" - ~/2 - 90" - t, - 90" - Acq 111 Where zyxwv T is the triple quantum creation time, t, its evolu- tion time. The TQF signal was selected using the appropriate phase cycling (48 steps).The 90° pulse width was 400 ps. Each spectrum was a result of 336 scans with line broad- ening of 10 Hz, a relaxation delay of 400 ms, and t, of 0.5 ms. Both legs of each patient were examined. Total sodium levels were obtained by measuring the ratio of the 23Na and the 'H water signals (17) using a 192