In vivo absolute quantification for mouse muscle metabolites using an inductively coupled synthetic signal injection method and newly developed 1H/31P dual tuned probe

Genetically engineered mouse models are becoming useful tools for studying various muscle disorders such as the mdx mouse model of Duchenne muscular dystrophy (1) and the dystrophin and utrophin double knockout mdx:utrophin-/- model (2,3). Studies that utilize genetically engineered mice usually rely on biopsy followed by histology as a means for obtaining metabolite concentrations. Muscle biopsy has been a gold standard to investigate both disease progression and treatment responses but is invasive so multi-time point procedures are not feasible. More important, muscle biopsy is a local procedure performed for a specific muscle type and has a substantial limit in examining the rest of muscle regions (4-9). Noninvasive MR measurements could enhance these studies by allowing longitudinal measurements during disease progression and therapy in individual mice. A surface coil and volume coils have been used to acquire 31P MR spectra for small animals (10,11). However, MR measurements in individual muscles are difficult to conduct in mice because the tissue volume is inherently small, so the signal-to-noise ratio (SNR) is typically very low for non-proton (or x-nucleus) MRS. Further, the results are usually reported as ratios, making interpretation ambiguous (12,13). In particular, absolute quantification is important for 31P MR, as none of the phosphorous containing metabolites is necessarily constant in pathology (14). A synthetic reference signal injection method has been developed to ease the burden of converting MR signals into quantitative units of concentration (15-19). This approach is similar to the ERETIC (Electronic Reference To In vivo Concentrations) method (20). The distinguishing feature of the method is that the synthetic reference signal is generated by inductive coupling rather than RF radiation. The method uses inductive coupling to inject artificial reference signals, which are called pseudo-signals that serve as calibration parameters for absolute quantification. All parameters downstream of the RF coil that affect quantification have equal effect on the pseudo-signal and the real signals. Any changes in these parameters, in particular coil loading and receiver gain, which might occur between subjects or in a single subject due to physiological perturbations, are transparent and have no effect on the quantification process. In this study, we applied the pseudo-signal injection method to determine 31P metabolite contents in mice. To improve the SNR, we developed a dual tuned 1H/31P probe optimized for MRI and MRS measurements in mouse hind limb muscles. The probe was equipped with two dual tuned 1H/31P solenoid coils, one for each leg, each of which was individually interfaced with an injector coil for pseudo-signal injections. 1H MRI was conducted to measure muscle volumes and 31P MRS was performed to quantify phosphorous metabolites. We provide a detailed description of the RF probe, demonstrating how it improves SNR, and provide quantitative measurements of average inorganic phosphate (Pi), phosphocreatine (PCr), and adenosine triphosphate (ATP) concentrations that agree with published values for normal mice. The approach we developed in this study would be applicable to longitudinal quantification of mouse muscle for any 1H/x-nucleus metabolites where SNR is expected low.