Free Mg2+ concentration in the calf muscle of glycogen phosphorylase and phosphofructokinase deficiency patients assessed in different metabolic conditions by 31P MRS

Background The increase in cytosolic free Mg2+ occurring during exercise and initial recovery in human skeletal muscle is matched by a decrease in cytosolic pH as shown by in vivo phosphorus magnetic resonance spectroscopy (31P MRS). To investigate in vivo to what extent the homeostasis of intracellular free Mg2+ is linked to pH in human skeletal muscle, we studied patients with metabolic myopathies due to different disorders of glycogen metabolism that share a lack of intracellular acidification during muscle exercise. Methods We assessed by 31P MRS the cytosolic pH and free magnesium concentration ([Mg2+]) in calf muscle during exercise and post-exercise recovery in two patients with McArdle's disease with muscle glycogen phosphorylase deficiency (McArdle), and two brothers both affected by Tarui's disease with muscle phosphofructokinase deficiency (PFK). Results All patients displayed a lack of intracellular acidosis during muscle exercise. At rest only one PFK patient showed a [Mg2+] higher than the value found in control subjects. During exercise and recovery the McArdle patients did not show any significant change in free [Mg2+], while both PFK patients showed decreased free [Mg2+] and a remarkable accumulation of phosphomonoesters (PME). During initial recovery both McArdle patients showed a small increase in free [Mg2+] while in PFK patients the pattern of free [Mg2+] was related to the rate of PME recovery. Conclusion i) homeostasis of free [Mg2+] in human skeletal muscle is strongly linked to pH as shown by patients' [Mg2+] pattern during exercise; ii) the pattern of [Mg2+] during exercise and post-exercise recovery in both PFK patients suggests that [Mg2+] is influenced by the accumulation of the phosphorylated monosaccharide intermediates of glycogenolysis, as shown by the increased PME peak signal. iii) 31P MRS is a suitable tool for the in vivo assessment of free cytosolic [Mg2+] in human skeletal muscle in different metabolic conditions;


Background
Human skeletal muscles contain approximately 35% of total human body magnesium, which is an essential cofactor in a number of cell reactions. Magnesium ions influence the equilibria of many reactions involved in cellular bioenergetics by interacting with phosphorylated molecules and interfere with the kinetics of ion transport across plasma membranes [1]. There is considerable evidence that Mg 2+ is actively transported and regulated, although the mechanisms are still largely unknown [2]. In skeletal muscle variations of cytosolic pH, phosphocreatine (PCr) and inorganic phosphate (Pi) concentrations influence the complex multi-equilibrium system of the molecular species binding magnesium ions. As a consequence [Mg 2+ ] changes considerably in different metabolic conditions such as rest, exercise and recovery, showing an increase matched by a decrease of intracellular pH during exercise and recovery [3].
We assessed the cytosolic pH and the [Mg 2+ ] by 31 P MRS at rest, during exercise and post-exercise recovery in the calf muscle of two patients with McArdle's disease with muscle glycogen phosphorylase deficiency (McArdle), and two brothers affected by Tarui's disease with muscle phosphofructokinase deficiency (PFK).
These two type of glycogenosis, being characterized by almost absent activity of enzymes involved in glycogenolysis (McArdle) and glycolysis (PFK) pathways, show in general limited/absent production of intracellular lactic acid, depending on the degree of enzyme deficit [4,5]. As consequence, patients with McArdle's and Tarui's disease, typically show a decrease or a lack of intracellular acidification during muscle exercise when studied by 31 P MRS [6,7]. We used these diseases as natural experimental models to study the pattern of free Mg 2+ during exercise and recovery in the absence of intracellular acidification to understand to what extent homeostasis of intracellular free Mg 2+ is linked to pH.

Patients
We studied 4 patients: two unrelated males both aged 42, with myo-phosphorylase deficiency (named MCArdle I and II respectively) and two brothers aged 18 and 10 years with phosphofructokinase deficiency (named PFK I and II respectively), as detected by histochemical/biochemical analysis of muscle.
Ten healthy volunteers (10 males age: 33 ± 15) were recruited as control subjects. Written informed consent was obtained from all subjects.

Protocol
MR spectra were acquired on a General Electric 1.5 T Signa System whole-body scanner. Radiofrequency pulses at 25.866 MHz with a pulse width of 400 µs and a transmitter power of 0.5 kW were transmitted by a surface coil (20.5 cm diameter; General Electrics) and the resonance signals were collected by a 7.5 cm receiving coil. A data table of 1024 complex points was collected for each FID. The band width was 2 kHz. The delay between transmission and reception was 0.5 ms and the dwell time was 250 µs. The stimulation-response sequence was repeated every 5000 ms (TR = 5000 ms). Magnetic field homogeneity was optimized by shimming the 1 H water spectrum (FWMH 0.25-0.35 ppm) The spectroscopic measurements were performed according to the quantification and quality assessment protocols defined by the EEC Concerted Research Project on "Tissue Characterisation by MRS and MRI", COMAC-BME II.1.3 [8].
Subjects lay supine with a 20.5/7.5 cm diameter transmitter/receiver surface coil centred on the maximal circumfer-31 P MRS spectra of calf muscle during exercise Figure 1 31 P MRS spectra of calf muscle during exercise. Endexercise 31 P MRS spectra of calf muscle acquired in patients and in a control subject reaching a PCr depletion of about 50%. PFK patients showed a marked phosphomonoester (PME) accumulation, although to a different extent.
ence of the right calf muscle. Muscle aerobic incremental exercise consisted of different levels of 1 minute each (12-FIDs) of plantar flexion against a pedal using a pneumatic ergometer [9]. All patients were asked to perform an exercise to reach a PCr depletion of about 50% at the end of exercise. Sixty-four FIDs at rest, and 12 FIDs for each level of work were averaged. During recovery 4-FIDs data blocks (20 s) were recorded for 60 s, while longer time blocks were collected thereafter. The area of each metabolite signal was fitted to a Lorentzian line shape using a time-domain fitting program AMARES/JMRUI [10], the PCr and Pi concentration were calculated by assuming a normal ATP concentration of 8 mM [11]. The cytosolic pH and [Mg 2+ ] are calculated from the chemical shift of Pi and β-ATP respectively, both measured from the resonance of PCr, using an equation which takes into account the mutual influence between pH and [Mg 2+ ] [3]. The simultaneous calculation of [Mg 2+ ] and pH was performed by the specific software package MagicMC, that we developed and made available on the internet [12].
Results 31 P MRS spectra of human skeletal muscle typically show the peak signal of: phosphomonoesters (PME) which represents the phosphorylated monosaccharide intermediates of glycogenolysis, inorganic phosphate (Pi), phosphocreatine (PCr) and the three phosphate groups α, β, γ of ATP.   Figure 3 report the PME pattern of the two PFK patients during exercise and recovery. PFK I patient shows a slower rate of both PME accumulation and recovery compared to PFK II patient.

Discussion
In the skeletal muscle variations of cytosolic pH, phosphocreatine and inorganic phosphate concentrations ] also persisted during recovery in PFK I patient who displayed a slower PME recovery. The PME peak in the 31 P MRS spectra corresponds to the phosphorylated monosaccharide intermediates of glycogenolysis. Therefore, due to the def- icit of the phosphofructokinase activity in Tarui's disease, the PME accumulation shown by these patients is likely due to the increase of fructose-6-phosphate, which represents an additional binding site for cytosolic Mg 2+ . As a consequence, we interpret the decrease of [Mg 2+ ] concomitant with the PME increase as due to the binding of Mg 2+ to fructose-6-phosphate. A previous study (6) reported that the abnormal PME accumulation of PFK patients during exercise was accompanied by a subnormal Pi accumulation. This finding was interpreted as a result of the incorporation of free Pi into phosphorylated glycolytic intermediates. However, both our patients did not show any Pi trap into PME, since we found that the sum of PCr and Pi was constant for the whole exercise duration, while the total phosphates signal increased proportionally to PME increase.

Conclusion
Our results show that: i) free [Mg 2+ ] is strongly linked to pH in skeletal muscle homeostasis as previously suggested by a study in healthy volunteers [3], and by computer simulation on a chemical model mimicking muscle cell cytosol [13]; ii) the decrease of free [Mg 2+ ] during exercise in both PFK patients suggests that [Mg 2+ ] is influenced by the accumulation of fructose-6-phosphate, an additional binding site for cytosolic Mg 2+ , as shown by the accumulation of the phosphomonoesters peak in the 31 P MRS spectra of these patients.
iii) 31 P MRS is a suitable tool for the in vivo assessment of free cytosolic [Mg 2+ ] in human skeletal muscle during rest, exercise and recovery; coordination of the study, SI participated in the study design, coordinated the study, and drafted the manuscript. All authors read and approved the final manuscript.