Free Mg2+ concentration in the calf muscle of glycogen phosphorylase and phosphofructokinase deficiency patients assessed in different metabolic conditions by 31P MRS
© Malucelli et al; licensee BioMed Central Ltd. 2005
Received: 10 January 2005
Accepted: 06 June 2005
Published: 06 June 2005
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.
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).
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.
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;
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 . There is considerable evidence that Mg2+ is actively transported and regulated, although the mechanisms are still largely unknown . 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 [Mg2+] 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 .
We assessed the cytosolic pH and the [Mg2+] by 31P 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 31P MRS [6, 7]. We used these diseases as natural experimental models to study the pattern of free Mg2+ during exercise and recovery in the absence of intracellular acidification to understand to what extent homeostasis of intracellular free Mg2+ is linked to pH.
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.
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 1H 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 .
Subjects lay supine with a 20.5/7.5 cm diameter transmitter/receiver surface coil centred on the maximal circumference 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 . 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, the PCr and Pi concentration were calculated by assuming a normal ATP concentration of 8 mM . The cytosolic pH and [Mg2+] 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 [Mg2+] . The simultaneous calculation of [Mg2+] and pH was performed by the specific software package MagicMC, that we developed and made available on the internet .
31P 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.
[Mg2+] and pH values at rest and end-exercise
Controls Mean (n = 10)
control range [min:max]
In the skeletal muscle variations of cytosolic pH, phosphocreatine and inorganic phosphate concentrations influence the complex multi-equilibrium system of the molecular species which bind magnesium ions. As a consequence free cytosolic [Mg2+] can change considerably in different metabolic conditions such as rest, exercise and recovery. It has been shown by 31P MRS that the increase of cytosolic free [Mg2+] occurring in skeletal muscle of healthy subjects during exercise and initial recovery is matched by a decrease in cytosolic pH, and the changes in cytosolic free [Mg2+] were mainly the result of the predominant effect of [H+]. . This result was attributed to mechanisms of binding competition existing between Mg2+ and H+ towards the molecules negatively charged present in the cell cytosol . However, the causal relationship between pH and [Mg2+] has not been proved yet, as it could be argued that muscular exercise per se elicits an increase in cytosolic free [Mg2+]. Therefore, we used patients with McArdle's and Tarui's disease as experimental models to study the pattern of [Mg2+] during exercise and recovery in the absence of intracellular acidification to understand to what extent homeostasis of intracellular free Mg2+ is linked to pH. Due to the rare nature of these disorders we were able to enrol just two patients for both diseases and therefore we had to deal with a small sample size. The results show that the increase in cytosolic [Mg2+] occurring in skeletal muscle during exercise is actually the consequence of the increase of H+ concentration and not of other mechanisms related to muscle contraction. In addition, we found that both PFK patients showed a reduction of [Mg2+] during exercise concomitant with the PME increase. The decrease of [Mg2+] also persisted during recovery in PFK I patient who displayed a slower PME recovery. The PME peak in the 31P MRS spectra corresponds to the phosphorylated monosaccharide intermediates of glycogenolysis. Therefore, due to the deficit 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 Mg2+. As a consequence, we interpret the decrease of [Mg2+] concomitant with the PME increase as due to the binding of Mg2+ 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. Therefore, the [Mg2+] decrease found in PFK patients cannot be ascribed to a diminished Pi build-up.
Moreover, PFK II patient displayed a larger decrease of [Mg2+] from rest to end-exercise compared to PFK I patient. Accidentally, PFK II patient also had a smaller PCr breakdown, nevertheless, this cannot be the cause of the larger decrease of [Mg2+], as a lower [PCr] corresponds to lower calculated [Mg2+] (3).
Our results show that:
i) free [Mg2+] is strongly linked to pH in skeletal muscle homeostasis as previously suggested by a study in healthy volunteers , and by computer simulation on a chemical model mimicking muscle cell cytosol ;
ii) the decrease of free [Mg2+] during exercise in both PFK patients suggests that [Mg2+] is influenced by the accumulation of fructose- 6-phosphate, an additional binding site for cytosolic Mg2+, as shown by the accumulation of the phosphomonoesters peak in the 31P MRS spectra of these patients.
iii) 31P MRS is a suitable tool for the in vivo assessment of free cytosolic [Mg2+] in human skeletal muscle during rest, exercise and recovery;
This work was supported by Ricerca Fondamentale Orientata (ex quota 60%) and by Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale – Cofin (ex quota 40%).
- Lawson RWJ, Veech RL: Effects of pH and free magnesium ion on the Keq of the creatine kinase reaction and other phosphate hydrolyses and phosphate transfer reactions. J Biol Chem. 1979, 254: 6528-6537.PubMedGoogle Scholar
- Masuda T, Dobson GP, Veech RL: The Gibbs-Donnan near-equilibrium system of heart. J Biol Chem. 1990, 265: 20321-20334.PubMedGoogle Scholar
- Iotti S, Frassineti C, Alderighi L, Sabatini A, Vacca A, Barbiroli B: In Vivo 31P-MRS assessment of cytosolic [Mg2+] in the human skeletal muscle in different metabolic condition. Magn Reson Imag. 2000, 18: 607-614. 10.1016/S0730-725X(00)00132-6.View ArticleGoogle Scholar
- McConchie SM, Coakley J, Edwards RHT, Beynon RJ: Molecular Heterogeneity in McArdle's disease. Biochim Biophys Acta. 1991, 1096: 26-32.View ArticleGoogle Scholar
- Tsujino S, Servidei S, Tonin P, Shanske S, Azan G, DiMauro S: Identification of three novel mutation in non-Ashkenazi Italian patients with muscle phosphofructokinase deficiency. Am J Hum Genet. 1994, 54: 812-819.PubMedPubMed CentralGoogle Scholar
- Bertocci LA, Haller RG, Lewis SF, Fleckenstein JL, Nunnally RL: Abnormal high-energy phosphate metabolism in human muscle phosphofructokinase deficiency. J Appl Physiol. 1991, 70: 1201-1207.PubMedGoogle Scholar
- Sahlin K, Areskog NH, Haller RG, Henriksson KG, Jorfeldt L, Lewis SF: Impaired oxidative metabolism increases adenine nucleotide breakdown in McArdle's disease. J Appl Physiol. 1990, 69: 1231-1235.PubMedGoogle Scholar
- EEC Concerted Research Project: Quality Assessment in in vivo NMR spectroscopy. Results of a Concerted Research Project of the European Economic Community (6 papers). Magn Reson Imag. 1995, 13: 115-176.Google Scholar
- Zaniol P, Serafini S, Ferraresi M, Golinelli R, Bossoli P, Canossi I, Aprilesi GC, Barbiroli B: Muscle 31 P-MR spectroscopy performed routinely in a clinical environment by a whole-body Imager/spectromemeter. Physica Medica. 1992, 8: 87-91.Google Scholar
- Magnetic Resonance User Interface Home Page. [http://carbon.uab.es/mrui]
- Harris RC, Hultman E, Nordesjö LO: Glycogen, glycolytic intermediates, and high-energy phosphates determined in biopsy samples of musculus quadriceps femoris of man at rest. Methods and variance of values. Scand J Clin Lab Invest. 1974, 33: 109-120.PubMedView ArticleGoogle Scholar
- Centro di Ricerca e Diagnostica Molecolare in vivo. [http://www.unibo.it/bioclin]
- Iotti S, Tarducci R, Gottardi G, Barbiroli B: Cytosolic Free [Mg2+] in the human calf muscle in different metabolic conditions: in vivo 31P MRS and computer simulation. Proceedings of the Society of Magnetic Resonance in Medicine (seventh scientific meeting and exhibition). 1999, Philadelphia, Pennsylvania USA, 3: 1540-22–28, May 1999Google Scholar
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