³¹Phosphorus Magnetic Resonance Spectroscopy Characterization of Muscular Metabolic Anomalies in Patients with Malignant Hyperthermia : Application to Diagnosis

This study was performed on 39 healthy persons (7 women and 22 men) referred to the laboratory for MH diagnosis. All were members of MH families. In vitro halothane-caffeine contracture tests were performed on muscle biopsy specimens in accordance with the protocol recommended by the European Malignant Hyperpyrexia Group (see In Vitro Contracture Tests). [9]The results were positive for 13 participants (3 women, 10 men; mean age, 40 yr; referred to as participants 27 to 39) who were classified as MHS, whereas 26 participants (4 women, 22 men; mean age, 35 yr; referred to as participants 1 to 26) were MH negative (MHN). The³¹P MRS tests were usually performed the day before the participants had the muscle biopsy.

A biopsy was excised from the vastus (n = 8) or biceps brachii (n = 31) muscles under local anesthesia. Biopsies were excised preferentially from biceps because recovery of muscle function was faster for the participants and we failed to observe any difference for the results of in vitro contracture tests.* The sample was dissected into four bundles approximately 2 cm long and 1 mm wide. Each test was performed in duplicate in accord with the European Malignant Hyperpyrexia Group protocol. [9]Each bundle was placed into an oxygenated (95% oxygen, 5% carbon dioxide) Krebs medium maintained at 37 [degree sign] Celsius. The bundles were stretched to a 2-g predefined tension and were continuously electrically stimulated to produce twitches. Stimulation was performed in the presence of increasing concentrations of caffeine, from 0.5 mM to 4 mM and then more than 32 mM. The second test was performed with 0.5 to 3% vol/vol halothane mixed into the gas flow. A result was considered as positive if a 0.2 g contracture (threshold) appeared for 0.5 or 2 mM caffeine and 0.5–2% vol/vol (0.44 mM) halothane. Participants were recognized as MHS if the results of both contracture tests (halothane and caffeine) were positive.

The MR spectra of forearm flexor muscles were recorded at 4.7 T throughout previously described rest-exercise-recovery protocols. [10]Details on experimental conditions are presented in the Appendix.

After 3 min of rest, exercise consisted of finger flexions at 1.5-s intervals for 3 min lifting a 6-kg weight for men and a 4-kg weight for women. The motion amplitude of the sliding weight was approximately 3 cm, and it was kept constant throughout the exercise protocol. There was no duty cycle because work was performed lifting and releasing the weight. Such an exercise has been chosen to ensure PCr consumption greater than 50% of rest level and significant pH decrease. [11]Exercise was successively performed under aerobic and ischemic conditions. The protocol under ischemia involved the application of a sphygmomanometer over the upper arm and inflated, just before the start of exercise, above the maximum arterial pressure to achieve circulatory occlusion during the exercise period. At the end of the exercise, the pressure was quickly released. The participant's arm was gently restrained with velcro straps throughout the protocol, which included 20 min of postexercise recovery under fully aerobic conditions. A resting period of at least 1 h was allowed between the two exercise periods. All participants performed both protocols in the same order.

Peak areas of phosphorus metabolite signals including PCr, Pi, adenosine triphosphate (ATP), and phosphomonoesters (mainly corresponding to glucose 6-phosphate and fructose 6-phosphate [12]) were obtained by curve fitting the spectum to Lorentzian line shapes. Relative concentrations of phosphorus metabolites were expressed in relation to PCr content measured at rest (100%). Intracellular pH was calculated from the chemical shift of Pi relative to PCr at -2.45 ppm with respect to 85% H³PO⁴. [13]The PCr recovery was fitted to an exponential function as follows [14]:Equation 1where t is time in min, rest and cons refer, respectively, to PCr content measured at rest and the amount of PCr consumed measured at end-of-exercise (% of PCr content at rest), and k is expressed in min. sup -1 In addition, the initial rate of PCr recovery (Vi) was calculated from the initial slope of the recovery curve:Equation 2.

For the sake of clarity, results for all figures are presented as means +/- SEM. To provide quantitative information about the distribution of each parameter, values in Table 1are presented as means +/- SD. Table 2summarizes values of metabolic parameters chosen a priori according to our previous work. [10] 

Several analyses of variance have been performed using general linear models. The procedure (GLM) of SAS software (SAS Institute, Cary, NC), with options REPEATED, LSMEANS, and CONTRAST, were used.

For each group, one-way analysis of variance with repeated measures was used to analyze pH time-dependent changes during exercise. An F test was performed to determine the overall effect of time on pH changes throughout exercise. Then multiple comparisons procedures (Scheffe contrasts) were used to compare each value of pH recorded throughout the exercise period with the resting pH value. The same procedure was applied for the analysis of pH recovery toward resting values.

Between-group (MHN vs. MHS) comparative analyses of metabolites and pH time-dependent changes during exercise and recovery were performed using two-way analyses of variance with repeated measures (with time as the repeated variable). Wilk's lambda tests were performed to analyze the effects of time and the interaction between time and group. Post hoc repeated comparisons of mean values of pH and metabolite concentrations for each min of exercise and recovery between groups were performed (Scheffe contrasts).

Probability values for testing hypotheses were significant at 0.05. When appropriate, sensitivity and specificity were calculated as previously described. [15] 

The results obtained retrospectively on participants 1 to 39 were subsequently applied prospectively on a separate group of 10 participants (2 women, 8 men; mean age, 37 yr; referred to as participants 40 to 49).

(Figure 1) shows results of in vitro contracture tests. Box plots clearly demonstrate the differences between MHS and MHN groups. For the MHS group, contractures recorded with halothane and caffeine were significantly greater than 0.2 g (threshold defined according to the protocol of the European Malignant Hyperpyrexia Group. [9] 

Values of metabolic parameters, chosen a priori according to our previous work, [10]were measured or calculated from³¹P-MR spectra recorded throughout rest-exercise-recovery protocols and compared for the two groups. Figure 2shows a series of typical MR spectra recorded on the flexor muscles of a healthy volunteer.

At the beginning of each protocol, under normoxia or ischemia, all metabolic parameters were stable, showing that the muscle metabolic state was identical before each test. No significant differences were observed between MHN and MHS participants for pH values, PCr:Pi and PCr:ATP (Table 1). No measurable signals in the phosphodiesters region (between 0 and 1 ppm) were observed on spectra recorded for MHS participants.

The PCr and pH time-dependent changes recorded throughout exercises (normoxic and ischemic) were qualitatively similar (Figure 3). We address the general metabolic changes associated with exercise, keeping in mind that the magnitude of those modifications was always larger under ischemic conditions, as expected. During the first minute of exercise, no significant pH decrease was observed for MHN participants (Table 1). Throughout the rest of the exercise, pH decreased continuously as a sign of anaerobic ATP production. The PCr decrease with respect to time revealed ATP production from PCr hydrolysis, whereas the Pi increase resulted from ATP hydrolysis during muscle contraction. Concerning the amount of PCr hydrolyzed during each minute of exercise, we observed a decrease over time, reaching a minimum at the third minute of exercise. An increase of phosphomonoesters level (phosphomonoesters accumulation) was noted as a result of the imbalance between glycogen phosphorylase and phosphofructokinase activities, as previously described. [12]A slight ATP decrease (about 20%) was observed for MHN participants.

Under both conditions of exercise (normoxic and ischemic), raw values of pH and PCr were significantly less for MHS participants, except for the PCr content measured at the end of the ischemic exercise (Figure 3). Whatever the conditions of oxygen availability, a significant pH decrease was measured at the onset of exercise in the MHS group (Table 1, Figure 3). This decrease in pH was significantly larger in MHS participants compared with controls (MHN group;Table 1, Figure 3). Taking into account the pH changes calculated for each minute of exercise (from the second minute of exercise), no significant difference was reached between groups or for both exercise conditions (normoxia and ischemia). The magnitude of PCr hydrolysis calculated at the beginning of both exercises (aerobic and ischemic) was also significantly larger for the MHS group. Throughout the remaining aerobic and ischemic exercises, the extent of PCr consumption calculated for each minute of exercises was similar in both groups. Finally, magnitudes of PCr changes measured at the end of exercise were significantly different between MHN and MHS groups for both exercises (Figure 3(A and C), Table 1). Phosphomonoesters accumulation and the slight ATP decrease (about 20%) were identical in MHN and MHS participants.

(Figure 4) shows recovery profiles of PCr and pH recorded under aerobic conditions after both types of exercises. At recovery, post-exercise acidosis was noted in the MHN group (Figure 4(B, D)). This additional acidosis, accounting for proton production associated with PCr resynthesis, was always recorded except after the ischemic exercise performed by the MHS group. Although pH values recorded at the first and second minute of aerobic exercise were significantly different between both groups, the extent of additional acidosis calculated as the difference between two consecutive points was not statistically different. The initial rate of PCr recovery, calculated from the monoexponential fit, did not reveal any differences between the MHN and MHS groups (Figure 4(A, C)). In both groups, the kinetics of recovery were faster for higher end-of-exercise pH values, as expected. [11] 

This analysis shows that anomalies recorded in the MHS group were mainly associated with pH and PCr time-dependent changes, whereas no significant differences were recorded for phosphomonoesters or ATP profiles. Neither the rest nor the recovery periods provided any metabolic information about a possible specific disorder of MHS muscle.

At the beginning of both exercises, an early intracellular acidosis associated with a larger extent of PCr consumption was systematically observed in the MHS group. For these two parameters, threshold values were calculated as follows: First, for each parameter and for each protocol, a “real” confidence interval (not issued from statistical analysis) was determined to encompass at least 96% of the recorded values in the MHN group, meaning that 25 of the 26 MHN participants were properly classified. Second, a parameter value was considered abnormal with reference to the value of the corresponding threshold (chosen as the lower or higher limit of the confidence interval, depending on the parameter; see Table 2). Finally, considering both protocols, a parameter value was considered abnormal when it was found out of the confidence interval for at least one protocol. As shown in Table 3and Table 4, 10 of 13 patients in the MHS group displayed an abnormal pH decrease at the beginning of one or both exercises, whereas only 2 of 26 MHS participants had this abnormality. Therefore, three MHS participants had false-negative results and two MHN participants had false-positive results. Accordingly, considering the intracellular acidosis recorded at the onset of both exercises (aerobic and ischemic), the sensitivity value was 77% and specificity was 93%.

Considering these low values, we tried to perform a retrospective analysis based on additional parameters. To formulate a diagnostic test, we focused on distinct anomalies in the time courses of various metabolic parameters and selected 19 parameters among the 24 usually measured or calculated for each participant (Table 2). [10]These parameters were chosen a priori to properly describe metabolic changes associated with the rest-exercise-recovery protocols. [10]For each parameter, a metabolic threshold was determined for the MHN group and classified as normal or abnormal, as described before. Then for each participant (MHN and MHS), a score was calculated (MRS score). This score corresponds to the number of abnormal values recorded among the 19 parameters (Table 3and Table 4). For both protocols, scores ranged from 0 to 2 for controls (Table 3), whereas it varied from 3 to 11 for MHS participants (Table 4). Based on this score, specificity and sensitivity of this test were calculated in the retrospective analysis. A participant was classified as MHS if more than two abnormalities were recorded among the 19 parameters. Accordingly, specificity and sensitivity of the retrospective test were both 100%. Compared with the results previously calculated for intracellular acidosis recorded at the first minute of exercise, we obtained a marked increase in sensitivity (from 77–100%) and specificity (93–100%). No false-negative or false-positive results were identified. Clearly the use of 19 parameters recorded throughout the aerobic and ischemic protocols provides the best results for the noninvasive determination of susceptibility to MH.

We tested this retrospective analysis in 10 new participants to probe its prospective value. These persons were examined by MRS without previous knowledge of their MH status. According to the MRS score (Table 5), two were declared MHS and eight MHN. In vitro contracture tests confirmed the 10 diagnoses obtained by MRS, illustrating the potential of the MRS method.

We found that exercise-induced metabolic changes differed between MHN and MHS participants and that an abnormal MRS score can indicate MH susceptibility.

The statistical analysis identifies, in agreement with a previous study, [6]several deviations affecting early changes of intracellular pH. This is indicative of an excessive contribution of glycogenolysis to meet energy demand. During exercise, ATP production from the glycolygenolytic pathway leads to proton production, whereas PCr hydrolysis together with proton efflux accounts for a decrease in proton concentration within muscle cells. [16] 

Consequently, pH changes are modulated by glycolysis activity, the amount of PCr hydrolyzed, and the capacity of muscle cells to handle proton production and elimination. Early acidosis recorded for the MHS group can then be associated with glycogenolysis hyperactivity, reduced PCr consumption, or altered proton efflux. Proton efflux is usually calculated during the recovery period, when pH changes are modulated by proton efflux and PCr resynthesis. [16]Considering that we did not record any differences between both groups throughout the recovery period, early acidosis is unlikely to be linked with anomalies of proton handling. If we account for the alkalinizing effect of PCr hydrolysis, the larger magnitude of PCr consumption recorded for the MHS group at the onset of exercise should have been associated with reduced intracellular acidosis. This further suggests that early acidosis could be considered a sign of abnormally high glycogenolytic contribution to energy production. This mechanism could result from a direct hyperactivation of glycogenolysis, due to an abnormally high Ca²+ concentration in the cytosol. It could also be regarded as an adaptative mechanism aiming at compensating for a deficient aerobic energy production.

Indeed, it could be suspected that impaired oxidative energy supply would lead to hyperactivation of anaerobic metabolism to meet energy demand, as previously described. [17]However, Table 1and Figure 4show that the kinetics of postexercise recovery, which depended exclusively on aerobic metabolism, [11]were not significantly different between the two groups, thereby excluding any aerobic deficiency. This observation clearly confirms that the early metabolic changes affecting pH are to be linked to an abnormally high activation of glycogenolysis. Malignant hyperthermia sensititivity is associated, in pigs and humans, with impaired Ca²+ release from sarcoplasmic reticulum, [1,18,19]and this early acidosis can be considered a result of a higher calcium level-induced glycogenolysis hyperactivation.

In contrast to previous findings, we did not record any significant abnormality either at rest or during recovery. [4,5,8]However, in those previous studies, maximal voluntary contraction was usually measured just before the exercise, and this could account for a PCr decrease (increase of the Pi:PCr ratio). In this case, abnormalities observed at rest could correspond to a higher sensitivity of MHS participants to PCr hydrolysis during moderate exercise (leading to a temporary Pi:PCr increase) rather than a permanent increase in the Pi:PCr ratio at rest, as is usually observed for patients with severe mitochondrial myopathy. [17] 

Considering the MHS subjects as a group, we did not observe any abnormality during the recovery period, which corresponds with the findings of Webster et al. [6]and contrasts with what was previously reported by Olgin et al. [5]The reasons for this discrepancy remain unclear.

Our statistical results clearly demonstrate, if we consider MHS patients as a group, that MH can be considered as a myopathic process associated with a glycogenolytic hyperactivation probably due to an abnormality of calcium cycling. The sensitivity calculated from pH changes during the first minute of both exercise periods was not entirely satisfactory because it detected only 10 of 13 MHS participants. The MRS score, corresponding to the sum of deviations recorded throughout normoxic and ischemic protocols, provides a better characterization because 100% sensitivity was reached both in the retrospective and the prospective analyses. However, we could question the specificity of the metabolic abnormalities detected. Early intracellular acidosis coupled to large PCr depletion has been observed in several cases of metabolic myopathies, including Brody's disease (deficiency of Ca²+ ATPase of sarcoplasmic reticulum) while early pH decrease has been noted in postviral infection syndrome. [20,21]We must emphasize that only an asymptomatic patient with a family history of MH and for whom a significant MRS score is recorded, is most likely to be MH susceptible. In addition, MRS is not advocated for screening the general population but rather for testing a distinct group of persons who are at risk because of a family history of undesirable MH events. Specificity should then be regarded as the capacity of the test to exclude MH susceptibility for a participant considered at risk and not for any participant selected randomly in the general population. The variability of MRS score, ranging from 3 to 11 for MHS patients, indicated a heterogeneity of metabolic profiles that corresponded to the multiple possible defects leading to MH. Indeed, abnormalities of calcium cycling within muscle cells might result from mutation in genes encoding the Ca²+ release channel or from other proteins that participate in the excitation-contraction process. The MHS locus has been found primarily on humans chromosome 19q, but a growing number of non-chromosome 19q MHS families has been reported, [22,23]including a localization on chromosome 3 [24]and chromosome 7. [25]In addition, we must keep in mind that the MHS phenotype has been identified from in vitro contracture tests in patients with other neuromuscular diseases. [2] 

We found that the selection of appropriate MRS parameters recorded throughout protocols including rest, exercise and recovery periods enable us (1) to noninvasively detect MH susceptibility and (2) to identify MHS as the manifestation of a latent myopathic process. The variability of MRS score illustrates the various abnormal metabolic profiles that can be recorded for MHS patients and corresponds with the multigenic origin of MHS and the possible association of MHS with other myopathies. [26]The diagnostic capacity of this score was tested prospectively in 10 new participants (two MHS and eight MHN) who were properly classified. The diagnostic potential of this MRS score must be evaluated in a larger group of persons.

The authors thank B. Ghattas and Professor J. P. Durbec for helpful discussions of statistical methods.

All magnetic resonance spectroscopy experiments were conducted on a Bruker-Biospec 47/30 spectrometer equipped with a horizontal magnet (bore diameter, 30 cm). To record³¹-phosphorus magnetic resonance (sup 31 P MR) spectra of forearm flexor muscles, the participants sat in a chair positioned by the magnet and inserted their arm horizontally into the magnet bore to position the flexor digitorum superficialis muscle over a 50-mm diameter, double-tuned (1-H,³¹P) surface coil. The forearm was placed approximately at the same height as the shoulder to ensure a good venous return and was gently restrained with velcro straps to minimize movements throughout the exercise period. Optimization of the field homogeneity was done by monitoring the 200.14-MHz signal from the muscle water and fat proton.³¹P MR data were acquired at 81.14 MHz after 55-micro s radiofrequency pulses applied at 2-s intervals. Pulsing conditions were as follows: repetition time, 2 s; spectral width, 10,000 Hz; collection of 4 10³data points. Spectra were time averaged over 1 min (32 scans) and sequentially recorded during 3 min of rest, 3 min of exercise, and 20 min of recovery. A 15-Hz line broadening function was applied before Fourier transformation. A micropipette filled with a solution of phenylphosphonic acid was positioned at the surface coil center to accurately monitor global changes in spectral intensity. A typical series of spectra recorded from a healthy volunteer is presented in Figure 2.

*Kozak-Ribbens G, Rodet L, Baeta A, Figarella Branger D, Pellissier JF, Cozzone PJ: Comparative Results of halothane/caffeine contracture tests from biceps and vastus muscle biopsies for the diagnosis of MH. Proceedings of the VIIth International Workshop on Malignant Hyperthermia, Hiroshima, Japan, 16–20 July 1994.