The IVCT with halothane and caffeine is generally accepted as the standard procedure for the diagnosis of MH. Despite the high sensitivity, few cases of fulminant MH and abortive MH under general anesthesia in patients tested as MHN were reported (5,6). The clinical presentation supports the view that contracture tests yielded a false negative result and a misleading diagnosis. Generally, determination of the sensitivity of MH testing is difficult because of the rarity of the trait in the population and the even rarer occurrence of fulminant MH episodes. Nevertheless, because of the possible lethal consequence of false negative results, it must be the aim to develop a test that can claim 100% sensitivity.
Studies concerning alternative diagnostic testing were performed with thrombocytes (7) and erythrocytes (8), analyzing intracellular calcium concentrations (9), and using magnetic resonance spectroscopy (10) and electromyography (11). A sufficient differentiation between MHS and MHN subjects, however, was not achieved.
Eventually, it was believed that a genetic approach would offer the best prospect for the development of a noninvasive test. Key proteins in the regulation of muscle calcium are the dihydropyridine receptor located in the transverse tubule and the ryanodine receptor (RYR1) of the sarcoplasmic reticulum. Contrary to the single amino acid mutation on chromosome 6 in porcine MH (12), more than 30 MH-associated point mutations have been found responsible for the RYR1 gene abnormality in MH-susceptible humans. For a maximum of up to 20 of these mutations, the causal relationship for MH could be shown in particular families, whereas only 15 were considered in European diagnostic guidelines (13,14). The complexity of the RYR1, one of the largest known proteins, with 2200 kDa corresponding to 5000 amino acids encoded by 106 exons increases the chance of detecting further mutations (15). Therefore, a universally applicable genetic screening test does not seem to be realistic in the near future. In addition, two MH-associated mutations have been identified encoding to the dihydropyridine receptor (16,17). Nevertheless, the accuracy of a DNA-based family diagnosis is essentially dependent on the IVCT phenotyping of the individual presenting with the clinical MH suspicion. This further stresses the need to optimize the standard IVCT.
Currently there are two drugs that are optionally accepted to be used for IVCT according to the EMHG guidelines (1,18). One is ryanodine; the other is 4-chloro-m-cresol (4-CmC), both specific activators of the calcium release from the sarcoplasmic reticulum via the RYR1. First, a comparison between a cumulative ryanodine IVCT and bolus administration was undertaken (19). Highly purified ryanodine was tested in various concentrations to estimate the differential power (20,21). Eventually the bolus test with 1 μmol/L highly purified ryanodine was authorized by the EMHG as an optional test (1). A European multicenter study showed reproducible results in each MH laboratory using 1 μmol/L highly purified ryanodine. However, no common diagnostic cut-off point could be defined until now because of variations among the test centers. The origin for these variations cannot be explained (18).
4-CmC induces definite concentration-dependent contractures in MHS muscles yet only weak contractures in MHN specimens (4,22). A multicenter study presented a good discrimination concerning the diagnostic groups, with a specificity of 99.0% and a sensitivity of 96.1% (23). The goal of reaching 100% differentiation between MHS and MHN could not be obtained.
CEP seems to be a promising test drug for diagnosis of MH susceptibility because it has been shown to trigger Ca2+-induced Ca2+-release in various nonexcitable cell types (24). In ryanodine type III isoform receptors it could be demonstrated that CEP enhances the binding of [3H]ryanodine 2.5-fold, whereas caffeine was much less effective in activating this receptor. Eventually a dose-dependent increase in the resting intracellular Ca2+-concentration in intact mice skeletal muscle fibers was demonstrated (25).
CEP and 4-CmC were reported to be advantageous to ryanodine as pharmacological test substances for pathophysiological skeletal muscle examinations. The latter binds to specific high or low affinity sites on the RYR1, has stimulatory as well as inhibitory effects, and binds irreversibly to the RYR1 (26). CEP and 4-CmC increase resting calcium in intact skeletal muscle fibers in a dose-dependent manner, specifically by activating the ryanodine receptor. In this regard CEP, the more lipophilic substance, was more potent.
In the present cumulative CEP experiment, human muscle contractures were attained at significantly smaller CEP concentrations and were significantly larger in the MHS compared with the MHN specimens. A 100% differentiation was obtained at 75 μmol/L CEP. MH diagnosis with cumulative CEP IVCT may be feasible and might be a promising optional diagnostic test. With regard to the small number of cases in the present study and the limited statistical power, it might be worthwhile to repeat this study in other countries with different genetic composition.
The 75 and 100 μmol/L CEP bolus administrations also showed a significant separation of the two diagnostic groups in all predefined variables, yet with overlaps. Therefore, these bolus tests are not indicated for the in vitro MH diagnosis.
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