Anesthesia & Analgesia:
Anesthetic Pharmacology: Research Report
Cumulative and Bolus In Vitro Contracture Testing with 4-Chloro-3-Ethylphenol in Malignant Hyperthermia Positive and Negative Human Skeletal Muscles
Gerbershagen, Mark Ulrich MD*; Fiege, Marko MD*; Weisshorn, Ralf MD†; Kolodzie, Kerstin MD†; Esch, Jochen Schulte am MD†; Wappler, Frank MD*
*Department of Anesthesiology, Hospital Köln-Merheim, University Witten/Herdecke, Cologne, Germany; †Department of Anesthesiology, University Hospital Hamburg-Eppendorf, Martinistraβe 52, 20246 Hamburg, Germany
Presented, in part, at the 76th Clinical and Scientific Congress of the International Anesthesia Research Society, San Diego, California, March 16–20, 2002 and at Euroanaesthesia 2003, Glasgow, Scotland, May 31–June 3, 2003.
Accepted for publication February 9, 2005.
Address correspondence and reprint requests to Mark Ulrich Gerbershagen, MD, Department of Anesthesiology, Hospital Köln-Merheim, University Witten/Herdecke, Cologne, Germany. Address e-mail to firstname.lastname@example.org.
In this study we evaluated the in vitro effects of 4-chloro-3-ethylphenol (CEP) using cumulative (12.5–200 μmol/L) or bolus (75 and 100 μmol/L) administrations, on muscle specimens from malignant hyperthermia (MH) susceptible and MH nonsusceptible patients, respectively. In the cumulative CEP in vitro contracture test, contractures were significantly greater in the MH susceptible compared with the MH nonsusceptible muscles in all concentrations between 25 and 100 μmol/L. There was no overlap between the diagnostic groups at 75 μmol/L of CEP, so this test appears to be feasible for diagnosis of MH susceptibility. The two bolus tests are not diagnostically useful, as overlaps between the diagnostic groups were observed.
Apart from an increasing awareness and the introduction of dantrolene, the diagnosis of malignant hyperthermia (MH) using the in vitro contracture test (IVCT) was predominantly responsible for the reduction of mortality from about 80% to <10%. The IVCT has a sensitivity of 99.0% and a specificity of 93.6% when applying the European MH Group (EMHG) protocol (1). The slightly different North American protocol was reported to have a sensitivity rate of 97% and a specificity rate of only 78% (2). Alternative diagnostic testing has been pursued to improve diagnostic sensitivity and specificity or to use tissue obtained by less invasive means than the current muscle biopsy testing.
Because the preservative 4-chloro-3-ethylphenol (CEP), a monocyclic hydrocarbon, was shown to differentiate in vitro between MH susceptible (MHS) and MH nonsusceptible (MHN) porcine muscle specimens (3), the aim of the present study was to examine the diagnostic potential in human skeletal muscle.
After approval of the institutional human investigation committee and obtaining written informed consent from the patients or, in the case of minors, from parents, respectively, muscle biopsies were obtained from 59 patients, 52 adults (aged 18–66 yr) and seven children (age 4–16 yr) with clinical suspicion of MH or relatives with MHS by biopsy. Among the patients were 3 families with 2 relatives each and 1 family with 3 relatives. Before starting the investigation, we obtained a complete medical and family history, electrocardiogram, and laboratory variables, including creatine kinase concentrations. In adults, muscle biopsies were taken under femoral nerve block and in children muscle biopsies were taken using trigger-free general anesthesia. Immediately after excision the specimens were placed in Krebs solution and equilibrated with carbogen.
The standard diagnostic IVCTs were performed according to the EMHG protocol (1). MH equivocal (MHE) patients were excluded from the study; MHE represents the IVCTs in which either the halothane or the caffeine test show a significant contracture. Surplus muscle specimens from 27 MHS and 30 MHN patients were investigated in 3 different CEP contracture tests.
Before the experiments a stock solution of CEP (Aldrich, Steinheim, Germany) was prepared in dimethyl sulfoxide. Test solutions were freshly prepared from the stock solution by diluting appropriate amounts in distilled water and heating to a temperature of 37°C. All tests were performed within 5 h after biopsy.
In the first experiment cumulative CEP organ bath concentrations were adjusted to 12.5, 25, 50, 75, 100, and 200 μmol/L. According to a previous porcine study we defined a time frame of 5 min before administration of the next CEP concentration (3). Contracture development and muscle twitch response were assessed.
The second and third experiments were bolus IVCTs in bath concentrations of 75 or 100 μmol/L. In accordance with earlier studies with optional test substances (4) we defined the following variables: time to the start of contracture development, time to the achievement of 2 and 10 mN contractures (min), and maximum contracture (mN). Contracture development and muscle twitch responses were recorded for 60 min.
Statistical analysis was performed using a computer-based statistical program (SPSS® 11.0; SPSS, Chicago, IL). Medians and total ranges were calculated. Group differences of the cumulative test were analyzed with the nonparametric Mann-Whitney U-test. The log-rank test was used for analysis of group differences of the bolus tests because not all specimens reached the defined levels. Intragroup differences were examined with Wilcoxon’s test.
Table 1 shows MHS and MHN patients’ demographic data and clinical characteristics. In the cumulative CEP IVCT 2 of 12 MHS muscle specimens showed a baseline contracture at a concentration of 12.5 μmol/L CEP with a maximum of 4.2 mN (Fig. 1). Seven muscles exhibited a contracture at a concentration of 25 μmol/L (median (interquartile range [range]; P value versus MHN): 1.4 (0.0–5.7 [0.0–23.5] mN; P = 0.014), and 11 exhibited a contracture at a concentration of 50 μmol/L (5.6 (2.5–26.1 [0.0–45.3]) mN; P = 0.000). All MHS specimens demonstrated baseline increases at 75 μmol/L (13.0 (7.8–27.3 [3.4–51.7]) mN; P = 0.000), 100 μmol/L (14.0 (9.7–25.0 [6.4–46.6]) mN; P = 0.005), and 200 μmol/L (22.9 (11.2–33.6 [7.7–55.7]) mN; P = 0.266).
In contrast, the specimens of MHN persons did not display contractures up to 50 μmol/L. Two of 12 preparations produced baseline increases at 75 μmol/L CEP with 1.2 and 2.5 mN, respectively. Baseline increases were seen in 11 MHN muscle preparations at CEP concentrations of 100 μmol/L (6.2 (1.7–12.0 [0.0–18.5]) mN) and in all specimens at 200 μmol/L (26.8 (20.5–37.8 [8.7–64.4]) mN). The contractures were significantly larger in the MHS compared with the MHN muscles in all CEP concentrations between 25 and 100 μmol/L. There was no overlap between the 2 diagnostic groups at 75 μmol/L of CEP.
The results of the bolus CEP administrations at the start and 2-mN and 10-mN contracture levels are shown in Table 2, and the maximum contracture is shown in Figure 2. All variables were reached significantly earlier in the MHS compared with the MHN group. There were overlaps, however, between the diagnostic groups in all variables.
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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