In Vitro Contracture Test Research Articles

Phenotyping malignant hyperthermia susceptibility by measuring halothane-induced changes in myoplasmic calcium concentration in cultured human skeletal muscle cells

Malignant hyperthermia (MH) is a potentially lethal disease triggered by volatile anaesthetics and succinylcholine in genetically predisposed individuals. Because of the heterogenetic nature of MH, a simple genetic-based diagnostic test is not feasible and diagnosis requires an invasive open muscle biopsy followed by the in vitro contracture test (IVCT). Our aim was to establish if measurements of halothane-induced increases in intracellular calcium ion concentration [Ca(2+)](i) in cultured human skeletal muscle cells can be used to phenotype MH susceptibility and if different mutations in the ryanodine receptor (RYR1) gene affect halothane-induced increases in [Ca(2+)](i). Primary cultures of human skeletal muscle cells were established from 54 individuals diagnosed by the IVCT according to the protocol of the European MH Group as: MH susceptible (n=22), MH negative (n=18) or MH equivocal (n=14). All individuals were screened for the presence of the most common mutations in the RYR1 gene. [Ca(2+)](i) was measured by fluorescent digital microscopy using fura-2/AM in 10 cells from each patient at five different halothane concentrations. The halothane-induced increase in [Ca(2+)](i) differed significantly between the three diagnostic groups. Different mutations of the RYR1 gene did not have a specific impact on halothane-induced increases in [Ca(2+)](i). Measurements of [Ca(2+)](i) in human skeletal muscle cells can be used to phenotype MH susceptibility; however, we did not observe a specific effect of any mutation in the RYR1 gene on the halothane-induced increase in [Ca(2+)](i).

Mutation screening in the ryanodine receptor 1 gene (RYR1) in patients susceptible to malignant hyperthermia who show definite IVCT results: identification of three novel mutations.

The ryanodine receptor of the skeletal muscle (RYR1) seems to be of outstanding importance in the pathogenesis of malignant hyperthermia (MH). It has been shown that point mutations in the RYR1 gene are strongly associated with the MH phenotype. A correctly determined phenotype is the basic prerequisite for adequate genetic MH screening. In this study we examined only those MH susceptible patients for the presence of potential RYR1 mutations who showed strong pathological muscle responses in the in vitro contracture test (IVCT). A total of 56 MHS index patients who complied with the following IVCT criteria were included in the molecular genetic investigation: Contracture forces > or =4 mN at a caffeine concentration of 2.0 mmol/l and > or =8 mN at a halothane concentration of 0.44 mmol/l. DNA sequences of exons 2, 6, 9, 11, 12, 14, 15, 17, 39, 40, 45, 46, 102 of the RYR1 gene were analysed by the direct sequencing technique. Furthermore, if an MH mutation was identified in an index patient, all relatives were screened for their family specific RYR1 defect. In 39 index patients an RYR1 mutation was detected: Arg163Cys (n = 2), Asp166Asn (n = 1), Gly341Arg (n = 2), Arg401His (n = 2), Arg614Cys (n = 12), Asp2129Glu (n = 1),Vol2168Met (n = 1), Thr2206Met (n = 9), Ala2428Thr (n = 1), Gly2434Arg (n = 2), Arg2435His (n = 1), Arg2452Trp (n = 1), Arg2454His (n = 4). Three new RYR1 mutations were identified. We found a potential MH mutation in a further 130 relatives of the 39 index patients. Thirty-seven individuals were classified as MHS exclusively by molecular genetic techniques and did not have to undergo the IVCT. The ascertained high rate of successful MH mutation screening (69.64%) is obviously associated with the more clearly defined MHS diagnosis in the IVCT. According to the EMHG guidelines for the molecular genetic detection of MH susceptibility, a positive MH disposition could be determined in numerous persons by a less invasive technique.

In-vitro-effects of cocaine in skeletal muscle specimens of patients susceptible to malignant hyperthermia

The abuse of cocaine can cause serious medical complications like tachycardia, rhabdomyolysis, and hyperthermia. Because of the clinical similarities, it has been suggested that cocaine might be a trigger of malignant hyperthermia (MH). Therefore, aim of this study was to investigate the in-vitro effects of cocaine in skeletal muscle specimens of MH susceptible (MHS) and normal (MHN) patients. 62 patients undergoing the in-vitro contracture test (IVCT) according to the protocol of the European MH Group (EMHG) for diagnosis of MH susceptibility were included in this study. In muscle specimens surplus to diagnostic requirements cocaine was added in order to achieve tissue bath concentrations of 0.01, 0.1 and 1.0 mM. The contracture development and twitch response have been registered. 21 patients were diagnosed as MHS and 36 patients as MHN. 5 patients tested as MH-equivocal (MHE) were excluded from the study. Following bolus administration of cocaine, no contracture development was observed in MHS, as well as MHN specimens. The muscle twitch decreased after cocaine administration significantly in both diagnostic groups. In contrast to the established MH trigger substances like volatile anaesthetics, cocaine produced no contracture development in MHS muscle specimens. Furthermore, cocaine produced a negative inotropic effect in all skeletal muscle preparations, which might be explained by local anaesthetic effects. Regarding these results, cocaine seems not to be a MH trigger agent.

Determination of a positive malignant hyperthermia (MH) disposition without the in vitro contracture test in families carrying the RYR1 Arg614Cys mutation.

Molecular genetic methods are used with caution for determining positive malignant hyperthermia (MH) disposition in clinical MH diagnosis because of the genetic variability of this disease. But under defined conditions, genotyping can have an advantage over the standardized in vitro contracture test (IVCT) in respect of invasive approach, specificity, patient compliance, and the work and expense involved in the method. We aim to demonstrate this using 10 families with the Arg614Cys mutation in the ryanodine receptor as an example. Fifty-one index patients who had been classified as MH-susceptible (MHS) in the IVCT (European MH protocol) after a clinical MH incident or suspected MH were screened for the Arg614Cys (C1840-->T) mutation in the RYR1 gene because this mutation is more common in German MH families (9%). The family members of those index patients, in whom a Arg614Cys mutation was detectable, were also screened for the presence of this mutation (n=136), and the results were subjected to a more detailed analysis including existing IVCT findings (n=71). The Arg614Cys mutation was identified in a total of 64 members of the 10 independent families. In 35 individuals in this group, there was a definite concordance between the MHS diagnosis in the IVCT and the presence of the Arg614Cys mutation. No individual phenotyped as MH-negative carried the mutation. On the basis of the guidelines of the EMHG for molecular genetic detection of MH susceptibility, 29 individuals who bore the Arg614Cys mutation were classified as MHS without the IVCT. If a causal mutation is detected in an MH family, the MHS diagnosis can be deduced without the invasive IVCT in all other mutation carriers. Despite inclusion of only one (Arg614Cys) of all known MH mutations, the study emphasizes the practical use of a genetic approach for determination of a positive MH diagnosis.

Homozygous and heterozygous Arg614Cys mutations (1840C-->T) in the ryanodine receptor gene co-segregate with malignant hyperthermia susceptibility in a German family.

The determination of susceptibility to malignant hyperthermia (MH) by genetic investigation is a controversial issue because of the genetic heterogeneity of this disorder. The requirement for such an approach in MH diagnosis is a strong correlation between MH-associated genetic abnormalities and phenotypic findings in the in vitro contracture test (IVCT). After a severe clinical MH crisis during general anaesthesia a patient was diagnosed by the IVCT in which susceptibility to MH was confirmed. Genetic screening for MH-related mutations in the RYR1 gene revealed the presence of a homozygous 1840C-->T base exchange (Arg614Cys substitution) in this patient. A specific search for this defect in 20 relatives led to the identification of a total of 11 Arg614Cys mutations. Of these, 10 were heterozygous (including both parents) and one was homozygous (sister). Further IVCTs were subsequently performed on the parents of the index patient, the homozygous sister and all relatives who did not carry the Arg614Cys in order to determine the genotype/phenotype correlation. After analysing these data, and because of the strong correlation between clinical, phenotypic, and genetic results in the index patient, we assigned the diagnosis 'MHS' to all the remaining Arg614Cys mutation carriers of that family without performing the IVCT.

Evidence for susceptibility to malignant hyperthermia in patients with exercise-induced rhabdomyolysis.

Malignant hyperthermia (MH), heat stroke, and exercise-induced rhabdomyolysis (ER) were suspected to be related syndromes. However, it is not known whether individuals with history of ER have an increased incidence of susceptibility to MH. To establish an association between ER and susceptibility to MH, the authors determined the MH status in patients with a history of MH-like episodes induced by physical stress. Twelve unrelated patients with ER, 18 patients with anesthesia-induced MH, and 28 controls were investigated with the in vitro contracture test (IVCT) according to the European MH Group protocol and the ryanodine contracture test. In addition, all patients were screened for genetic mutations, and histology was performed on muscle specimens. Ten ER patients had positive IVCT results, one patient had a negative test result, and one patient showed equivocal responses. Samples from patients with positive IVCT results showed pronounced contractures after exposition to ryanodine, as opposed to specimens from patients with negative IVCT results, which developed contractures slowly. Three ER patients had mutations at the ryanodine receptor gene. All anesthesia-induced MH patients had positive IVCT results, two of them presented the C1840T mutation. The control patients had normal contracture test results and no typical MH mutations. Histologic examination determined no specific myopathies in any patient. Regarding these results, the authors recommend performing muscle biopsies for histologic examination and IVCT in patients with ER. In addition, the patient should be seen by a neurologist and screened for genetic abnormalities to shed light on the genetics of MH.

Between-Center Variability of Results of the In Vitro Contracture Test for Malignant Hyperthermia Susceptibility

The in vitro contracture test (IVCT) remains the standard test for the diagnosis of malignant hyperthermia (MH) susceptibility. The aim of this study was to investigate whether results of the IVCT varied between two diagnostic centers. The study took place at the national MH centers in Denmark and Sweden. Forty-three patients investigated for MH gave informed consent to have four extra muscle specimens excised. These were sent to the other center and immediately used for a parallel IVCT, according to the protocol of the European MH Group. Results of the IVCTs performed in the two centers on muscle samples from the same patients were compared. Each patient was assigned a diagnosis according to the result obtained in the “mother-center.” Identical diagnostic results were obtained for 56% of the patients. The differing diagnostic outcomes were almost exclusively seen in cases with contractures of <5 mN (0.5 g) and abnormal results in only one or two muscle strips. We suggest different criteria for the interpretation of results for clinical and scientific purposes. The clinical criteria should remain unchanged. The scientific designation of susceptibility should be used in cases with contractures of ≥5 mN and abnormal results in at least 75% of the tested muscle strips. Implications The diagnostic outcomes of tests for malignant hyperthermia susceptibility were compared between two laboratories by using muscle tissue from the same patients. Identical outcomes were found for 56% of the patients. Almost all diverging outcomes were seen in cases with a few small contractures near the cutoff limit. Different diagnostic criteria for clinical and scientific purposes are suggested.

Between-Center Variability of Results of the In Vitro Contracture Test for Malignant Hyperthermia Susceptibility

The in vitro contracture test (IVCT) remains the standard test for the diagnosis of malignant hyperthermia (MH) susceptibility. The aim of this study was to investigate whether results of the IVCT varied between two diagnostic centers. The study took place at the national MH centers in Denmark and Sweden. Forty-three patients investigated for MH gave informed consent to have four extra muscle specimens excised. These were sent to the other center and immediately used for a parallel IVCT, according to the protocol of the European MH Group. Results of the IVCTs performed in the two centers on muscle samples from the same patients were compared. Each patient was assigned a diagnosis according to the result obtained in the "mother-center." Identical diagnostic results were obtained for 56% of the patients. The differing diagnostic outcomes were almost exclusively seen in cases with contractures of <5 mN (0.5 g) and abnormal results in only one or two muscle strips. We suggest different criteria for the interpretation of results for clinical and scientific purposes. The clinical criteria should remain unchanged. The scientific designation of susceptibility should be used in cases with contractures of > or =5 mN and abnormal results in at least 75% of the tested muscle strips. The diagnostic outcomes of tests for malignant hyperthermia susceptibility were compared between two laboratories by using muscle tissue from the same patients. Identical outcomes were found for 56% of the patients. Almost all diverging outcomes were seen in cases with a few small contractures near the cutoff limit. Different diagnostic criteria for clinical and scientific purposes are suggested.

Inositol 1,4,5-trisphosphate synthesis in mononuclear white blood cells of malignant hyperthermia-susceptible and normal human beings, following in vitro exposure to halothane, caffeine and ryanodine.

Despite a plethora of findings associated with the pathophysiology of malignant hyperthermia (MH), the in vitro contracture test (IVCT) is the only reliable test for diagnosis of this heterogeneous syndrome in man. An increase of 1,4,5-IP3 (inositol 1,4,5-trisphosphate), a second messenger involved in cellular calcium homeostasis, has been observed in muscle tissue of MH susceptible (MHS) patients. The aim of this study was to evaluate if the known differences of 1,4,5-IP3 content in muscle tissue might be reproduced in mononucleated white blood cells (MWBCs). Subsequently, MWBCs of 23 healthy controls and 12 patients with a clinical suspicion for MH disposition were isolated and screened for 1,4,5-IP3 content. An IVCT according to the protocol of the European Malignant Hyperpyrexia Group (EMHG) was performed on muscle specimens of 12 patients. Eight MHN and four MHS individuals were diagnosed. Additionally, 1,4,5-IP3 synthesis in MWBCs was detected following in vitro exposure to IVCT test substances halothane (2%), caffeine (1-30mM), and ryanodine (1-5 microM). A broad inter-individual variability of 1,4,5-IP3 content was observed in MWBCs of all volunteers, but no differences were detected between MHS and MHN individuals. These findings are in strong contrast to those observed in muscle tissue. In vitro exposure of isolated MWBCs to halothane, caffeine and ryanodine yielded no statistically significant differences between groups. A time- and concentration-dependent increase in cellular 1,4,5-IP3 content could be induced in some but not all individuals of both groups. Since no correlation was obtained between induction of 1,4,5-IP3-synthesis following in vitro exposure of MWBCs to MH test substances and MH disposition, this study was terminated. We conclude from our data that the detection of 1,4,5-IP3 synthesis in MWBCs is not suitable for diagnosis of MH disposition. It remains questionable whether an altered 1,4,5-IP3 metabolism in MWBCs is involved in pathologic cascades of MH. Therefore, other cell tissues should be evaluated in further studies to clarify the role of the 1,4,5-IP3 metabolism in MH.

Inositol 1,4,5-trisphosphate synthesis in mononuclear white blood cells of malignant hyperthermia-susceptible and normal human beings, following in vitro exposure to halothane, caffeine and ryanodine

Despite a plethora of findings associated with the pathophysiology of malignant hyperthermia (MH), the in vitro contracture test (IVCT) is the only reliable test for diagnosis of this heterogeneous syndrome in man. An increase of 1,4,5-IP3 (inositol 1,4,5-trisphosphate), a second messenger involved in cellular calcium homeostasis, has been observed in muscle tissue of MH susceptible (MHS) patients. The aim of this study was to evaluate if the known differences of 1,4,5-IP3 content in muscle tissue might be reproduced in mononucleated white blood cells (MWBCs). Subsequently, MWBCs of 23 healthy controls and 12 patients with a clinical suspicion for MH disposition were isolated and screened for 1,4,5-IP3 content. An IVCT according to the protocol of the European Malignant Hyperpyrexia Group (EMHG) was performed on muscle specimens of 12 patients. Eight MHN and four MHS individuals were diagnosed. Additionally, 1,4,5-IP3 synthesis in MWBCs was detected following in vitro exposure to IVCT test substances halothane (2%), caffeine (1-30 mM), and ryanodine (1-5 μM). A broad inter-individual variability of 1,4,5-IP3 content was observed in MWBCs of all volunteers, but no differences were detected between MHS and MHN individuals. These findings are in strong contrast to those observed in muscle tissue. In vitro exposure of isolated MWBCs to halothane, caffeine and ryanodine yielded no statistically significant differences between groups. A time- and concentration-dependent increase in cellular 1,4,5-IP3 content could be induced in some but not all individuals of both groups. Since no correlation was obtained between induction of 1,4,5-IP3-synthesis following in vitro exposure of MWBCs to MH test substances and MH disposition, this study was terminated. We conclude from our data that the detection of 1,4,5-IP3 synthesis in MWBCs is not suitable for diagnosis of MH disposition. It remains questionable whether an altered 1,4,5-IP3 metabolism in MWBCs is involved in pathologic cascades of MH. Therefore, other cell tissues should be evaluated in further studies to clarify the role of the 1,4,5-IP3 metabolism in MH.

Effects of the phosphodiesterase-III inhibitor enoximone on skeletal muscle specimens from malignant hyperthermia susceptible patients

Study Objectives: To study the in vitro effects of the phosphodiesterase-III inhibitor enoximone on skeletal muscle specimens from malignant hyperthermia susceptible (MHS) and normal (MHN) patients. Design: Prospective study. Setting: Malignant hyperthermia (MH) laboratory at a university hospital. Patients: 47 patients with clinical suspicion for MH undergoing in vitro contracture test (IVCT) for diagnosis of MH susceptibility. Interventions: Biopsies of M. quadriceps femoris were performed in adult patients with a 3-in-1 nerve block and in children with trigger-free general anesthesia. Measurements and Main Result: Patients were first classified as MHS or MHN by the IVCT according to the protocol of the European MH Group (EMHG). Patients with equivocal results (MHE) or with neuromuscular diseases were excluded from the study. Enoximone was added to the organ bath to surplus vital muscle specimens in single bolus concentrations of 0.4, 0.6, 0.8, or 1.6 mmol/L. The in vitro effects of enoximone on muscle contractures and twitch were measured. Seventeen patients were classified as MHS and 30 as MHN by the EMHG criteria. Enoximone induced contractures in skeletal muscles in a dose-dependent manner. Contractures of MHS compared to MHN muscle specimens were significantly larger at all concentrations used in this study. No overlap in maximum contractures was seen between MHS and MHN muscles at a bath concentration of 0.6 mmol/L enoximone only. Conclusions: Diagnosis of MH by an IVCT test with a single bolus administration of enoximone seems to be possible using a concentration of 0.6 mmol/L. The findings of this study may indicate an involvement of the phosphodiesterase-III and cAMP system in pathogenesis of MH. Further in vivo investigation should determine the trigger potency of enoximone in MH susceptible individuals.

Genetics and pathogenesis of malignant hyperthermia.

Malignant hyperthermia (MH) is a potentially life-threatening event in response to anesthetic triggering agents, with symptoms of sustained uncontrolled skeletal muscle calcium homeostasis resulting in organ and systemic failure. Susceptibility to MH, an autosomal dominant trait, may be associated with congenital myopathies, but in the majority of the cases, no clinical signs of disease are visible outside of anesthesia. For diagnosis, a functional test on skeletal muscle biopsy, the in vitro contracture test (IVCT), is performed. Over 50% of the families show linkage of the IVCT phenotype to the gene encoding the skeletal muscle ryanodine receptor and over 20 mutations therein have been described. At least five other loci have been defined implicating greater genetic heterogeneity than previously assumed, but so far only one further gene encoding the main subunit of the voltage-gated dihydropyridine receptor has a confirmed role in MH. As a result of extensive research on the mechanisms of excitation-contraction coupling and recent functional characterization of several disease-causing mutations in heterologous expression systems, much is known today about the molecular etiology of MH.

5-HT2 receptor antagonist-mediated inhibition of halothane-induced contractures in skeletal muscle specimens from malignant hyperthermia susceptible patients.

Administration of 5-HT2 receptor agonists induced malignant hyperthermia (MH) in susceptible pigs. Furthermore, the 5-HT2 receptor antagonist ritanserin prevented 5-HT-induced porcine MH. It has been shown that 5-HT2 receptor agonists induce marked contractures in skeletal muscle specimens from MH susceptible (MHS) but not in specimens from normal patients. The purpose of this study was to investigate the effects of ritanserin on halothane-induced contractures in muscle specimens from MHS patients. Twenty-five patients aged 8-56 years (29.5+/-13.6) classified as MHS by the in vitro contracture test (IVCT) with halothane and caffeine according to the protocol of the European MH Group participated in this study. Muscle specimens were pretreated with ritanserin 10 micromol/l (n= 14), 20 micromol/l (n=14) and 100 micromol/l (n=12) for 10 min and subsequently halothane was added incrementally (0.11-0.22-0.44 mmol/l) to the tissue bath as described in the European MH protocol. The results of the halothane contracture test were used as control. Following administration of halothane, muscle contractures reached a maximum of 16.9+/-4.2 mN. Ritanserin led to a significant inhibition of halothane-induced contractures in MHS muscles. Following pretreatment with ritanserin, halothane-induced contracture maximum was significantly smaller with 7.5+/-3.1 mN after 10 micromol/l ritanserin, 4.9+/-1.5 mN after 20 micromol/l ritanserin and 0.5+/- 0.2 mN after 100 micromol/l ritanserin than without pretreatment. Administration of ritanserin induced at all concentrations a decrease in muscle twitch height. Increase in muscle twitch following halothane was reduced in a concentration-dependent manner by ritanserin. The presented findings indicate that 5-HT might be involved in the mechanisms of halothane-induced MH in humans. Further studies have to determine the pathophysiological role of the 5-HT system in MH, and whether ritanserin could be an alternative for treatment or prevention of halothane-induced MH.

A case of discordance between genotype and phenotype in a malignant hyperthermia family.

Malignant hyperthermia (MH) is an inherited autosomal dominant pharmacogenetic disorder and is the major cause of anaesthesia-induced death. Malignant hyperthermia susceptibility is usually diagnosed by the in vitro contracture test (IVCT) performed on fresh muscle biopsies exposed to caffeine and halothane, respectively. Around 50% of affected families are linked to the ryanodine receptor (RYR1) gene. The human RYR1 gene maps to chromosome 19q13.1 and encodes a protein that associates as a homotetramer and acts as a calcium-release channel from the sarcoplasmic reticulum. To date, 17 mutations have been identified in the coding region of the RYR1 gene and appear to be associated to the MH-susceptible phenotype. Here we describe a rare case of discordance between genotype (characterised by the presence of the Arg614Cys mutation in the RYR1 gene) and MH-normal typed phenotype. Although the IVCT remains a very reliable procedure for the assessment of MH status, genetic data can provide in some cases an additional aid to clinical diagnosis.

Evaluierung von Maligne-Hyperthermie-Episoden mit der Clinical Grading Scale

The Clinical Grading Scale (CGS) was introduced to predict malignant hyperthermia (MH) susceptibility in adverse anaesthetic events. Because many of the clinical symptoms that occur during MH episodes are nonspecific, the CGS was designed as a tool to estimate the qualitative likelihood of MH. The purpose of this study was to compare the results of the CGS with the established in vitro contracture test (IVCT). 92 patients with a personal history for MH were tested for MH susceptibility with the IVCT according to the protocol of the European MH Group. All patients were also evaluated with the CGS. Clinical indicators for the CGS are rigidity, muscle breakdown, respiratory acidosis, temperature increase and cardiac involvement. There are additional indicators in case of a family history for MH. For each indicator 3-15 points are added to build a raw score; this raw score corresponds to a MH rank in the CGS that describes the likelihood of MH in the suspected event. The higher the raw score rank, the higher the likelihood of MH and vice versa. From 92 patients, 32 (35%) were diagnosed as MH-susceptible (MHS) with the IVCT, 47 (51%) were MH-normal (MHN), and 13 (14%) were MH-equivocal (MHE). One patient with MH-rank 1 (MH almost never) in the CGS was diagnosed as MHS; on the other hand no patient with MH-rank 6 (MH almost certain) in the CGS was diagnosed as MHN. However, the majority of patients (72%) were assigned to ranks 3 and 4 (MH somewhat less than likely/MH somewhat greater than likely). The qualitative likelihood of MH could therefore not be clearly estimated. Our study shows that the MH-rank of the CGS corresponds poorly with the results of the IVCT. In any case the evaluation of an MH suspicious event depends on the availability of data of that event. It is often difficult to obtain sufficient data, especially if the event occurred a long while ago. In these cases the MH rank may underestimate the likelihood of MH susceptibility. On the other hand, overestimation is also possible because some of the scoring indicators depend on the anaesthesiologist's judgement only. At present, the use of the CGS is neither validated nor clinically feasible. The CGS cannot replace IVCT.

4-Chloro-m-cresol test--a possible supplementary test for diagnosis of malignant hyperthermia susceptibility.

In vitro contracture test (IVCT) for diagnosis of MH in our laboratory has a sensitivity of 100% and a specificity of 93%. The results are equivocal in 10-15%, and supplementary tests may thus be required. We have tested the hypothesis that 4-chloro-m-cresol (4-cmc) may be useful for a supplementary test. Muscle from 41 consecutive patients from 7 families undergoing diagnostic muscle biopsy with IVCT was exposed in vitro to increasing concentrations of 4-cmc (25, 50, 75, 100, 150, and 200 mumol l-1), and the force development recorded. Diagnosis of MH susceptibility was made with standard halothane and caffeine tests and included as results MHS (MH susceptible), MHN (MH negative), and MHE (equivoval result). At all concentrations of 4-cmc, the increase in baseline force was significantly greater in the MHS group compared to the MHN group (P < 0.05). Muscle from 15 MH-susceptible (MHS) patients responded to 4-cmc with increasing force at a threshold concentration of 75 mumol l-1 or less, whereas muscle from 23 MH-non-susceptible (MHN) patients had thresholds of 100 mumol l-1 or more. The accuracy of the chlorocresol test was thus 100% (95% confidence limits 90.75-100%) at a threshold of 75 mumol l-1. Amplitude of contractures at 2 mmol l-1 caffeine was not different from contractures at 75 mumol l-1 of 4-cmc in either the MHS or the MHN group (P > 0.05). In vivo concentrations of chlorocresol from clinical use of insulin and somatropin are estimated to be 20 times less than the threshold concentration and thus these drugs seem safe in MH patients. 4-chloro-m-cresol may be a suitable aid to clarify puzzling results of standard testing of MH susceptibility.

4-chloro-m-cresol-induced contractures of skeletal muscle specimen from patients at risk for malignant hyperthermia

4-chloro-m-cresol (4-CmC), commonly used as preservative, has been shown to induce contractures in skeletal muscle specimens from individuals susceptible to malignant hyperthermia (MH). It has been suggested that a defect of the calcium release channel of the skeletal muscle sarcoplasmic reticulum (ryanodine receptor) in MH susceptible (MHS) patients could be responsible for this phenomenon. 4-CmC was found to be a potent activator of ryanodine receptor-mediated Ca2+ release. The aim of this study was to determine the in vitro effects of 4-CmC on muscle specimens from MHS and normal (MHN) patients, and whether contracture testing with different concentrations of 4-CmC could result in a more precise discrimination between MHS and MHN. In this prospective study muscle biopsies were obtained from 40 patients with clinical suspicion of MH. The patients were first classified by the in vitro contracture test (IVCT) according to the European MH protocol. After MH classification, surplus muscle specimens were subjected to the 4-CmC study. Cumulative administration of 4-CmC (25, 50, 75, 100, 150, and 200 mumol/l) produced contractures in a concentration-dependent manner. However, contractures developed significantly earlier and were greater in MHS (n = 17) than in MHN specimens (n = 23). After bolus administration of 50, 75, and 100 mumol/l 14-CmC MHS specimens developed distinct muscle contractures. In contrast, in MHN specimens only 100 mumol/l 4-CmC produced contractures. All contracture levels following bolus administration of 100 mumol/l 4-CmC were attained significantly earlier in MHS than in MHN. There was no overlapping in the range of times between both groups. In vitro contracture testing with 4-CmC seems to be a specific method to distinguish between MHS and MHN patients. However, the question whether 4-CmC is an MH-triggering agent is not completely solved. 4-CmC is a preservative within a large number of commercially available preparations (e.g. insulin, hormones, etc.). Regarding the results of contracture testing with 4-CmC it has been suggested that 4-CmC possibly represents a high-risk agent for MHS individuals. To reduce the risk of MH in susceptible patients due to administration of chlorocresols, we recommend avoiding preparations containing the preservative 4-CmC.

Classification of malignant hyperthermia-equivocal patients by 4-chloro-M-cresol.

To clarify the contracture response to 4-chloro-m-cresol (4-CmC) in malignant hyperthermia (MH) equivocal (MHE) muscle, we studied the effect of cumulative concentrations of 4-CmC. In vitro contracture test (IVCT) was performed in 35 probands according to the European MH test protocol. Surplus muscle bundles were exposed to 4-CmC (25-200 micromol/L), maintaining each concentration for 4 and 8 min. After 4 min exposure, the contracture increase of MH susceptible (MHS) (n = 7) muscle specimens was significantly (P = 0.05) greater at 50 micromol/L compared with either MHE halothane sensitive (MHEh) (n = 13) or MH normal (MHN) (n = 15) classified patients. Statistically significant differences (P < 0.05) were also found at 75 micromol/L. Exposure for 8 min yielded significant differences at 50 micromol/L only between MHS and MHEh. MHEh muscles revealed a dose-response curve similar to that found in MHN specimens. MHS muscles showed a significantly higher sensitivity to 4-CmC than either MHEh or MHN, and, in the probands tested so far, MHEh and MHN muscles seem to identically respond to 4-CmC, which seems to indicate a normal response in MHEh probands, implying no MH susceptibility. Therefore, 4-CmC might reduce the frequency of MHEh diagnosis based on standard halothane-caffeine IVCT. However, since MHE individuals may also represent an aberrant genetic status, with MH causing defects linked to unknown mutations, it is premature to consider 4-CmC as a solution to the diagnostic uncertainty of the true status of MHE probands. Presently, 4-CmC may provide supplementary information for a more precise phenotypic categorization of MHE individuals.

Results of in vitro contracture tests for the diagnosis of malignant hyperthermia susceptibility in monozygote twins.

Malignant hyperthermia susceptibility is a pharmacogenetic disorder in which susceptible individuals may develop a potentially life-threatening hypermetabolism when exposed to certain anaesthetic agents. The most common diagnostic method is the in vitro contracture test (IVCT) of skeletal muscle biopsies. There is a wide variation in the size of contractures between susceptible individuals and the reproducibility of the test in humans has not been evaluated. We have performed the IVCT in 4 monozygote pairs of twins, which gave us on opportunity to study the reproducibility. The clinical diagnoses were consistent in all twin pairs, although slight differences in contractures and thresholds were seen. In this material the reproducibility of the IVCT was found to be satisfactory.

Inositol 1,4,5-trisphosphate in blood and skeletal muscle in human malignant hyperthermia

The in vitro contracture test (IVCT) is the only available diagnostic method at present for evaluation of malignant hyperthermia (MH) susceptibility. However, the disadvantage of the IVCT is that it is invasive. Several studies suggest that an altered inositol phosphate system is involved in the development of MH. A greater concentration of inositol 1,4,5-trisphosphate (1,4,5-IP3) was found in MH susceptible (MHS) than in normal (MHN) skeletal muscles. In this study the concentrations of 1,4,5-IP3 in blood samples and skeletal muscle specimens of identical patients were measured in an attempt to define susceptibility to MH. Muscle biopsies were obtained from 34 patients with clinical suspicion of MH. Patients were first classified as MHS (n = 19), MHN (n = 8) or MH equivocal (MHE; n = 7) by the standard IVCT. For detection of 1,4,5-IP3 concentrations, blood samples were obtained and an additional muscle specimen was excised. After sample preparation, concentrations of 1,4,5-IP3 were measured using radioimmunoassay. In blood samples, concentrations of 1,4,5-IP3 were similar in all individuals tested for MH susceptibility and in control patients not tested for MH susceptibility (n = 44). In skeletal muscle, 1,4,5-IP3 concentrations were significantly higher in MHS than in MHE or MHN patients, respectively. Each MHS sample contained more 1,4,5-IP3 than the highest concentration measured in MHN muscle. Defining arbitrary thresholds for 1,4,5-IP3 concentration in skeletal muscles in order to discriminate between MHS and MHN status, it was possible to assign three MHE patients to MHS and four to MHN. This study supports the hypothesis that an altered inositol phosphate system might be involved in MH. However, measurement of 1,4,5-IP3 concentration in a simple blood sample preparation is not reliable for MH susceptibility screening.