COMPARISON OF IN VITRO CONTRACTURE TESTING WITH RYANODINE, HALOTHANE AND CAFFEINE IN MALIGNANT HYPERTHERMIA AND OTHER NEUROMUSCULAR DISORDERS

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1 British Journal of Anaesthesia 99; 7: 97- COMPARISON OF IN VITRO CONTRACTURE TESTING WITH RYANODINE, HALOTHANE AND CAFFEINE IN MALIGNANT HYPERTHERMIA AND OTHER NEUROMUSCULAR DISORDERS P. M. HOPKINS, F. R. ELLIS AND P. J. HALSALL SUMMARY In vitro exposure of living skeletal muscle to ryanodine has been proposed as a potentially specific test for malignant hyperthermia (MH). In this study we have compared in vitro contracture responses to halothane, caffeine and ryanodine in skeletal muscle specimens obtained from patients attending for diuynosis of susceptibility of MH and also from six patients having muscle biopsy for diagnosis of other neuromuscular disorders. A/though the ryanodine contracture test was not specific for MH, the results suggest it may greatly aid (in conjunction with the standard halothane and caffeine contracture tests) the accurate phenotyping of individuals that is essential for the further genetic analysis of MH. (Br. J. Anaesth. 99; 7: 97-) KEY WORDS Hyperthermia.malignant in vitro test. Pharmacology, ryanodine. Both of the two widely used procedures for the diagnosis of malignant hyperthermia (MH) susceptibility using in vitro contracture tests (IVCT) with living skeletal muscle [,] use separate exposure to halothane and to caffeine. The regimen of the North American MH Group also allows for a combined caffeine-halothane test [], but recently this has been invalidated because of a high incidence of false positive results [, ]. The separate-exposure halothane and caffeine tests also have imperfections. Results of IVCT performed in patients with a low risk of MH ("controls") suggest a false positive rate of approximately % using the European procedure [] and 9 % using the North American procedure []. In our series of more than tests, % of patients with an abnormal response to halothane reacted normally to caffeine, indicating the caffeine test lacks total sensitivity. Both procedures are associated also with lack of specificity, in that muscle from patients with myopathies other than MHS develop abnormal contracture responses [6, 7]. It was for these reasons, and with the need for the most accurate phenotyping of MH susceptibility for successful genetic analysis of the condition, that we investigated the potential of the plant alkaloid, ryanodine, for use in IVCT. The early results of our ryanodine contracture test were encouraging and were presented as a preliminary report [8]. We now present results from patients attending for diagnosis of their MH status, on whose muscle IVCT have been done using halothane, caffeine and ryanodine. In a similar way, we also investigated muscle specimens from six patients with other neuromuscular disorders. PATIENTS AND METHODS Patients uluriding for irrvcstigation of MH status Muscle specimens were obtained by open biopsy of the vastus medialis under regional anaesthesia ( in block) or general anaesthesia (thiopentone, fentanyl and nitrous oxide in oxygen) and maintained and used for diagnostic IVCT according to the European MH Group procedure []. MH status was assigned in the usual way using suitably viable specimens (twitch response > g): abnormal responses to halothane (>. g contracture with ^ % halothane) and caffeine (>.g contracture with caffeine < mmol litre" ) indicates MH susceptible (MHS); abnormal response to only one of either halothane or caffeine indicates MH equivocal (MHE); normal responses to both drugs indicate non-susceptibility (MHN). Any muscle fascicle surplus to diagnostic requirements was used for a ryanodine contracture test (RCT). The first 6 of these were performed as described previously [8] with ryanodine added in increments at -min intervals to achieve muscle bath concentrations of.,.8,.6 and nmol litre" (RCT version ). For the remaining 9 patients, additional ryanodine concentrations of. and 6. umol litre" were included (RCT version ). The RCT was modified in an attempt to produce a clear distinction in ryanodine threshold concentration between MHS and MHN groups. Initially, the threshold value of ryanodine was determined as that concentration of ryanodine at which a contracture of. g developed. This was soon changed to the concentration of ryanodine at which a sustained contracture commenced. The results presented here use this later definition for threshold value of ryanodine. For P.M.HOPKINS, M.B., B.S., F.R.C.ANAES.; F.R.ELLIS, PH.D., F.R.C.ANAES. ; P. J. HALSALL, M.B., CH.B. ; Malignant Hyperthermia Investigation Unit, University Department of Clinical Medicine (Anaesthesia), St James's University Hospital, Leeds LS9 7TF. Accepted for Publication: September 7, 99. Downloaded from at Pennsylvania State University on March 6, 6

2 98 BRITISH JOURNAL OF ANAESTHESIA RCT: MHS + MHE 8 CD E Z RCT:MHN Ryanodine threshold concn (umol litre" ) Ryanodine threshold time (min) Ryanodine threshold concn ( imol litre" ) Rvanodine threshold time (min) FIG.. Ryanodine thresholds using RCT version for MHS ( ) and MHE () patients and for MHN patients. Ryanodine was added in increments at -min intervals to achieve muscle bath concentrations of.,.8,.6 and umol litre". The threshold value of ryanodine was determined as that concentration of ryanodine at which a sustained contracture commenced. If no contracture had developed within min of exposure to ryanodine umol litre", this concentration was maintained until a contracture did develop and the contracture threshold expressed as the time (in minutes) after exposure to ryanodine umol litre". Downloaded from at Pennsylvania State University on March 6, 6 version of the RCT, where no contracture had developed within min of exposure to ryanodine imol litre", this concentration was maintained until a contracture did develop and the contracture threshold expressed as the time (in minutes) after exposure to ryanodine umol litre". Patients with other neuromnscular disorders Six patients attending for muscle biopsy for histological diagnosis of a clinical neuromuscular disease were included. Surplus tissue was used for halothane, caffeine and ryanodine contracture tests that were performed as for the MH patients. The ryanodine used (ryanodine dehydroryanodine, 98 % pure) was obtained from Agri Systems Chemicals (Philadelphia, U.S.A.). RESULTS Patients attending for investigation of MH status RCT version (fig. ). Of the 6 patients investigated, 7 were classified as MHS, as MHN and six as MHE. All the MHS muscle fascicles developed a contracture within min of exposure to the greatest concentration of ryanodine ( (imol litre" ). No MHN fascicle developed a contracture by this stage, the earliest contractures occurring after 7 min of ryanodine (imol litre". The MHE fascicles had lower thresholds than the MHN fascicles. RCT version. Results of the RCT version of 6 MHS, MHN and MHE patients are shown in figure. The one MHS fascicle with a ryanodine

3 RYANODINE CONTRACTURE TEST FOR MH 99 RCT: MHS RCT: MHN RCT: MHE Ryanodine threshold concn (nmol litre" ) Ryanodine threshold concn ((irnol litre" ) Downloaded from at Pennsylvania State University on March 6, 6 Ryanodine threshold concn ( imol litre" ) FIG.. Ryanodine thresholds using RCT version for MHS patients, MHN patients and MHE patients. Ryanodine was added in increments at -min intervals to achieve muscle bath concentrations of.,.8,.6,., 6. and junol litre". The threshold value of ryanodine was determined as that concentration of ryanodine at which a sustained contracture commenced.

4 BRITISH JOURNAL OF ANAESTHESIA TABLE I. Results of halotharu, caffeine and ryanodine (version ) contracture tests in individual MHE patients. Halothane test; threshold concentration of halothane and maximum force of contracture obtained at % halothane in the static test; DH = abnormal response to only the dynamic halothane test. For description of static and dynamic tests see [/]. * Poorly viable specimen (twitch response < g), the result from which should be interpreted with caution MHE Threshold concn (%). Halothane Maximum contracture (g) DH DH Threshold concentration Caffeine (mmol litre" ) > > Ryanodine (umol litre-') * * threshold greater than. umol litre" was poorly viable (twitch response =. g). The halothane, caffeine and ryanodine thresholds for the important group of patients classified as MHE are shown in table I. The relationships between the thresholds in individual patients for halothane, caffeine and ryanodine (version ) contracture tests (HCT, CCT and RCT respectively), as indicated by the Spearman rank correlation coefficient (rs) were: HCT vs RCT, rs =.77 (P <.); CCT vs RCT, rs =.76 (P <.); HCT vs CCT, rs =.787 (P <.). Patients with other neuromuscular disorders Histological diagnosis and details of results of halothane, caffeine and ryanodine contracture tests are shown in table II. There was insufficient muscle for a caffeine contracture test in one patient. DISCUSSION The results of the diagnostic investigations for MH susceptibility in the patients studied here are typical for our laboratory, with % classified as MHS, % MHE and % MHN. When the European MH Group procedure was agreed, there was a consensus that it was far more important, from a clinical perspective, to avoid false negative rather than false positive diagnoses. As one of the purposes of this process was to enable validation of results and research between European laboratories, it was necessary to set criteria for recognition of true MH positive and true MH negative patients that became, respectively, the MHS and MHN categories. It was perhaps unfortunate, and the cause of considerable confusion since, that those patients not meeting the criteria for MHS or MHN were labelled "equivocal". While this group of patients, probably, does include those with false positive results (true equivocals), the European Group's study of low risk patients indicated a false positive rate of about % []. This suggests that the majority of the MHE patients are susceptible to MH. In our laboratory, all MHE patients respond abnormally to halothane and it is our contention that this large MHE group is, therefore, a reflection of the lack of sensitivity of the caeffeine contracture test rather than a lack of specificity of the halothane contracture test. It should be noted, however, that in some centres MHE status results from an abnormal caffeine response with normal halothane response. However, these points were only of academic importance with respect to the original goals of the European MH Group. Clinically, MHE patients usually were (and still are) regarded as susceptible to MH, but only MHS and MHN patients are entered in research programmes. Since 98 there have been major and rapid advances in the field of molecular biology that have resulted in the real prospect of identifying the gene defect(s) responsible for MH. The nature of the required linkage studies was described elegantly by Levitt and colleagues [9], but the critical point is that their accuracy is dependent TABLE II. Halothane, caffetne and ryanodine contracture test results in six patients with various neuromuscular disorders. RCT version was used for patients Nos ; RCT version used for patients Nos and 6. The threshold value of ryanodine was determined as that concentration ofryanodine at which a sustained contracture commenced. If no contracture had developed within rm'n of exposure to ryanodine fmwl litre'', this concentration was maintained until a contracture did develop and the contracture threshold expressed as the time (in minutes) after exposure to ryanodine fmwl litre"' Downloaded from at Pennsylvania State University on March 6, 6 Contracture (g) Patient No. Diagnosis Halothane (force at %) Caffeine (force at mmol litre"') Ryanodine threshold Concn (umol litre-') Time (min) 6 Motor neurone Facioscapulohumeral dystrophy Type lib fibre atrophy Acute myositis Acute denervation Polymyositis

5 RYANODINE CONTRACTURE TEST FOR MH entirely on the accurate phenotyping of as many individuals as possible from MH families by IVCT. For these studies it is essential, therefore, that the numbers of false positive, false negative and doubtful IVCT diagnoses are minimized. To achieve this, it is clear that the MHE classification must be redressed. Experience with the European MH Group procedure has demonstrated that positive results in two distinct tests increase the statistical confidence of the diagnosis of susceptibility to MH. This principle logically might be extended by the addition of a third test. Examination of the results obtained using RCT version indicates that this warrants consideration for use as such a third test. If a ryanodine threshold of. umol litre" or less is taken as an abnormal result, the RCT successfully identified the MHS patients (excluding the result obtained with poorly viable muscle). Furthermore, if one assumes that the MHN group are true negatives (which is highly likely), the RCT has a false positive rate of.8% (99 % confidence interval of -. % to. %). The most exciting aspect of the RCT, however, is that it appears to identify two populations within the MHE group, which suggests it does have the potential to distinguish positive and negative patients within this group. From this study, it is possible to conclude the following about individual MHE patients: a ryanodine threshold of.6 umol litre" indicates MH susceptibility with near certainty; a ryanodine threshold of. umol litre" indicates a probability of at least 8 % of MH susceptibility; a ryanodine threshold of umol litre" indicates almost certainly non-susceptibility to MH. Although no MHS patient (with viable specimen) had a ryanodine threshold greater than. umol litre", an insufficient number of MHS patients has been studied to permit the conclusion that MHE patients with a ryanodine threshold of 6. umol litre" are not susceptible. However, the lower limit of the 99% confidence interval for the probability that patients with a ryanodine threshold of 6. umol litre" are not susceptible is 8%. MacKenzie and colleagues [] have suggested that the threshold force of contracture for the halothane contracture test is too low. From the results shown in table I, it can be seen that it would be unwise to increase the threshold force of contracture at % halothane to more than. g as, of the five patients with muscle reaching no greater force, three had a ryanodine threshold of. umol litre", while one had a normal response to ryanodine (threshold of umol litre" ). The specimen from the fifth such patient for the RCT was poorly viable. Of course, if must be noted that the RCT may be unreliable with poorly viable specimens and we would recommend that, as stipulated in the European MH Group procedure for halothane and caeffeine tests, muscle specimens must have a twitch response of at least g. The IVCT results from muscle of patients with other neuromuscular disorders (table II) confirm earlier studies [6, 7] which demonstrated that neither halothane or caffeine contracture tests are specific for MH. The present results also show that the RCT is also not specific for MH. That the most abnormal response with the RCT was seen in denervated muscle suggests the RCT is able to detect some forms of diseased muscle (primary and secondary conditions), rather than only those caused by an abnormal ryanodine receptor protein (sarcoplasmic reticulum calcium release channel). Results of the RCT, therefore, are consistent with the hypothesis that MH is caused by an abnormal ryanodine receptor protein gene [, ], but equally consistent with MH being a genetically heterogeneous condition []. ACKNOWLEDGEMENT We thank Mr P. L. Allam and Ms A. Clough for technical assistance. REFERENCES. European Malignant Hyperpyrexia Group. A protocol for the investigation of malignant hyperpyrexia (MH) susceptibility. British Journal of Anaesthesia 98; 6: Larach MG, for the North American Malignant Hyperthermia Group. Standardization of the caffeine halothane muscle contracture test. Anesthesia and Analgesia 989; 69: -.. Larach MG, Landis JR, Bunn JS, Diaz M, The North American Malignant Hyperthermia Registry. Prediction of malignant hyperthermia susceptibility in low-risk subjects. Antsthesiology 99; 76: Ellis FR, Halsall PJ, Hopkins PM. Is the "K type" halothane-caffeine responder susceptible to malignant hyperthermia? British Journal of Anaesthesia 99; 69: OrdingH, for the European Malignant Hyperthermia Group. Experience with the European protocol for contracture testing. Journal of the Neurological Sciences 99; 98S:. 6. Lehmann-Horn F, Iaizzo PA. Are myotonias and periodic paralyses associated with susceptibility to malignant hyperthermia? British Journal of Anaesthesia 99; 6: Heiman-Patterson T, Rosenberg H, Fletcher JE, Tahmoush AJ. Halothane-caffeine contracture testing in neuromuscular diseases. Muscle and Nerve 988; : ^7. 8. Hopkins PM, Ellis FR, Halsall PJ. Ryanodine contracture: a potentially specific in vitro diagnostic test for malignant hyperthermia. British Journal of Anaesthesia 99; 66: Levin RC, Meyers D, Fletcher JE, Rosenberg H. Molecular genetics and malignant hyperthermia. Anesthesiology 99; 7: -.. MacKenzie AE, Allen G, Lahey D, Crossan M, Nolan K, Mertler G, Worton RG, MacLennan DH, Korneluk R. A comparison of the caffeine halothane muscle contracture test with the molecular genetic diagnosis of malignant hyperthermia. Anesthesiology 99; 7: -8.. MacLennan DH, Duff C, Zorzato F, Fujii J, Phillips M, Korneluk RG, Frodis W, Britt BA, Worton RG. Ryanodine receptor gene is a candidate for predisposition to malignant hyperthermia. Nature (London) 99; : Healy JMS, Heffron JJA, Lehane M, Bradley DG, Johnson K, McCarthy TV. Diagnosis of susceptibility to malignant hyperthermia with flanking DNA markers. British Medical Journal 99; : -8.. Ball SP, Dorkins H, Ellis FR, Hall JL, Halsall PJ, Hopkins PM, Stewart AD. Is muscle biopsy for malignant hyperthermia diagnosis still necessary? British Journal of Anaesthesia 99; 69: P-P. Downloaded from at Pennsylvania State University on March 6, 6