[Malignant hyperthermia. General, clinical and experimental aspects].
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Animals↗
Biomedical subjects
M Steinfath
Publications and source records attributed to M Steinfath.
Automated image analysis for array hybridization experiments.
MOTIVATION: Image analysis is a major part of data evaluation for array hybridization experiments in molecular biology. The program presented here is designed to analyze automatically images from hybridization experiments with various arrangements: different kinds of probes (oligonucleotides or complex probes), different supports (nylon filters or glass slides), different labeling of probes (radioactively or fluorescently). The program is currently applied to oligonucleotide fingerprinting projects and complex hybridizations. The only precondition for the use of the program is that the targets are arrayed in a grid, which can be approximately transformed to an orthogonal equidistant grid by a projective mapping. RESULTS: We demonstrate that our program can cope with the following problems: global distortion of the grid, missing of grid nodes, local deviation of the spot from its specified grid position. This is checked by different quality measures. The image analysis of oligonucleotide fingerprint experiments on an entire genetic library is used, in clustering procedures, to group related clones together. The results show that the program yields automatically generated high quality input data for follow up analysis such as clustering procedures. AVAILABILITY: The executable files will be available upon request for academics.
Algorithms↗
Evidence for susceptibility to malignant hyperthermia in patients with exercise-induced rhabdomyolysis.
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
Adolescent↗
New tools for oligonucleotide fingerprinting.
Oligonucleotide fingerprinting is an attractive, high-throughput complement to tag sequencing methods to determine the spectrum and abundance of genes in cDNA libraries. This method currently relies on the sequential hybridizations of short, radioactively labeled DNA oligonucleotides to clone arrays. Here, we describe a new environment that substantially improves this technology. Fluorescently labeled peptide nucleic acid (PNA) oligonucleotides are used as hybridization probes. Hybridization results are recorded with a large-field, high-resolution laser scanner developed for this purpose. Automated image analysis allows easy handling of large numbers of hybridization images. Signal interference effects, which limit the gridding density in the radioactive approach, are strongly reduced. The sensitivity of the fluorescence detection demonstrated permits the convenient use of nylon membranes. Hybridization data quality is improved, and its generation is substantially accelerated, simplified, and less expensive.
Autoanalysis↗
Information theoretical probe selection for hybridisation experiments.
MOTIVATION: The choice of probes is an important feature of hybridisation experiments. In this paper we present an algorithm that optimises probes with respect to a training set of sequences based on Shannon entropy as a quality criterion. The practical motivation for our algorithm is oligonucleotide fingerprinting, a method for the simultaneous identification of sequences (cDNA or genomic DNA) by their hybridisation tags according to a set of short probes such as octamers, although the algorithm is of course not restricted to that application. RESULTS: We can show that our method is superior to the selection of probes according to their frequencies, which is a widely used strategy, and to randomly chosen probe sets. The quality of probe sets is assessed by a simulation pipeline that entails the set of probes as a simulation parameter. The performance of probe sets trained on sequences from different organisms shows additionally that probes should be chosen with regard to the organism under analysis. Case studies are presented on how constraints (G+C-content, complexity of the individual probes) influence the selection process. AVAILABILITY: A description of the oligonucleotide fingerprinting pipeline is published on our web-page http://www.molgen.mpg.de/ approximately ag_onf/met.htm. An executable of the algorithm and probe lists designed for human and rodents can be downloaded from the ftp-site ftp://ftp.molgen.mpg.de/pub/mpimg/probe_design/.
Algorithms↗
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.
Adolescent↗
[Interactions of intravenous anesthetics with cerebral alpha-2-adrenoceptors].
OBJECTIVE: Up to date the exact mechanisms of action of anaesthetics on structures of the central nervous system have not been elucidated. Agents acting on central alpha 2-adrenoceptors have been demonstrated to possess potent sedative properties. Beside the classical agonists, general anaesthetics that are currently in clinical use e.g. etomidate have also been shown to act on alpha 2-adrenoceptors. Thus, the aim of this study was to study the effect of the intravenous anaesthetic agents propofol and ketamine on the binding of the specific alpha 2-adrenoceptor agonist paraiodoclonidine at cerebral alpha 2 adrenoceptors. METHODS: After approval of the local animal care committee brain tissue of six Wistar rats was removed and frozen at -80 degrees C. The tissue was sectioned in 15-20 microns thick slices. After washing in TRIS-buffer (pH = 7.5) the slices were incubated with 2.5 nM 125I-paraiodoclonidine (PIC) for determination of specific binding. Unspecific binding was determined in presence of 200 microM unlabeled clonidine. Displacement experiments were performed in presence of propofol (11-530 microM) and ketamine (52-1100 microM) and evaluated autoradiographically. Average exposure time was three days. All films were then analysed by densitometry. Statistical significance calculated by Student's t-test was assumed at a level of p < 0.05. RESULTS: Only at the highest concentration of propofol and ketamine an effect on PIC binding was noticeable. Neither propofol nor ketamine was able to displace PIC completely from cerebral alpha 2-adrenoceptors. CONCLUSION: In contrast to other anaesthetics such as etomidate which was demonstrated to displace PIC completely from alpha 2-adrenoceptors, propofol and ketamine exerted only minor effects on the binding of PIC even at concentrations that exceeded clinical concentrations. These data suggest that an additional site of action of these anaesthetics at cerebral alpha 2-adrenoceptors beside the known actions on GABA receptors or NMDA receptors is unlikely.
Adrenergic alpha-Agonists↗
Preselection of shotgun clones by oligonucleotide fingerprinting: an efficient and high throughput strategy to reduce redundancy in large-scale sequencing projects.
Large-scale genomic sequencing projects generally rely on random sequencing of shotgun clones, followed by different gap closing strategies. To reduce the overall effort and cost of those projects and to accelerate the sequencing throughput, we have developed an efficient, high throughput oligonucleotide fingerprinting protocol to select optimal shotgun clone sets prior to sequencing. Both computer simulations and experimental results, obtained from five PAC-derived shotgun libraries spanning 535 kb of the 17p11.2 region of the human genome, demonstrate that at least a 2-fold reduction in the number of sequence reads required to sequence an individual genomic clone (cosmid, PAC, etc.) can be achieved. Treatment of clone contigs with significant clone overlaps will allow an even greater reduction.
Chromosomes, Human, Pair 17↗
Depletion of endogenous dopamine stores and shift in beta-adrenoceptor subtypes in cardiac tissue following five weeks of chronic denervation.
Surgical ablation of extrinsic cardiac nerve fibers results in a chronically denervated state of the left ventricle of the heart. The present study was performed to elucidate the effect of a period of 5 weeks of chronic denervation on cardiac catecholamine levels in general and dopamine in particular. Moreover, the possible effect on cardiac beta-adrenoceptor subtypes was investigated. Experiments were performed on adult dogs. In addition to adrenaline and noradrenaline the tissue levels of dopamine were found to be severely depressed. A significant shift from beta1- to beta2-adrenoceptor subtype was observed, while the total beta-adrenoceptor density remained unaffected. The present findings indicate that catecholamine synthesis in chronically denervated hearts is impaired upstream of dopamine and that a shift in beta-adrenoceptor subtype occurs already within a relatively short period of five weeks of denervation, and suggest that the lack of endogenous catecholamines influence the relative expression levels of the two subtypes of beta-adrenoceptors present in cardiac tissue.
Animals↗
Tropisetron or ondansetron compared with placebo for prevention of postoperative nausea and vomiting.
In a prospective, randomized, double-blind, placebo-controlled, multicentre study, the efficacy of prophylactic tropisetron (2 mg) or ondansetron (4 mg) for the prevention of post-operative nausea and vomiting after abdominal or non-abdominal surgery with general balanced anaesthesia was studied in 842 ASA I-III patients. In patients undergoing abdominal surgery, ondansetron and tropisetron reduced the frequency of emetic episodes compared with the placebo (29%, 30% vs. 42% respectively). In men, neither tropisetron nor ondansetron had an effect different from the placebo, whereas in women both drugs led to lower rates of emetic episodes and nausea. In comparison with abdominal surgery, fewer patients in the non-abdominal surgery subgroup had emetic episodes (42% vs. 23% in the placebo group). However, neither tropisetron nor ondansetron was significantly different from the placebo in this patient subgroup. In conclusion, for patients at increased risk of post-operative nausea and vomiting, a prophylactic therapy at the lowest effective dose with tropisetron or ondansetron may be useful.
Abdomen↗
Ritanserin attenuates the in vitro effects of the 5-HT2 receptor agonist DOI on skeletal muscles from malignant hyperthermia-susceptible patients.
STUDY OBJECTIVES: To study the in vitro effects of the serotonin2 (5-HT2) receptor agonist 1-(2.5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) in skeletal muscle specimens from malignant hyperthermia-susceptible (MHS) and normal (MHN) patients following pretreatment with the 5-HT2 receptor antagonist ritanserin. DESIGN: Prospective study. SETTING: Malignant hyperthermia (MH) laboratory at a university hospital. PATIENTS: 41 patients undergoing in vitro contracture test for diagnosis of MH susceptibility. INTERVENTIONS: Skeletal muscle biopsies in adult patients were performed with a 3-in-1 nerve block with 40 ml prilocaine 1%. In children, general anesthesia was induced with 50 micrograms/kg alfentanil intravenously (i.v.) and 2 to 2.5 micrograms/kg propofol i.v. and maintained with a continuous infusion of propofol (< or = 150 micrograms/kg/min) and nitrous oxide (66%) in oxygen. MEASUREMENTS AND MAIN RESULTS: Patients were first classified as MHS or MHN by the in vitro contracture test according to the European MH protocol. Surplus muscle specimens of 21 MHS and 20 MHN patients were used in this study. At first, DOI was added to the organ bath at a concentration of 0.02 mM. In the second part of the study, muscle specimens were preincubated with ritanserin 0.01 mM for 10 minutes before DOI 0.02 mM was added to the bath. Muscle specimens from all patients developed contractures after administration of DOI. The onset of contractures was significantly faster in MHS muscles, and the magnitude of contracture was significantly greater than in MHN. The muscle twitch decreased significantly in both groups after DOI. After pretreatment with ritanserin, start of contracture was significantly delayed in MHS muscles. MHN muscles failed to develop contractures. The maximum level of contracture was significantly reduced in MHS. Muscle twitch decreased also in both MHS and MHN groups. CONCLUSIONS: The findings may indicate that stimulation of 5-HT2 receptors is involved in MH induction. Furthermore, 5-HT2 receptor antagonists could possibly be effective in preventing MH. Additional studies are required to determine if administration of 5-HT2 receptor antagonists could be of additional value in the treatment or prevention of anesthetic-induced MH.
Adolescent↗
[4-chloro-m-cresol-induced contractures of skeletal muscle specimen from patients at risk for malignant hyperthermia].
PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Adolescent↗
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.
Adolescent↗
Late intraoperative clonidine administration prevents postanesthetic shivering after total intravenous or volatile anesthesia.
Postoperative administration of clonidine is an effective treatment for shivering. However, the ability of this drug to stop postanesthetic shivering when administered intraoperatively remains controversial. Furthermore, the relative efficacy of clonidine during isoflurane and propofol anesthesia remains unknown. We therefore evaluated the incidence of postanesthetic shivering in patients given clonidine during nitrous oxide/isoflurane or propofol anesthesia. Because clonidine is an analgesic, we also evaluated postoperative pain and analgesic requirements. We studied 60 patients undergoing elective ear or nose surgery. General anesthesia was induced with 2.0 mg/kg propofol, 1.5 micrograms/kg fentanyl, and 0.1 mg/kg vecuronium. General anesthesia was maintained with isoflurane and 70% nitrous oxide in one group of patients; in the other, a continuous infusion of propofol (8 mg.kg-1.h-1) was administered (without nitrous oxide). Five minutes before tracheal extubation, patients in each group were randomly assigned to receive saline, placebo, or 3 micrograms/kg clonidine intravenously. Postanesthetic shivering was evaluated by a blind investigator. Postoperative pain was assessed using a visual analog scale. Postoperative shivering was observed in 53% of the patients given isoflurane without clonidine and in 13% of the patients given propofol without clonidine. No patient given clonidine shivered. Clonidine administration significantly reduced postoperative pain. The incidence of postanesthetic shivering was significantly less after propofol anesthesia than after isoflurane/nitrous oxide anesthesia. However, a late intraoperative bolus administration of 3 micrograms/kg clonidine prevents postoperative shivering in patients given either type of anesthesia.
Adult↗
[The pharmacologic basis of postoperative pain therapy. Epidural opioid administration].
The administration of epidural opioids is alternatively used in the management of postoperative analgesia. However, the administration is associated with side effects, including respiratory depression, somnolence and pruritus. A rational opioid selection between the hydrophilic and lipophilic opioids morphine, hydromorphone, alfentanil, fentanyl and sufentanil is discussed in this mini-review. Thus, the administration of the lipophilic opioid sufentanil might has some advantages. Notwithstanding, epidural opioid administration alone offers no marked clinical advantages compared to the intravenous route. In future, reduced doses of lipophilic opioids and local anaesthetics like bupivacaine 0.05-0.1% may provide benefits over the use of either drug alone and may offer marked clinical advantages over the intravenous route of opioids alone. The same holds true for alpha 2-adrenoceptor agonists as adjuvants. However, multicenter dose-ranging studies are necessary to determine both the ideal concentrations of the drug combinations and the general outcome. Moreover, we must also determine cost effectiveness for our postoperative analgesic techniques.
Analgesia, Epidural↗
Chronic treatment with carbachol sensitizes the myocardium to cAMP-induced arrhythmia.
BACKGROUND: The present study investigated biochemical and functional consequences of chronic activation of the inhibitory Gi alpha-coupled adenylyl cyclase pathway in the heart. METHODS AND RESULTS: Rats (220 to 260 g) were treated with 4-day infusions of the M-cholinoceptor agonist carbachol (9.6 mg/kg per day) or vehicle. An additional group that received the beta-adrenoceptor agonist isoprenaline (2.4 mg/kg per day) served as control. The main finding was that chronic infusion of carbachol led to a marked increase in isoprenaline- or forskolin-induced arrhythmia in electrically driven papillary muscles (in vitro). Compared with control, the potency of isoprenaline and forskolin to induce arrhythmia in cardiac preparations from carbachol-treated rats was increased 36- and 2.2-fold and the efficacy was increased 7.3- and 2.3-fold, respectively. The potency of carbachol to antagonize the isoprenaline- and forskolin-induced arrhythmia was decreased 30-fold. These changes were accompanied by a decrease in left ventricular M-cholinoceptor density by 15% (P < .05) and a decrease in pertussis toxin-sensitive G proteins (Gi alpha) by 26% (P < .05) without a decrease in the corresponding mRNAs. beta-Adrenoceptor density and basal and stimulated adenylyl cyclase activity remained unchanged. In contrast, isoprenaline infusion induced a decrease in arrhythmogenic potency of forskolin (P = NS), which was accompanied by a decrease in beta-adrenoceptor density, an increase in Gi alpha protein and mRNA levels, and a decrease in basal and stimulated adenylyl cyclase activity. CONCLUSIONS: Chronic parasympathetic activation sensitizes the myocardium to cAMP-induced arrhythmia. These changes may be due to quantitative alterations in functional Gi alpha.
Adenylate Cyclase Toxin↗
Effects of metoprolol on myocardial beta-adrenoceptors and Gi alpha-proteins in patients with congestive heart failure.
OBJECTIVE: In human heart failure downregulation of beta-adrenoceptors and upregulation of Gi-protein alpha-subunits (Gi alpha) results desensitization of the myocardial beta-adrenergic signal transduction pathway and reduced positive inotropic effects of catecholamines. Metoprolol treatment has been shown to restore the reduced beta-adrenoceptor density in dilated cardiomyopathy. The main objective of the present study was to investigate whether metoprolol also decreases the elevated inhibitory Gi alpha levels in patients suffering from congestive heart failure. METHODS: Total Gi alpha was determined by pertussis toxin-catalysed ADP ribosylation and beta 1- and beta 2-adrenoceptor densities by radioligand binding in right ventricular myocardial biopsies of 18 patients with dilated or ischaemic cardiomyopathy (NYHA II-IV) before and after 3 months of therapy. Nine controls were treated with conventional therapy only [diuretics, digitalis, nitrates, angiotensin-converting enzyme (ACE) inhibitors], and nine received the beta 1-selective blocker metoprolol in addition (mean 98 +/- 12 mg daily). RESULTS: In biopsies from patients treated with metoprolol, Gi alpha significantly decreased to 74% of predrug value and total beta-adrenoceptor increased by a selective increase in beta 1-adrenoceptors (44.7 vs 34.0 fmol mg-1 protein). These effects were accompanied by significantly increased oxygen uptake at the anaerobic threshold (8.65 vs 6.95 ml . kg-1 . min-1). In the control group no significant changes in biochemical and clinical parameters occurred. CONCLUSION: Metoprolol partly reverses Gi alpha-upregulation and beta-adrenoceptor downregulation in heart failure, which might contribute to the clinical improvement of patients treated with beta-blockers.
Adrenergic beta-Antagonists↗