Introduction
Malignant hyperthermia, a potentially lethal hypermetabolic syndrome of skeletal muscle, may be induced in susceptible individuals by volatile anaesthetics or depolarizing muscle relaxants. Enhanced intracellular Ca2+ release in the course of a malignant hyperthermia episode alters mitochondrial energy turnover leading to a massive increase in oxygen consumption and overwhelming production of carbon dioxide and heat and finally to lactic acidosis [1,2] [3,4]
Recently, a minimally invasive metabolic test was proposed to differentiate between malignant hyperthermia susceptible (MHS) and malignant hyperthermia nonsusceptible (MHN) individuals [5–7]
The present pilot study was designed to test a new approach to trigger application by a multiply perforated microtubing catheter and a rapid injection procedure in order to reach a homogeneous trigger distribution and a maximum concentration in the interstitial tissue of the skeletal muscle. We hypothesized that a more simplified investigation protocol with rapid bolus injection of 80 mmol l−1 caffeine and 10 vol% halothane dissolved in soybean oil sufficiently differentiates between MHS, MHN and controls without side effects.
Methods
Individuals
Twenty healthy individuals (ASA physical status I or II), aged 18–70 years, were examined. The study was approved by the local ethics committee (Government of Unterfranken, Würzburg, Germany), and all participants gave their prior informed written consent. Out of 13 individuals who were previously investigated by IVCT according to the criteria of the European malignant hyperthermia group, six patients were diagnosed as MHS and seven were MHN [3] Table 1 ). Furthermore, seven control individuals without a history of malignant hyperthermia or myopathy were randomly studied. Patients with known myopathies or a BMI of greater than 35 kg m−2 were excluded from the investigation.
Table 1: In-vitro contracture test results
Experimental protocol
Patients were positioned in a supine position. The right leg was immobilized by a leg splint. The skin and subcutaneous tissue over the lateral vastus muscle was infiltrated with 100 mg mepivacain 1% (Fresenius, Bad Homburg, Germany). Two introducer cannulas (Insyte G14; Becton Dickinson, Heidelberg, Germany) were placed under ultrasound guidance (SonoSite, 180 Plus; SonoSite Corporation, Bothell, Washington, USA) at an angle of 30° cephalad in the distal portion of the lateral vastus muscle at a distance of at least 30 mm from each other. A microdialysis probe (MAB7; Microbiotec, Stockholm, Sweden) with an attached microtube, a 23-G epidural catheter with three perforations located within 1 cm of the end of the distal tubing (Pajunk, Geisingen, Germany) for injection of trigger substances, and a p CO2 probe (Paratrend 7+; Diametrics Meduical Inc., High Wycombe, Buckinghamshire, UK) were inserted in each cannula. The tip of the perforated microtube was adjusted to 5 mm distal of the microdialysis probe and of the p CO2 probe. Microdialysis probes were perfused with Ringer's solution (Braun, Melsungen, Germany) at a rate of 1 μl per minute.
To verify function and accurate placement of the p CO2 probes, an in-vivo sham test with 500 μl Ringer's solution was performed. A sharp drop in the intramuscular p CO2 level after injection and its return to physiological levels was used to confirm correct positioning.
Following at least 30 min of equilibration and baseline measurement, single boluses of 500 μl caffeine 80 mmol l−1 (Merck, Darmstadt, Germany) or 500 μl 10% (vol/vol) halothane (Fluothane; AstraZeneca, Wedel, Germany) were injected. Lactate samples were collected every 15 min, whereas carbon dioxide pressure was recorded every minute. Caffeine solution was prepared under sterile conditions by the hospital's pharmacy (University of Würzburg) by dissolving analytical grade caffeine in Ringer's solution. Halothane was dissolved in soybean oil (Intralipid; Baxter, Unterschleissheim, Germany) in a sterile and gas tight cylinder 60 min prior to injection. As shown in prior studies [5]
During the investigation, heart rate, noninvasive blood pressure and peripheral oxygen saturation were measured. For pain evaluation, a visual analogue scale (VAS; 0, no pain; 10, maximal pain) was used. To monitor local metabolic response, myoglobin and creatine kinase in the serum were measured before, after and 24 h after the experiment. Venous blood for gas analysis (pH, p CO2 , base excess) was drawn 75 min after the trigger injection to analyse the systemic metabolic status.
Microdialysis Flexible microdialysis probes with an 80 mm shaft, a 10 mm polyethersulphone membrane and a cut-off weight of 15 kDa (MAB7) were used for collection of dialysate. Dead space of the tubing was 12.6 μl. The inlet tube of the probe was connected to a micro-syringe (Hamilton, Gastight Syringe; Reno, Nevada, USA) and perfused with Ringer's solution at a rate of 1 μl min−1 using a high-precision pump (PHD 2000 Syringe Pump; Harvard Apparatus, Holliston, Massachusetts, USA). The dialysate was collected at 15 min intervals and analysed immediately after the experiment.
For lactate analysis, samples were incubated with a lactate reagent solution (Sigma Chemicals, Deisenhofen, Germany) containing lactate oxidase (400 U l−1 ), horseradish peroxidase (2400 U l−1 ) and chromogenic precursors at a buffered pH of 7.2. Lactate oxidase converts the lactic acid into pyruvic acid and hydrogen peroxide. The peroxidase catalyses the reaction of hydrogen peroxide with the chromogenic precursors leading to a coloured dye with a maximum absorption at 540 nm that can be measured by a spectrometer (HP 8453-ultraviolet-visible spectrometer; Hewlett-Packard, Böblingen, Germany). Its intensity is proportional to the concentration of lactate up to 13 mmol l−1 with a sensitivity of 0.2 mmol l−1 . Prior to sample measurement, a calibration curve with standard solutions (lactate 5, 10, 20 and 40 mmol l−1 ) (Sigma Chemicals) was established [8]
p CO2 measurementThe p CO2 probe is an optical system that uses phenol red as an indicator. The intensity of phenol red is dependent on hydrogen ion concentration within the probe and corresponds to the surrounding p CO2 level. The absorption of phenol red can be measured at a wavelength of 550 nm and is proportional to the p CO2 level within the tissue. After in-vitro calibration (Paratrend 7+ Calibration Device; Diametrics Meduical Inc.), the probe measures p CO2 levels between 1.3 and 21.3 kPa with an accuracy of +/−0.4 kPa. The response time at a body temperature of 37°C is 15 s.
Statistic analysis
The patient's availability to take part in the study was used to randomize the groups. The availability was independent from the malignant hyperthermia diagnosis. Because of the small number of individuals, data were supposed to be nonparametrically distributed and are shown as the median with the interquartile range (i.e. 25 and 75% quartiles). The Kruskal–Wallis test and Mann–Whitney U -test were used to test for differences between MHS, MHN and control subjects. Changes in the course of creatine kinase and myoglobin due to the study were analysed by the Wilcoxon test. A P value of less than 0.05 was considered to be significant.
Results
The study groups did not differ concerning age, weight and height (Table 2 ). Two MHS individuals were kindreds.
Table 2: Patients' characteristics. Data are median (range) or numbers of patients
Cardiovascular monitoring
During the investigation, the vital parameters did not differ between the MHS, MHN and the control groups. Heart rate and mean arterial blood pressure were within physiological range throughout the whole experiment. Peripheral oxygen saturation was above 95% throughout the investigation in every individual.
p CO2 measurementsAfter placement of the cannulas, p CO2 equilibrated within 30 min at comparable values in MHS [4.9 kPa (4.4–5.3)], MHN [4.9 kPa (4.3–5.2)] and control [5.2 kPa (4.9–5.6)] individuals. Following caffeine injection, the rate of p CO2 increase was significantly higher in MHS than in MHN or in control individuals (Table 3 ). The rate of p CO2 increase was calculated using the p CO2 level at the time of injection, the highest p CO2 level and the time difference between the two values.
Table 3: p CO2 levels following trigger injection
Maximum p CO2 was not different in all three groups. Following halothane injection, the rate of p CO2 increase was significantly steeper in the MHS group than in the MHN and control groups. Interestingly, maximum p CO2 following halothane injection was not statistically different between the investigated groups.
Lactate measurements
After placement of the cannulas, the measured lactate concentration equilibrated at similar levels within 30 min in the MHS, MHN and control subjects (Table 4 ).
Table 4: Lactate levels before and after injection of trigger substances
Caffeine as well as halothane injection led to a significant lactate increase in the MHS group. The maximum lactate levels were observed 30 min after halothane and 45 min after caffeine injection (Figs 1 and 2 ).
In the MHN and control groups, the lactate levels recovered completely within 75 min. In the MHS group, the lactate decreased 45 min after trigger application but did not reach baseline levels.
Metabolic and psychometric monitoring
Venous blood gas analysis 75 min after intramuscular trigger application was normal and showed no signs of systemic hypermetabolism in any group.
Serum creatine kinase values were higher in the MHS group than in the MHN and control groups at all times (Fig. 3 ). In the MHS group, creatine kinase increased significantly within 24 h after the test compared with baseline levels. No significant increase was seen in the other two groups.
Myoglobin was not significantly different between groups before and after the experiment. A myoglobin level of 290 μg l−1 was measured in an MHS patient before the experiment (Fig. 4 ).
The pain level reported during placement of the cannulas was comparable between the groups. MHS individuals characterized the pain sensation after trigger injection as being similar to muscle cramping. The muscle itself was regularly soft on palpation. All individuals were free of pain, able to walk and discharged home immediately after the experiment.
Discussion
The results of the study indicate that local application of caffeine and halothane induces an increase in local metabolism in MHS and for halothane also in MHN individuals without severe systemic or local side effects. The extent of hypermetabolism in MHS individuals can easily be measured by p CO2 and lactate levels. Interestingly, intramuscular injection of halothane induced a hypermetabolic reaction even in MHN individuals.
Under resting conditions, the basal intramuscular lactate level in skeletal muscle is about 2.5 mmol l−1 [9] [10] microdialysis of up to 8 mmol l−1 following halothane injection may reflect therefore an interstitial concentration of 24 mmol l−1 . In MHS pigs, similar systemic lactate concentrations were reported during a malignant hyperthermia crisis [11]
In prior studies, significant differences for maximal p CO2 levels were observed between MHS and MHN individuals but could not be seen in our protocol [5] p CO2 concentration. Compared with prior protocols with a trigger application over a couple of minutes, we assume that more muscle tissue was exposed to the triggering agent in a shorter period of time.
Both lactate and p CO2 levels in MHS individuals indicate a local hypermetabolic reaction. It can be assumed that this is due to an activation of contractile filaments with an increase in energy consumption or a direct stimulation of intracellular energy production via increased Ca2+ levels. The cramping sensation that patients complained about supports this hypothesis. Even in the MHN and control groups, an increase of measured lactate and p CO2 could be elicited with an increase in dosage. This indicates that trigger substances can induce a local hypermetabolism in patients without genetic malignant hyperthermia predisposition. This finding is supported by a previous investigation in MHS and MHN pigs. With increasing concentrations of caffeine and halothane, muscle metabolism was increased even in MHN pigs, reflecting a dose-dependent rise of lactate and p CO2 levels [10] −1 induces a contracture in isolated skeletal muscle even in muscle of nonsusceptible, that is, healthy patients. Caffeine induces a Ca2+ release from the sarcoplasmic reticulum. In patients with malignant hyperthermia susceptibility, this initial increase of intracellular Ca2+ leads to an uncontrolled hypermetabolism [11,12]
Intramuscular lactate and p CO2 levels may be influenced by many factors. Interindividual response or different causative mutations may react differently towards triggering agents. Furthermore, local factors, such as the fat/muscle ratio, are likely to have a certain influence on our results apart from an unknown trigger concentration within the muscle. The exact concentration that reaches the muscle varies with distribution and dilution of caffeine or halothane in the tissue. Different lactate and p CO2 levels might be the result of different trigger concentrations in the muscle.
Intramuscular trigger application in MHS individuals still raises concerns about local or systemic side effects. In earlier studies [5,10]
Neither MHS nor MHN or control patients showed any systemic side effects. No haemodynamic or metabolic differences were detected between the groups. Owing to the small total dose of the injected triggering agent, a systemic reaction is highly unlikely.
On the contrary, there might be a risk for muscle damage up to compartment syndrome and rhabdomyolysis following local trigger application. Owing to dilution with interstitial fluid, the exact intramuscular concentration of the applied agents is unknown but is probably far lower than the applied concentration in the perfusate. Caffeine itself is an approved drug for intramuscular injection. Doses that are 10-fold higher than the one we used are applied to treat arterial hypotension. However, injection of pure halothane leads to severe cell damage. Emulsified in a lipid carrier, the cell damage is absent. This was documented by investigating pulmonary damage in intravenous anaesthesia in pigs [13]
Creatine kinase levels increased in all groups 24 h after the trigger application, indicating local muscle damage. However, MHS individuals had a higher increase in creatine kinase levels as an indication for a greater vulnerability without reaching clinical significance. MHS individuals are known to have a higher basal creatine kinase level than healthy individuals [14]
Conclusion
The results demonstrate that local trigger application does not cause a systemic hypermetabolic reaction, and that the intramuscular injection of halothane and caffeine induces a p CO2 increase and a rise in lactate levels in MHS and healthy individuals. The reaction towards triggering agents in MHS individuals is significantly more intense.
The measurement of lactate and p CO2 after local intramuscular application of triggering substances may be a promising principle for a metabolic test to diagnose malignant hyperthermia susceptibility. Such a test would be easy to handle and because of its minimal invasiveness less costly and stressful for patients. However, further studies to find an ideal halothane concentration have to be conducted to allow a clear diagnostic identification of MHS individuals.
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