Daniel Massik Winner 2007 (Schuster)

A Minimally-Invasive Metabolic Test detects Probands at Risk for Malignant Hyperthermia

F. Schuster, T. Metterlein, S. Negele, N. Roewer, M. Anetseder

Department of Anesthesiology, University of Würzburg, Germany

 

Background:

In malignant hyperthermia (MH) susceptible individuals, volatile anesthetics and depolarizing muscle relaxants may lead to a potentially lethal hypermetabolic syndrome due to intramuscular impairment of intracellular calcium homeostasis [1]. The standard procedure to diagnose MH susceptibility is the in-vitro-contracture test requiring a muscle biopsy. However, this approach is invasive, expensive, consists of a complex test procedure, and is applicable mainly to persons with a high MH probability. In addition, a muscle biopsy may be associated with side effects like haematoma, infection or persistent paraesthesia [2,3]. The genetic prevalence of the MH gene of 1 in 10,000 is higher than previously thought but at the same time genetic screening detects causative mutations for MH in less than 50% of MH susceptible families so far [4]. Hence, for patients without a known causative MH mutation, an alternative and less invasive test is needed.

Recently, a minimally-invasive metabolic test with local trigger injection and lactate measurement by microdialysis technique was shown to differentiate between MH susceptible (MHS) and non-susceptible (MHN) pigs [5].

In the current study, we postulate that local lactate measurement following intramuscular injection of small amounts of caffeine 80 mM and halothane 4 vol% allows a diagnostic assignment of MHS, MHN and control probands without clinically relevant side-effects.

Methods:

Experimental set-up

With approval of the local ethic committee and written informed consent of the probands, two microdialysis probes with an attached microtubing for caffeine or halothane injection were placed ultrasound guided in the lateral vastus muscle of 8 MHS, 7 MHN and 7 control probands and perfused with Ringers’ solution at a flow of 1 µL/min. Following an equilibration period of 30 min, single boli of 200 µL caffeine 80 mM and halothane 4 vol% dissolved in soy bean oil were injected locally via the microtubing catheter into the muscular tissue. Lactate was measured spectrophotometrically in 15 min intervals. Systemic hemodynamic (ECG, BP, SpO2) and metabolic parameters (pH, BE, pCO2v) of the probands were monitored. Data as mean and standard deviation; Kolmogorov-Smirnov-test for parametric distribution; analysis of variance and non-dependent t-test for differences between groups; dependent t-test for serum creatine kinase analysis. P < 0.05.

Microdialysis Setup

Flexible microdialysis catheters with an 80 mm shaft and a semi-permeable membrane of  10 mm were used.  The inlet tubing of the catheter was connected to a syringe mounted on a high-precision pump and perfused with Ringer’s solution at a flow rate of 1 µL/min. After insertion of the catheter in the muscular tissue, unbound molecules diffuse along a concentration gradient bi-directionally via the membrane from the interstitium in the dialysate or  vice versa. Samples were collected and analyzed by an enzymatic spectrophotometric assay for lactate.

Results:

Baseline lactate values did not differ between the study groups. Caffeine injection increased local lactate in MHS significantly higher than in MHN and in the control group respectively (2.5 ± 1.4 mM vs. 0.8 ± 0.3 mM / 0.7 ± 0.4 mM). Similarly, intramuscular halothane induced a significant lactate increase in MHS compared to MHN and control individuals (6.5 ± 3.5 mM vs. 1.1 ± 0.9 mM / 1.7 ± 1.1 mM). In one MHS proband, halothane increased lactate to 10.2 mM, while caffeine did not increase lactate above a suggested caffeine threshold of 1.6 mM. In a second MHS proband, halothane did not increase above a suggested halothane threshold of 2.8 mM but caffeine increased to 2.0 mM clearly above the threshold of 1.6 mM. In a third MHS proband, halothane measurement failed due to technical reasons but caffeine increased lactate to 5.4 mM. In one MHN and in two control probands, a relevant increase of lactate (2.8 mM; 3.4 mM; 2.8 mM) was observed following halothane (figure 1).

Haemodynamic and metabolic parameters did not differ between MHS, MHN and control individuals.

In comparison to basal values, serum creatine kinase increased significantly 24 hours after the test in the MHS group (258 ± 138 U/L to 490 ± 124 U/L).

All participants were free of pain and mobile after the investigation. According to a questionnaire 4 weeks later, no side effects occurred.

Discussion:

This functional metabolic test with intramuscular injection of caffeine and halothane induces a temporary hypermetabolic response in MH susceptible skeletal muscle but not in MHN or control individuals. The differentiation between the diagnostic groups using a combined caffeine-halothane test yields a preliminary sensitivity of 100% and a specificity of 78.5% at a lactate threshold of 1.6 mM following caffeine and 2.8 mM following halothane. The failed increase in two MHS individuals following intramuscular caffeine or halothane injection might be explained by methodological problems, i.e. catheter dislocation or insufficient tissue concentration of the injected triggers, or by an individual physiological response to trigger application [6]. Furthermore, due to a defect of one microdialysis probe, lactate measurement was not possible in one MHS and in one control participant following halothane. Interestingly, a hypermetabolic response was also observed in one non-susceptible individual and in two control probands after halothane, supporting the assumption of a dose-dependent metabolic reaction to the applied trigger agents [5,7]. 

The significant serum creatine kinase increase in MHS individuals reflects a local, but clinically irrelevant muscular tissue damage.

 

We suggest that this metabolic test may be helpful in diagnosing patients in MH families without known causative mutations. Its application may follow the same criteria like with genetic analysis, i.e. a positive result would avoid muscle biopsy, a negative result calls for an in-vitro contracture test. A multicentre study might be helpful to define sensitivity and specificity.

 

Figure 1: Individual maximum lactate [mM] following intramuscular injection of 200 µL caffeine 80 mM or halothane 4 vol% respectively; n = 8 for malignant hyperthermia susceptible (MHS) probands; n = 7 for malignant hyperthermia non-susceptible (MHN) probands; n = 7 for control probands; preliminary diagnostic threshold following halothane injection at 2.8 mM lactate and following caffeine at 1.6 mM lactate.

References:

  1. Gronert GA, Antognini JF, Pessah IN: Malignant Hyperthermia, Anesthesia 5th edition. Edited by Miller RD. Philadelphia, Churchill Livingstone, 2000, pp 1033-1052
  2. The European Malignant Hyperpyrexia Group. A protocol for the investigation of malignant hyperpyrexia (MH) susceptibility. Br J Anaesth 1984;56:1267-9
  3. Larach MG, for the North American Malignant Hyperthermia Group. Standardization of the Caffeine Halothane Muscle Contracture Test. Anesth Analg 1989;69:511-5
  4. Girard T, Urwyler A, Censier K, Mueller CR, Zorzato F, Treves S: Genotype-phenotype comparison of the Swiss malignant hyperthermia population. Hum Mutat 2001; 18: 357-358
  5. Schuster F, Schöll M, Hager M, Müller R, Roewer N, Anetseder M: Dose-response relationship and regional distribution of lactate following intramuscular injection of halothane and caffeine in malignant hyperthermia susceptible pigs. Anesth Analg 2006; 102: 468-4
  6. McGrath CJ, Lee JC, Rempel WE: Halothane testing for malignant hyperthermia in swine: dose-response effects. Am J Vet Res 1984; 45:1734-672
  7. Schuster F, Anetseder M, Metterlein T, Müller R, Roewer N: Direct intramuscular injection of halothane induces hypermetabolism in MH non-susceptible and control volunteers. Anesthesiology 2005: 103: A –1121