An In-Vivo Metabolic Test for Detecting Malignant Hyperthermia Susceptibility in Humans: A Pilot Study

Abstract

INTRODUCTION: 

In vitro contracture testing to diagnose malignant hyperthermia (MH) susceptibility requires a muscle biopsy, which may be associated with severe side effects for the patient. After investigation of several different protocols, we present a less invasive metabolic test that involves IM injection of caffeine and halothane, and subsequent measurement of interstitial lactate to differentiate between MH susceptible (MHS) and MH non-susceptible (MHN) individuals.

METHODS: 

Two microdialysis probes with attached microtubing for trigger injection were inserted into the lateral vastus muscle of eight previously diagnosed MHS patients (representing three genetic variants Gly2434Arg, Thr2206Met, and Arg614Cys), seven MHN patients, and seven control individuals. After equilibration and lactate baseline recording, a single bolus of 200 μL caffeine 80 mM and a suspension of 200 μL halothane 4%V/V in soy bean oil (triggers) were injected locally. Lactate was measured spectrophotometrically. Data are presented as medians and interquartile ranges.

RESULTS: 

Although baseline lactate values were similar in the investigated groups before trigger injection, caffeine increased local lactate in MHS patients significantly more (2.0 [1.8–2.6] mM) than in MHN (0.8 [0.6–1.1] mM) or in control individuals (0.8 [0.6–0.8 mM]). Similarly, halothane lead to a significant lactate increase in MHS compared to MHN and control individuals (8.6 [3.7–8.9] mM vs 0.9 [0.5–1.1] mM and 1.7 [0.9–2.3] mM, respectively). However, a relevant increase of lactate was observed in one MHN and in two control individuals. Systemic hemodynamic and metabolic variables did not differ between the investigated groups.

DISCUSSION: 

Metabolic monitoring of IM lactate after local caffeine and halothane injection may allow less invasive testing to detect MH susceptibility, without systemic side effects.

In Brief

IMPLICATIONS: Local metabolic monitoring of lactate after intramuscular caffeine and halothane injection allows the differentiation between malignant hyperthermia susceptible and non-susceptible individuals and may prove to be a less expensive and invasive test.

Malignant hyperthermia (MH) is an autosomal dominant inherited myopathy associated with abnormal intracellular calcium regulation. Deterioration of calcium homeostasis in susceptible individuals exposed to volatile anesthetics and/or depolarizing muscle relaxants leads to muscle contracture, metabolic failure, lactic acidosis, and heat production.¹ The standard procedure for diagnosing MH susceptibility is the halothane-caffeine contracture test requiring a muscle biopsy that is associated with several risks to the patient, i.e., postoperative bleeding, dysesthesia or wound infection.² Important progress was made within the last years by screening for causative MH mutations allowing a genetic diagnosis in 30% to 50% of MH families.³ However, for patients without known MH mutation, a functional but less invasive test is needed.

Microdialysis, originally developed for monitoring cerebral neurotransmitters, has become a widely used technique to monitor pharmacologically active concentrations of exogenous or endogenous compounds in the target tissue of humans and animals.⁴ After insertion of the probe, small unbound molecules, like lactate, diffuse along their concentration gradient across a semipermeable membrane from the interstital space into the perfusate.⁵ Recently, the described technique has successfully been used in a metabolic test at different concentrations with lactate measurement after IM MH trigger injection to differentiate between MH susceptible (MHS) and non-susceptible (MHN) animals.^6,7

In the present investigation, we studied MHS, MHN and control participants by microdialysis technique, postulating that IM injection of caffeine and halothane increases local lactate levels more in MHS than in MHN or control individuals and that this test allows diagnostic differentiation.

METHODS

Participants

After approval by the local ethics committee (Government of Unterfranken, Würzburg, Germany) 15 patients with a personal or family history of MH, a previously performed in vitro contracture test and a mutation analysis of the skeletal muscle ryanodine receptor gene (Gly2434Arg, Thr2206Met, and Arg614Cys) were investigated. According to the diagnostic criterions of the European Malignant Hyperthermia Group, eight participants were classified MHS and seven participants MHN.² Furthermore, seven control individuals were included in the study who had not undergone in vitro contracture testing nor genetic analysis and had no family history of MH. All individuals were healthy (ASA physical status I-II) and gave written informed consent. Individuals with evidence for myopathys other than MH or a Body Mass Index >35 kg/m² were excluded from the investigation.

Experimental Protocol

Participants were placed in supine position with their leg in a splint. After locating the lateral vastus muscle, the skin was disinfected, draped and anesthetized by exclusively subcutaneous infiltration with mepivacaine 1% 100 mg. Two 18-gauge introducer cannulae (Becton Dickinson, Helsingborg, Sweden) were placed under ultrasound guidance (SonoSite 180 Plus, SonoSite, Bothell, WA) 10–15 cm proximal to the knee into the lateral vastus muscle. Two flexible microdialysis probes (CMA 70, CMA, Stockholm, Sweden) with a molecular cut-off of 15.000 Dalton and an attached microtubing catheter (Pajunk, Geisingen, Germany) for caffeine or halothane injection placed 5 mm proximal to the tip of the probes were then inserted. The inlet of the microdialysis catheter was connected to a high-precision microinjection pump (Hamilton Gastights Syrings, Reno, NV; PHD 2000 Syringe Pump, Harvard Apparatures, Holliston, MA) and perfused with Ringer's solution (B. Braun, Melsungen, Germany) at a flow rate of 1 μL/min. After implanting the probes in the tissue, interstitial molecules diffuse through the semipermeable membrane along a concentration gradient into the perfusate until equilibrium is reached. After equilibration for at least 30 min and measurement of baseline lactate levels, single boli of either 200 μL caffeine 80 mM (Merck, Darmstadt, Germany) or 200 μL of a suspension of halothane 4%V/V (Fluothane, AstraZeneca, Wedel, Germany) in soy bean oil (Intralipid, Baxter, Unterschleiβheim, Germany) were injected synchronously via the microtubing catheter to the tip of the microdialysis probe.

Six dialysate samples were collected in 15 min intervals and lactate was measured spectrophotometrically after enzymatic conversion. In detail, 10 μL of the dialysate was incubated with lactate oxidase 400 U/L, peroxidase 2400 U/L and buffer at a pH of 7.2 (Trinity Biotech, Wicklow, Ireland) converting a chromogene dye linear to the lactate concentration of the sample. The absorption of the dye is measured spectrophotometrically at λ = 540 nm (HP 8453-UV-Visible spectrophotometer; Hewlett Packard, Waldbronn, Germany).⁸

Vital signs were monitored throughout the experiment by electrocardiography, arterial blood pressure, and peripheral oxygen saturation. The participants estimated the intensity of pain during catheter placement and after IM drug injection by using a visual analog scale (0: no pain; 10: maximum pain). Serum creatine kinase and myoglobin samples were drawn before, at the end, and 24 hours after the study. The systemic metabolic status was analyzed by peripheral venous blood gas samples before and at the end of the investigation. For evaluation of patient's satisfaction, a questionnaire was sent out to the participants 4 weeks after the test, allowing the participant to answer questions regarding pain at the injection site, local side effects, e.g., hematoma or infections, and personal limitations.

Statistical Analysis

A quasi-randomization was given by the availability of the participants independently from the MH diagnosis. Due to the small sample size, data were assumed to be nonparametrically distributed and therefore displayed as median and interquartile range (25% and 75% quartile). Kruskal-Wallis test and post hoc Mann-Whitney-U-test were applied to detect differences between MHS, MHN, and control individuals. Wilcoxon test was used to test for differences between lactate values within the investigated groups and to analyze differences of serum creatine kinase and myoglobin before as compared to immediately after and 24 hours after the test. For evaluation of the questionnaire, Mann-Whitney-U-test was applied. A P < 0.05 was considered statistically significant.

RESULTS

MHS and MHN individuals did not differ regarding age (39 [34–46] years vs 31 [25–46] years) and Body Mass Index (28 [25–29] kg/m² vs 25 [24–27] kg/m²), whereas control participants were younger compared to MHS and MHN (25 [24–25] years; Body Mass Index 24 [21–26] kg/m²). Biometric and individual data of the investigated probands are shown in Table 1.

Table 1: Biometric Data, Proband Status and Individual Maximum Lactate Levels During the Investigation Following Intramuscular Caffeine 80 mM and Halothane 4%V/V Injection

Table 1: (Continued)

IM Lactate After Caffeine 80 mM and Halothane 4%V/V Injection

Prior to trigger injection, the baseline lactate level did not differ between MHS, MHN and control individuals. After local injection of caffeine, lactate significantly increased in individuals susceptible to MH, but not in MHN or control participants. Similarly, halothane injection resulted in a significant increase of lactate in MHS compared to MHN and control individuals (Table 2). In MHS participants, lactate peaked at 15 to 45 min after caffeine or halothane injection and then decreased slowly (Fig. 1 and 2). In one MHS individual, halothane induced a lactate increase to 10.2 mM, whereas caffeine did not increase lactate above baseline. In a second participant, halothane did not increase above a suggested diagnostic threshold for halothane at 2.8 mM, but caffeine increased lactate to 2.0 mM at a defined threshold of 1.6 mM. Due to technical reasons lactate measurement failed in another MHS individual and in one control participant after halothane injection. Interestingly, in 1 MHN (2.8 mM) and in 2 control (3.4 mM; 2.8 mM) individuals, relevant increases of lactate were observed after halothane (Table 1).

Table 2: Baseline Lactate Levels Before Trigger Injection and Maximum Lactate Levels During the Investigation Following Intramuscular Injection of 200 μL Caffeine 80 mM and Halothane 4%V/V

Figure 1.: IM lactate values (mM) after local injection of 200 μL caffeine 80 mM in malignant hyperthermia susceptible (MHS), non-susceptible (MHN) and in control individuals. Data are presented as median and interquartile range (25% and 75% quartile); time = 0 min = trigger injection time; n = 8 for MHS; n = 7 for MHN; n = 7 for control; *: P < 0.05 for differences between MHS and MHN. #: P < 0.05 for differences between MHS and control. +: P < 0.05 for differences compared to the pre-injection value within the MHS group.

Figure 2.: IM lactate values (mM) after local injection of 200 μL halothane 4%V/V in malignant hyperthermia susceptible (MHS), non-susceptible (MHN) and in control individuals. Data are presented as median and interquartile range (25% and 75% quartile); time = 0 min = trigger injection time; n = 8 for MHS; n = 7 for MHN; n = 7 for control; *: P < 0.05 for differences between MHS and MHN. #: P < 0.05 for differences between MHS and control. +: P < 0.05 for differences compared to the pre-injection value within the MHS group.

To maintain a sensitivity of 100% related to the in vitro contracture test, preliminary empirical lactate thresholds were defined at 1.6 mM after caffeine and at 2.8 mM after halothane injection. Based on these thresholds, specificity reaches 79%, whereas the positive predictive value gains 89% and the negative predictive value 100%.

Cardiovascular, Metabolic and Psychometric Variables

Vital signs (heart rate, arterial blood pressure, peripheral oxygen saturation) and venous blood gas measurements in MHS, MHN, and in the control individuals were within normal range and did not differ among the groups during the investigation.

Myoglobin did not differ before, after and 24 hours after the experiment in any group: MHS: 75 [52–120] ng/mL vs 100 [74–109] ng/mL vs 88 [81–125] ng/mL; MHN: 34 [22–45] ng/mL vs 34 [22–36] ng/mL vs 37 [28–42] ng/mL; control: 40 [27–51] ng/mL vs 41 [37–46] ng/mL vs 29 [25–34] ng/mL. Creatine kinase significantly increased 24 hours after the investigation compared to pretest values in MHS from 273 [162–308] U/L to 285 [133–319] U/L to 509 [416–524] U/L, but not in MHN (129 [91–146] U/L vs 111 [88–132] U/L vs 134 [85–159] U/L) or in control (151 [101–211] U/L vs 124 [85–184] U/L vs 131 [61–202] U/L) individuals.

Evaluation of individual pain intensity estimated by a visual analog scale was similar among the groups during placement of the microdialysis probes (MHS: 3 [2–4]; MHN: 2 [1–5]; control: 2 [1–3]). However, caffeine and halothane injection were considered significantly more painful in MHS and in the control than in MHN individuals (MHS: 4 [3–5]; MHN: 1 [0–2]; control: 4 [2–4]). All participants were free of pain, able to walk, and discharged home immediately after the test.

The questionnaire sent 4 weeks after the investigation was returned by eight MHS, six MHN, and seven control individuals. Retrospectively, pain sensation was comparable among the groups during placement of the microdialysis probes, whereas IM trigger injection was described significantly more painful and long lasting in MHS patients (Table 3). No participants complained of any serious local or systemic side effects, i.e., hematoma or erythema. Furthermore, wound healing was uneventful. All MHS and MHN individuals participating in this study voted in favor of the metabolic test compared to muscle biopsy.

Table 3: Results of the Questionnaire

DISCUSSION

The results of the presented study indicate that (a) baseline lactate levels were similar among MHS, MHN and control participants at rest during this experiment, (b) local injection of caffeine and halothane lead to a significantly larger increase of interstitial lactate in MHS compared to MHN and control individuals, reflecting a hypermetabolic reaction of skeletal muscle, and (c) lactate measurement by microdialysis technique after IM trigger injection is a suitable less invasive test to detect individuals at risk for MH without serious side effects.

Microdialysis has been proven suitable for sampling interstitial target tissue concentrations of metabolic variables, i.e., lactate at steady-state conditions or stimulated by administered drugs without systemic involvement.^4,9 In the present study, we established a standardized and easy-to-perform test protocol to diagnose susceptibility to MH in humans by using a microdialysis technique.

Caffeine and halothane, have been successfully used for in vitro MH testing, induce contractures in skeletal muscle bundles of MHS, but not in MHN patients, reflecting the metabolic response to an excessively increased sarcoplasmic calcium release.² Furthermore, both substances have been previously used for safe local IM injection in humans.^10,11 To avoid severe cell damage, halothane must be emulsified in a lipophilic carrier.¹² In this context, an alteration of skeletal muscle metabolism by the solvent soy bean oil was excluded.⁶ Since the induced hypermetabolism is limited to an area <10 mm around the probe and due to expected dilutional effects of the administered drugs in the tissue, serious systemic or local side effects caused by the injected drugs are unlikely.⁶ Indeed, histological evaluation of muscle biopsies in rats after caffeine 80 mM injection revealed only unspecific morphological changes analogous to hydropic swelling.¹³ In the present study, MHS individuals complained of local pain sensations after trigger injection comparable to a muscle cramp, which lasted about 30 min and then disappeared. Palpation of participants' muscle was unremarkable and not indicative of cramping, whereas in animal studies a local contracture after trigger injection was detected sporadically by ultrasound. Serum creatine kinase increase in susceptible individuals 24 hours after the investigation indicates local muscle damage that is similar to minor surgical procedures.¹⁴ Nevertheless, all individuals were pain-free, mobile and discharged home immediately after the test. Wound healing was uncomplicated and no participant complained of serious local side effects, i.e., infection or any other limitations.

Since most MHS individuals do not suffer from muscular symptoms in daily life, it is not surprising that baseline lactate values before caffeine and halothane injection were similar between MHS and MHN or control individuals. The increase of muscular lactate in MHS patients after caffeine or halothane injection, however, reflects an excessive metabolic response of skeletal muscle, comparable to the significant increase of muscular venous outflow of lactate in the course of a MH episode.¹⁵

Although only eight MHS individuals were investigated, lactate increased at least in one of both tests in susceptible individuals, allowing a diagnostic assignment with a preliminary sensitivity of 100% and a specificity of 79% at defined lactate thresholds of 1.6 mM after caffeine and 2.8 mM after halothane, respectively. The failed increase in two MHS patients after caffeine or halothane injection might be explained either by methodological problems, i.e., incorrect placement of the microdialysis catheter or insufficient drug concentration in the tissue, or by an individual physiological response to trigger application similar to MH equivocal results in the in vitro contracture test. Furthermore, due to a defect of one microdialysis probe, lactate measurement was not possible in the MHS individual #1107 and in the control participant C5 after halothane injection. Interestingly, halothane also led to a hypermetabolic reaction in one MHN individual and in two control probands. This finding has been observed after local administration of 10%V/V halothane in MHN and control volunteers¹¹ and might be explained as an individual reaction to the administered trigger drug in accordance with the proposed dose-response relationship with lactate increase even in MH animals.⁶

In general, this metabolic test is a functional in vivo test measuring the metabolic response on exposure to caffeine and halothane. With respect to this quantitative biologic test and the heterogeneity of MH, a gray zone with overlapping is expected, similar to the in vitro contracture test.¹⁶ Therefore, repeated measurements with caffeine and halothane may help to increase sensitivity, comparable to the European Malignant Hyperthermia Group testing protocol, e.g., if lactate increases in one of two tests with caffeine and halothane, susceptibility to MH is confirmed.

The presented metabolic test carries a very low risk for hematoma, local infection and sensory deficit, is an easily standardized procedure that can be repeated if necessary, and is by far not as costly as the contracture test. In contrast to genetic analysis, due to its functional characteristics, the test is independent of the locus of a MH causative mutation. With genetic analysis, more than 150 MH associated mutations were described and 28 MH causative mutations cover 30% to 50% of MH families.¹⁷ For epidemiologic and economic reasons, primary genetic screening in any MH suspect seems likely to be impossible.

With respect to diagnostic guidelines for MH,¹⁸ metabolic testing does have the potential to become a less invasive test in the near future in MH families without known causative mutation. Its application may follow the same criteria as with genetic analysis, a positive result would avoid muscle biopsy, whereas a negative test result should be verified by an in vitro contracture test.

In conclusion, this metabolic test as a first step in the development of a less invasive test, allows differentiation of MHS and MHN individuals without relevant local side effects. We suggest using the presented test protocol as an additional method to detect patients at risk for MH. A multicenter trial with a large number of participants may help to define diagnostic thresholds as well as sensitivity and specificity.