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Anesth Analg 2006;102:468-472
© 2006 International Anesthesia Research Society
doi: 10.1213/01.ane.0000189553.57833.11

ANESTHETIC PHARMACOLOGY

The Dose-Response Relationship and Regional Distribution of Lactate After Intramuscular Injection of Halothane and Caffeine in Malignant Hyperthermia-Susceptible Pigs

Frank Schuster, MD, Hendrik Schöll, MS, Martin Hager, MD, Rainer Müller, MD, Norbert Roewer, MD, and Martin Anetseder, MD

Department of Anesthesiology, University of Wuerzburg, Wuerzburg, Germany

Address correspondence and reprint requests to PD Dr. Martin Anetseder, Department of Anesthesiology, University of Wuerzburg, Oberduerrbacher Straße 6, D-97080 Wuerzburg, Germany. Address e-mail to Anetseder_M{at}klinik.uni-wuerzburg.de.


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
We hypothesized that IM halothane and caffeine injection increases local lactate concentration dose-dependently in malignant hyperthermia-susceptible (MHS) and nonsusceptible (MHN) pigs and that the hypermetabolic reaction measured by regional distribution of lactate and carbon dioxide is limited to a small muscle volume. Microdialysis probes were placed in the hindlimbs of 7 MHS and 7 MHN pigs and perfused with Ringer's solution. After equilibration, boluses of increasing halothane and caffeine concentrations were injected. For the second hypothesis regarding regional distribution, microdialysis probes were positioned in 7 MHS and 6 MHN pigs at the injection site for halothane and caffeine and at a distance of 10 mm and 25 mm. Lactate was measured in the dialysate by spectrophotometry. In addition, Pco2 was measured in the halothane experiments. Halothane and caffeine increased IM lactate dose-dependently in MHS pigs significantly more than in MHN pigs. Lactate and Pco2 were increased only at the injection site but not at 10 mm and 25 mm distance. MH susceptibility leads to a leftward shift of the dose-response curve for IM lactate after local injection of halothane and caffeine. The increase of lactate and carbon dioxide levels after local MH trigger injection is limited to a small area around the probe.


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Volatile anesthetics and succinylcholine may induce malignant hyperthermia (MH) in susceptible individuals, resulting in a potentially lethal hypermetabolic syndrome of skeletal muscle as a result of uncontrolled sarcoplasmic calcium release via the ryanodine receptor. In the course of MH, myoplasmic calcium activates contractile filaments leading to muscle contracture and enhanced mitochondrial energy turnover with a massive increase of oxygen consumption, carbon dioxide, and heat production, as well as early lactic acidosis (1). MH susceptibility is primarily diagnosed by an in vitro contracture test with halothane and caffeine after a muscle biopsy (2,3). Genetic diagnosis is currently limited to fewer than 50% of MH families, and thus alternative and minimally invasive methods to detect MH susceptibility continue to be sought (4,5).

Microdialysis is a minimally invasive technique for measuring dialyzable compounds like lactate in the interstitial tissue fluid. Once the probes are implanted in the tissue, small unbound molecules in the interstitial space are able to diffuse across a semipermeable membrane into the perfusate. Samples can either be analyzed online or collected for post hoc analysis (6). Local IM injection of MH trigger drugs, such as halothane (7) or caffeine (5), allows provocative metabolic testing for MH without systemic side effects. However, it is unclear whether skeletal muscle metabolism is modulated dose-dependently in MH susceptible (MHS) and in MH nonsusceptible (MHN) individuals. In addition, the regional distribution of lactate modulation in the surrounding muscle tissue caused by local trigger application is unknown.

We hypothesized that local halothane and caffeine injection enhances IM lactate concentration dose-dependently but more in MHS than in MHN pigs and that the local IM distribution of lactate and carbon dioxide increase is limited to a small area around the stimulating probe.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
With approval of the local animal care committee, anesthesia was induced in 14 MHS and 13 MHN Pietrain pigs weighing 28–35 kg with midazolam (0.15 mg/kg; Hoffmann-La Roche AG, Grenzach-Wyhlen, Germany) and fentanyl (2 µg/kg; Janssen-Cilag, Neuss, Germany) via a 22-gauge ear vein catheter. After tracheal intubation (6.5-mm inner diameter endotracheal tube, Rüsch, Kernen, Germany), animals' lungs were mechanically ventilated (Servo 900D; Siemens, Erlangen, Germany) with 65% nitrous oxide and 35% oxygen. Ventilator settings were adjusted to maintain an end-tidal Pco2 of 33–37 mm Hg (respiratory rate: 10–12/min; tidal volume: 10–15 mL/kg; positive end-expiratory pressure: 5 mm Hg) throughout the preparation and experimental period. Anesthesia was maintained using a midazolam/fentanyl infusion and bolus administration if necessary. A cannula was placed in a saphenous artery for monitoring arterial blood pressure and blood gases. Vital signs were monitored continuously by peripheral oxygen saturation, electrocardiogram, and rectal temperature.

In 7 MHS and 7 MHN animals, introducer cannulae were placed under ultrasound guidance (SonoSite, 180 Plus, Bothell, WA) in the adductor muscles. Microdialysis probes (MAB 7; Microtech, Stockholm, Sweden) with an attached microtubing catheter for halothane or caffeine injection were inserted and perfused with Ringer's solution (B Braun, Melsungen, Germany) at 1 µL/min. The tip of the microtubing was adjusted to 5 mm proximal to the tip of the microdialysis probe. After 30 min of equilibration, a single bolus of 100 µL halothane 0, 1, 2.5, 5, and 10 vol% (Fluothane; AstraZeneca, Wedel, Germany) dissolved in soy bean oil (Intralipid; Baxter, Unterschleissheim, Germany) or 500 µL caffeine 0, 1, 10, 40, and 80 mM (Merck, Darmstadt, Germany) dissolved in Ringer's solution was injected via the microtubing catheter next to the membrane of the microdialysis probes.

In a second series of experiments in 7 MHS and 6 MHN pigs, cannulae for central halothane and caffeine injection and at a distance of 10 mm and 25 mm away were placed under ultrasound guidance in the gracilis muscle of each leg. Microdialysis probes and microdialysis probes plus microtubing for trigger drug application were inserted and perfused with Ringer's solution at 1 µL/min. In the halothane cannulae of the MHS animals, additional Pco2 probes (ParaTrend 7+; Diametrics Medical Inc., High Wycombe, Buckinghamshire, UK) were placed. After equilibration, 200 µL halothane 10 vol% or 1000 µL caffeine 80 mM, respectively, were injected via the microtubing catheter at the central probes.

Lactate was measured spectrophotometrically from the dialysate after enzymatic conversion. Pco2 was recorded in 1-min intervals. Systemic hemodynamic variables were monitored throughout the experiment.

Flexible microdialysis probes with an 80 mm shaft and a 10 mm semi-permeable polyethylensulfone membrane with a molecular cut-off of 15000 Dalton were used. The microdialysis tubings were connected to syringes in a high-precision micro pump (Hamilton Gastights Syringes, Reno, NV; PHD 2000 Syringe Pump, Harvard Apparatus, Holliston, MA; CMA/100, CMA, Solna, Sweden) and perfused with Ringer's solution. Dialysate was collected at 15-min intervals and lactate was measured by a spectrophotometer immediately after the experiment. In detail, after incubation with lactate oxidase 400 U/L, peroxidase 2400 U/L, and buffer at a pH of 7.2 (Sigma Chemicals, Diesenhofen, Germany), a chromogene dye is converted proportionally to the concentration of lactate. The absorption of the dye is measured spectrophotometrically at {lambda} = 540 nm (HP 8453-UV-Visible spectrophotometer; Hewlett-Packard, Böblingen, Germany). In preliminary experiments, the in vitro recovery of the microdialysis membranes for lactate was 70%–80% at a flow rate of 1 µL/min. Before sample analysis, a standard curve for lactate was established by using lactate 20 mM, 40 mM, and 80 mM (Sigma Chemicals, Deisenhofen, Germany) to assure accuracy in every measurement series (8).

Data are shown as median and quartiles. The Mann-Whitney U-test was used to test for differences in the dose-response experiments with halothane and caffeine in MHS versus MHN pigs. The Wilcoxon test was used to detect differences of lactate between the halothane and caffeine injection site and results at 10 mm and 25 mm distance. A value of P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
During both sets of experiments, systemic hemodynamic and metabolic variables did not differ between MHS and MHN pigs before and 60 min after IM halothane or caffeine injection (Table 1).


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  		Table 1. Systemic Hemodynamic Variables of the Investigated Malignant Hyperthermia Susceptible and Nonsusceptible Pigs Before (0) and 60 min (60) After Intramuscular Application of Halothane and Caffeine

 

The injection of plain Ringer's solution or soy bean oil did not influence local lactate concentrations significantly, whereas IM halothane and caffeine injection increased local lactate dose-dependently in MHS more than in MHN animals. Halothane 2.5 vol% and 5 vol% increased lactate concentrations significantly in MHS animals compared with MHN. IM caffeine 10 mM and 40 mM injection lead to a significant increase of lactate concentrations in the MHS animals compared with the MHN group (Table 2).


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  		Table 2. Maximum Intramuscular Lactate Concentrations After Local Injection of 100 µL Halothane Dissolved in Soy Bean Oil at 0, 1, 2.5, 5, and 10 vol% and 500 µL caffeine at 0, 1, 10, 40, and 80 mM

 

After insertion of the microdialysis probes, basal IM lactate concentrations did not differ between study groups. IM injection of 200 µL halothane 10 vol% as well as 1000 µL caffeine 80 mM increased maximum lactate concentrations significantly at the central microdialysis probe compared with the 10-mm and 25-mm distance measurements of the MHS and MHN animals (Table 3). Similarly, basal IM Pco2 concentrations were increased significantly to a maximum only at the central probe but not at 10 mm and at 25 mm distance after halothane injection in MHS and MHN pigs (Table 4).


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  		Table 3. Basal Lactate Concentration and Maximum (max) Lactate Concentrations After Application of 200 µL Halothane 10 vol% in Soy Bean Oil and 1000 µL Caffeine 80 mM at the Injection Site, and at a Distance of 10 mm and 25 mm

 

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  		Table 4. Basal Pco2 and Maximum (max) Pco2 Concentrations After Application of 200 µL Halothane 10 vol% in Soy Bean Oil at the Injection Site, and at a Distance of 10 mm and 25 mm

 


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The results of the present study indicate that a) IM injection of soy bean oil and Ringer's solution does not modulate interstitial lactate concentrations, b) MH susceptibility induces a leftward shift of the dose-response curve of lactate after halothane and caffeine injection, and c) the regional IM distribution of lactate and carbon dioxide increase after local MH trigger application is limited to an area <10 mm around the stimulating probe.

In vivo microdialysis has been established as an excellent tool in experimental as well as in clinical pharmacology to measure interstitial target drug concentrations and metabolic variables (9). Based on a diffusion controlled process, dialyzable substances pass along a concentration gradient between two compartments separated by a semipermeable membrane into the perfusate of the microdialysis probe (6). This allows investigation of skeletal muscle metabolism without systemic involvement.

In the present study, interstitial metabolic changes were measured by microdialysis after local application of MH trigger drugs in MHS and MHN pigs.

An increased calcium release in predisposed individuals mainly via a mutated sarcoplasmic ryanodine receptor is widely accepted as a pathogenic mechanism for MH (10). The enhanced energy turnover during an MH episode finally results in hypoxia, hypercapnia, lactic acidosis, and heat production (1). A significantly increased muscular venous outflow of lactate reflects the metabolic deterioration in the course of MH (11). In this context, IM lactate measurement seems to be a suitable method to study MH hypermetabolism in vivo.

IM injection of caffeine 80 mM was shown to increase IM carbon dioxide concentration in MHS individuals (5). Similarly to caffeine, halothane mediates sarcoplasmic calcium release in skeletal muscle fibers (12) and increases local lactate concentrations (13). Halothane, dissolved in a lipophilic carrier like soy bean oil, allows IV anesthesia comparable to the inhaled route without associated organ failure (14), whereas pure IV injection causes severe cell damage (15).

Several findings support a dose-dependency for triggering MH. In the barnyard test, MHS in swine was diagnosed by inducing muscle rigidity with inhalation of halothane. On withdrawal of the volatile anesthetic, the pigs recovered quickly (16). In MHS humans, a variable intraindividual muscle contracture response in the diagnostic contracture test was reported (17). In addition, it remains unclear why some MHS individuals only develop single symptoms e.g., masseterspasm, rhabdomyolysis (18), or no significant clinical signs at all, while in other patients a fulminant MH is induced after the first exposure to trigger drugs. These facts point towards a dose-dependency of MH trigger drugs, although the threshold dose differs between and even within an individual depending on as yet unknown factors. In the present study, IM injection of varying halothane and caffeine concentrations revealed a left shift of the dose-response curve for lactate in MHS individuals compared with MHN individuals, suggesting local hypermetabolism and anaerobic glycolysis in MHS pigs. With maximal concentrations of halothane (10 vol%) and caffeine (80 mM), lactate increased even in MHN pigs and was not significantly different from MHS pigs. This finding supports the assumption of a dose-response relationship for MH.

To use the microdialysis technique as a minimally invasive method to detect MHS, serious local and systemic side effects caused by local trigger application must be excluded for patient safety. Interstitial microdialysis diffusion characteristics are well established (19) and demonstrate a steep decrease of drug concentration around the microdialysis probe. Although this diffusion model has not been proven for halothane or caffeine in skeletal muscle, it seems unlikely that the drug doses used herein, applied IM, would initiate the syndrome of MH. In the present study, there was no increase in local lactate or Pco2 concentrations at 10 mm and at 25 mm distance from the injection site after IM halothane and caffeine injection.

In conclusion, the present study clearly demonstrates that local IM lactate increase is modulated dose-dependently by IM injection of halothane and caffeine. The dose response curve for MHS pigs is shifted to the left. Furthermore, the local application of MH triggers does not lead to a spreading MH-like reaction and the increase of lactate and Pco2 by the trigger doses applied herein is limited to an area <10 mm around the injection site.


   Footnotes
 
This study was supported by departmental funding only.

Presented, in part, in a letter to the editor in The Lancet 2003;362:494-5.

Accepted for publication September 6, 2005.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Gronert GA, Antognini JF, Pessah IN. Malignant hyperthermia. In: Miller RD, ed. Anesthesia 5th ed. Philadelphia: Churchill Livingstone, 2000: 1033–52.
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  3. Larach MG, for the North American Malignant Hyperthermia Group.Standardization of the Caffeine Halothane Muscle Contracture Test. Anesth Analg 1989;69:511–5.[Free Full Text]
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  6. Ungerstedt U. Microdialysis: principles and applications for studies in animal and man. J Intern Med 1991;230:365–73.[ISI][Medline]
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  14. Musser JB, Fontana Jl, Mongan PD. The anesthetic and physiologic effects of an intravenous administration of halothane lipid emulsion (5% vol/vol). Anesth Analg 1999;88:671–5.[Abstract/Free Full Text]
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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press