4-Chloro-m-cresol Triggers Malignant Hyperthermia in Susceptible Swine at Doses Greatly Exceeding Those Found in Drug Preparations

After approval from the Institutional Animal Care and Use Committee of the University of Minnesota, the following experiments were conducted on pure-bred Pietrain swine with the a mutation of arginine 615 in the ryanodine receptor (University of Minnesota Rosemount Animal Facility). [20]Susceptibility to malignant hyperthermia was verified by in vitro contracture testing (according to the protocol recommended by the North American Malignant Hyperthermia Registry).

The animals (weight: 38.5 kg +/- 3.6 kg) were fasted (12-14 h) and subsequently anesthetized with a mixture of intramuscular tiletamine and zolazepam (Telazol, Fort Dodge, IA) and xylazine (TranquiVed, Vedco, St. Joseph, MO), 0.25 mg/kg of each; intubated; and mechanically ventilated. While probes were positioned for invasive monitoring, the animals were anesthetized with 70% N²O and thiopental. Paralysis was achieved during induction with a pancuronium bolus (6 +/- 2 mg) followed by a continuous during surgery of 30 +/- 12 mg. Mechanical ventilation was adjusted to maintain ET^CO(2) at 40 +/- 2 mmHg (Nellcor gas analyzer, Hayward, CA). The following physiological parameters were monitored throughout the studies: end-tidal CO², heart rate, arterial blood pressure, blood chemistry (arterial oxygen [Pa^O(2)] and carbon dioxide (Pa^CO(2)] tensions, pH, and K⁺, Na⁺, and HCO³levels) and temperatures (esophageal, rectal, vastus lateralis, liver, kidney, masseter, and tympanic). [21] 

During all surgical procedures, the animals were maintained at esophageal temperatures of 38.2 +/- 0.6 [degree sign]C. This was accomplished utilizing forced-air warming (Bair Hugger, Model 500, Augustine Medical, Eden Prairie, MN). [21]The rates and volumes of the inspired gases, which were not warmed or humidified, were not adjusted from the beginning of the control period or throughout the rest of the study. The arterial blood Pa^O(2), Pa^CO(2), pH, and K⁺, Na⁺, HCO³, and hematocrit levels were determined from fresh arterial samples using a blood gas analyzer with temperature compensation (Model BGE 1304, Instrumentation Laboratories, Lexington, MA).

A cannula was inserted into either the brachial or femoral artery for pressure monitoring using a SpaceLabs model 1020 (SpaceLabs, Chatsworth, CA) and blood sampling. The ear vein catheter (18-gauge Jelco, Johnson and Johnson Medical, Arlington, TX) served as the primary route for intravenous fluid administration (780 +/- 160 ml/h of Ringer's lactate solution). The heart rate was recorded using a five-lead electrocardiogram (SpaceLabs model 1020). Temperatures were recorded in the distal esophagus and rectum using Mon-a-Therm probes (Mallinckrodt Anesthesia Products, St. Louis, MO). [21]The electronic thermocouples were connected to a calibrated Iso-Thermex thermometer (Columbus Instruments, Columbus, OH), accurate to 0.1 [degree sign]C. All data were collected using LabVIEW software (National Instruments, Austin, TX) on a Macintosh computer (Apple Computer, Cupertino, CA).

Following the collection of the last set of control measurements (for a minimum of 30 min), 4-chloro-m-cresol (Pfaltz and Bauer, Waterbury, CT) was administered. The 4-chloro-m-cresol was prepared in normal saline to a concentrations of 5 mg/ml. This initial preparations required warming to approximately 50 [degree sign]C with constant stirring to facilitate dissolution. It was then allowed to cool to room temperature. To minimize acute cardiovascular effects, the 4-chloro-m-cresol solution was injected gradually (over a 1- to 10-min period, depending on the concentration; see Figure 1).

The animals were divided into two groups to which different doses of 4-chloro-m-cresol were given intravenously (see Figure 1). Group 1, the low-dose group, received 4-chloro-m-cresol boluses of 0.14, 0.14, 0.28, 0.57, 1.14, 2.27, and 4.54 mg/kg (12.5, 12.5, 25, 50, 100, 200, and 400 [micro sign]mol; n = 3), and group 2, the high-dose group, received boluses of 1.14, 2.27, 4.54, 9.08 (n = 4), and/or 18.18 (n = 1) mg/kg (100, 200, 400, 800, and/or 1,600 [micro sign]mol). These administered boluses resulted in equivalent cumulative doses of 0.14, 0.28, 0.57, 1.14, 2.27, 4.54, 9.08 mg/kg (12.5, 25, 50, 100, 200, 400, 800 [micro sign]mol) in the first group and 1.14, 3.41, 7.95, 17.04, 35.22 mg/kg (100, 300, 700, 1,500, 3,100 [micro sign]mol) in the second group. These concentrations were derived using an estimated blood volume of 80 ml/kg. If an animal did not trigger an episode of malignant hyperthermia by the end of its predesignated concentration run, halothane (0.87%) and succinylcholine (2 mg/kg) were administered, and in all such cases, episodes of malignant hyperthermia were initiated.

Plasma levels of 4-chloro-m-cresol were determined using high-performance liquid chromatography. The system consisted of a Spectra-Physics (San Jose, CA) model SP8810 pump; a Rheodyne (Cotati, CA) model 7010 injection valve with a 20-[micro sign]l sample loop; an Inertsil ODS-2 250 x 4.6-mm, 5-[micro sign]m column (MetaCham Technologies, Torrance, CA); and a Waters Chromatography (Milford, MA) model 481 spectrophotometer. The chromatographs were acquired and calculated using a Waters Baseline (810) chromatography workstation. The wavelength used was 217 nm. The mobile phase was 45% acetonitrile and 55% H²O, to which was added 2.0 ml/l glacial acetic acid and the pH of which was adjusted to 8.0 with sodium hydroxide. A stock standard was prepared from 97% pure 4-chloro-m-cresol to a concentration of 10 mM in 50% acetonitrile/water. Dilutions of this standard were prepared in water and diluted 1:1 with acetonitrile for injection into the column. The isolated plasma volumes were deproteinized with an equal volume of acetonitrile mixed vigorously and centrifuged to obtain a clear supernatant liquid, which was then injected into the chromatographic column. To estimate the concentrations of 4-chloro-m-cresol, the appropriate peak spectral areas associated with this agent in the plasma samples were subsequently compared with peak areas obtained for known standards. Drug recoveries done by adding known quantities of 4-chloro-m-cresol to blank porcine plasma yielded a mean recovery of 94.9% (n = 8).

Percent plasma hemolysis was determined by diluting 200 [micro sign]l of plasma into 2.0 ml of distilled water. The absorbance was read in a Beckman (Fullerton, CA) model 24 spectrophotometer in 1.0-cm quartz cuvettes against a water reference. The wavelength used was 540 nm, which is near the absorbance maximum for oxyhemoglobin. Reference standards were prepared by diluting 100 [micro sign]l of porcine whole blood into 20.0 ml of distilled water or 0.005% saponin in water, which caused complete hemolysis (Sigma Chemical, St. Louis, MO). Blood of several different hematocrits was used and a reference standard curve of 100% hemolysis was obtained using a linear regression. The absorbance of the reference blood was adjusted to compensate for the lower dilution of the unknown plasma. The standard curves for both water and saponin were virtually identical; therefore they were combined to yield a single reference line with a correlation coefficient of 0.991. Percent hemolysis was determined by dividing the absorbance value of the unknown plasma by the absorbance value of 100% hemolyzed whole blood of the same hematocrit and multiplying by 100.

In vitro testing was performed using rectus abdominis muscle and the protocol recommended by the North American Malignant Hyperthermia Registry on equipment previously described. [22,23]Additional muscle samples were obtained from normal swine (n = 4), which included both mongrel and "near" Pietrain (90-95% genetically similar to pure-bred) animals. All muscle samples were intact fiber bundles from the rectus abdominis muscle, dissected during the abdominal surgery and immediately placed in oxygenated Krebs solution for transport to an adjacent laboratory. Halothane was given as a 3% bolus for 10 min and contractures greater than 700 mg were necessary for a positive diagnosis. Caffeine was given in increasing doses of 0.5, 1, 2, 4, 8, and 32 mmol at 4-min intervals. A contracture greater than 200 mg at or below a concentration of 2 mmol was necessary for a positive diagnosis. For 4-chloro-m-cresol, increasing doses of 6.25, 12.5, 25, 50, 100, 200, 400, 800, and 1,600 [micro sign]mol were given at 4-min intervals.

The beginning of the 4-chloro-m-cresol administration was considered elapsed time 0. Data were analyzed using either repeated-measures analysis of variance or two-tailed, paired Student t tests with Bonferroni corrections as appropriate. Data are presented as mean +/- SD; P < 0.05 was considered statistically significant.

Episodes of malignant hyperthermia were not triggered in response to administration of low doses of 4-chloro-m-cresol, but transient cardiovascular reactions (e.g., tachycardia, arrhythmias, and hypotension) were observed (Figure 2). Subsequently, episodes in these animals were triggered when halothane (1 MAC) and succinylcholine (2 mg/kg) were given. However, animals administered the higher doses of 4-chloro-m-cresol all elicited fulminant episodes of malignant hyperthermia, when equivalent cumulative concentrations were 7.95 mg/kg (700 [micro sign]mol, n = 3) or in one case 17.04 mg/kg (1,500 [micro sign]mol; Figure 2).

The severity of the transient cardiovascular responses (tachycardia, arrhythmias, and hypotension) increased with increasing concentrations of 4-chloro-m-cresol. To minimize such acute cardiovascular effects, the 4-chloro-m-cresol solution was injected gradually (over a 1- to 10-min period, depending on the concentration; see Figure 1). Even then, in some cases, the mean arterial pressures dropped below 60 mmHg during the infusion of bolus doses greater than 400 [micro sign]M. Interestingly, all hemodynamic values returned to baseline within 5 min after completion of injection, with the exception being the initiation of fulminant episodes of malignant hyperthermia following exposure to high levels of 4-chloro-m-cresol (Figure 2).

The initiated malignant hyperthermia episodes in all cases resulted in death (n = 7). Arterial pH, Pa^O(2), Pa^CO(2), and K⁺level showed no statistical significant changes until 5 min into the fulminant malignant hyperthermia episode. The time in which it took the end-tidal CO²to reach 70 mmHg, from initial triggering, also showed no significant difference between the two groups of animals, even though the administration of halothane and succinylcholine was required for initiation in the group 1 animals (Figure 3). Proliferation rates determined for changes in Pa^CO(2) were similar between the two groups of animals (Figure 3). The Pa^CO(2) levels, for both groups, stayed well within control values until cumulative doses of 4-chlor-m-cresol exceeded 17.04 mg/kg (1,500 [micro sign]mol). Nevertheless, in all animals once an episode of malignant hyperthermia was initiated the Pa^CO(2) rose dramatically (see Table 1). Rises in core temperature were observed for the high-dose animals in which the 4-chloro-m-cresol did initiate episodes of malignant hyperthermia (Table 2).

Concentrations of 4-chloro-m-cresol in the blood rapidly decreased (Table 1). For example, 5 min after administration of the 7.95-mg/kg (700-[micro sign]mol) dose in the group 2 animals, (high dose) the mean plasma level was 19.31 [micro sign]mol +/- 4.35 (n = 4; see Table 1). These levels decreased so rapidly that, 15 min after the same dose, the mean plasma levels were 9.49 +/- 4.8 (n = 4; data not shown). Plasma concentrations obtained 5 min after the highest cumulative dose and subsequent levels measured thereafter again illustrate the rapid decline of 4-chloro-m-cresol in plasma (Figure 4). It was determined that the relative half-life of plasma concentrations of 4-chloro-m-cresol in these susceptible animals was approximately 20 min. It should be noted that concentrations of 4-chloro-cresol were found to be maintained in incubated fresh whole blood for up to 3 h and that no degradation within the blood was identified.

Visible hemolysis (i.e., detectable to the eye) was observed in plasma samples following 4-chloro-m-cresol concentrations > 3.41 mg/kg (300 [micro sign]mol). However, spectrophotometric analysis detected that significant hemolysis had occurred even following the administration of the 1.14-mg/kg (100-[micro sign]mol) cumulative dose (0.07 +/- 0.01%; see Table 1). The animals administered the lower doses elicited a maximum hemolysis of 0.98 +/- 0.23% 5 min after the maximum cumulative dose of 4-chloro-m-cresol was given; i.e., 9.08 mg/kg (800 [micro sign]mol). The hemolysis was even greater in the high-dose group, which provided a maximum hemolysis of 3.26 +/- 0.90% 5 min after the 17.04-mg/kg (1,500-[micro sign]mol) injection. These values became statistically significant from the predrug control values following the 4.54-mg/kg (400-[micro sign]mol) bolus in the low-dose group and at the 7.95-mg/kg (700-[micro sign]mol) dose in the high-dose group. Hemolysis occurred in whole blood alone as tested using fresh heparinized porcine blood: Cumulative doses of 4-chloro-m-cresol were added and incubated for 20 min at 37 [degree sign]C, and estimated hemolysis was found to be only slightly higher than in corresponding in vivo samples.

All muscle bundles obtained from the swine susceptible to malignant hyperthermia elicited positive in vitro contracture tests to both halothane and caffeine. Figure 5shows the in vitro comparison test of susceptible and normal bundles to caffeine and 4-chloro-m-cresol. All muscle bundles produced contractures >or= to 200 mg at 2 mmol caffeine, compared with 8 mmol in normal bundles (Figure 5). With 4-chloro-m-cresol, 100% of the susceptible bundles produced contractures >or= to 200 mg at 50 [micro sign]mol, compared with 1,600 [micro sign]mol in normal bundles (Figure 5). More specifically, 20 of 24 bundles exposed to 4-chloro-m-cresol produced contractures >or= to 200 mg at bath concentrations < 50 [micro sign]mol. In terms of diagnostic differentiation, caffeine provides a 4- to 16-fold concentration difference, and 4-chloro-m-cresol provides at least a 32-fold diagnostic concentration difference. Three percent halothane contracture testing was also performed (data not shown). During exposure to 3% halothane bath concentration, all the muscle bundles from the susceptible swine (n = 33) and none of the normal bundles (n = 10) had contractures > 700 mg.

Although 4-chloro-m-cresol can trigger fatal episodes of malignant hyperthermi in susceptible swine, the concentrations necessary should be considered nonpharmacologic. It is highly unlikely that an episode of malignant hyperthermia would occur through exposure to 4-chloro-m-cresol under normal clinical circumstances. For example, concentrations found in one available preparation of succinycholine, Midarin (Welcome, Pomezia, Italy), are low, and if one administered a 2-mg/kg dose of either a 20- or 50-mg/ml preparation, estimated blood concentrations would range between 0.01 and 0.06 [micro sign]M (see Table 3for additional product information).

An important finding in the present study was that 4-chloro-m-cresol is rapidly redistributed or metabolized in the swine susceptible to malignant hyperthermia, and thus plasma levels never each expected levels (e.g., 5 min following injection, plasma levels are approximately 2-4% of the expected levels). It should be noted that it was observed that 4-chloro-m-cresol is not metabolized within whole blood. One possible explanation for the rapid decline in plasma levels is the known ease of absorbability of cresols into fat, through the skin, into the respiratory tract, and into the gastrointestinal tract. [5,7,8]Although the rate and extent of absorption of cresols have not been studied specifically, studies have shown that both gastrointestinal and dermal absorption are rapid and extensive. [8]Cresols are also distributed in all the major organs. [5,7,8]The primary metabolic pathway for cresols is conjugation with glucuronic acid and inorganic sulfate. Minor metabolic pathways for cresols include hydroxylation of the benzene ring and side-chain oxidation. The main route of elimination of cresols from the body is renal elimination in the form of conjugates. [8] 

Other forms of cresols have also been suspected to be reactive, but previous work has shown that the meta form of chlorocresol is the most potent for inducing the release of intracellular Ca²⁺. [1]The in vivo bolus doses of 4-chloro-m-cresol required to initiate episodes of malignant hyperthermia in susceptible swine were found to be extremely high (> 9.08 mg/kg or 800 [micro sign]mol), and it was noted that such concentrations also cause cardiovascular aberrations and were cytotoxic for red blood cells (i.e., resulting in significant hemolysis). Such a hemolytic response is normally not observed following the administration of other known triggering agents: i.e., volatile anesthetic agents or succinylcholine administered at concentrations that will elicit episodes of malignant hyperthermia in such animals. It was noteworthy that the measured plasma levels at which episodes of malignant hyperthermia were induced were similar to those that elicit skeletal muscle contracture responses in vitro. More specifically, the 4-chloro-m-cresol levels were approximately 50 [micro sign]mol for contracture responses, > 200 mg for muscles bundles obtained from the susceptible animals. This compares well with the in vivo plasma levels (52 [micro sign]mol) obtained 5 min after the administration of the 17.04 mg/kg (1,500 [micro sign]mol) bolus (Table 1). Therefore, it is unlikely that the clinical use of substances containing a chlorocresol agent as a preservative will initiate an episode of malignant hyperthermia in a susceptible individual, because the ultimate circulating plasma levels would be extremely low. Hence, much like caffeine, patients susceptible to malignant hyperthermia should readily tolerate low levels of these agents within their bodies.

It is well established that succinylcholine is a trigger of episodes of malignant hyperthermia in susceptible swine. [26,27]The underlying mechanism by which succinylcholine initiates episodes of malignant hyperthermia remains unclear. Nevertheless, even in those preparations of succinylcholine that use chlorocresols as preservatives it is unlikely that the low concentrations of these agents would have an important contribution as to the onset of proliferation of a subsequently initiated epidose. As with other known triggers of malignant hyperthermia episodes in susceptible swine, once an event was initiated by high in vivo concentration of 4-chloro-m-cresol the proliferation rate of the event was rapid and similar to those previously reported for animals with core temperatures of approximately 38 [degree sign]C. [21,24-26] 

In previous reports it has been noted that unless certain experimental and physiologic criteria are not controlled during studies performed to initiate episodes of malignant hyperthermia in susceptible swine, such events will not proliferate. [21,24-26]Specifically, it has been shown that hypotension minimized the ability to trigger episodes with either succinylcholine or volatile anesthetics. Similarly, it was recently reported by our laboratory that the core temperature of the animal at the time of exposure to triggering agents is vital to control. [21]It should be noted that in the present experiments all animals maintained a high mean arterial pressure, and their core temperatures were maintained at or slightly above the normal core temperature in swine, 38 [degree sign]C. Such parameters were controlled to insure that the potential triggering response following various bolus concentrations of 4-chloro-m-cresol would not be attenuated.

The in vitro 4-chloro-m-cresol responses of the isolated skeletal muscle bundles from the susceptible animals were clearly different from normal muscle, and these results were consistent with previous findings for human muscle. [15-19]From a potential diagnostic standpoint, it is interesting to consider that caffeine contracture testing provided a 4- to 16-fold concentration difference, and 4-chloro-m-cresol provided at least 32-fold diagnostic concentration difference between normal and susceptible responses. Thus, the use of a dose-response, 4-chloro-m-cresol contracture test may be more reliable to administer than the caffeine test: e.g., this may decrease center-to-center variability. Nevertheless, it is possible that this strong differentiating response may be unique for the swine mutation and thus may not be as predictable as the presently used caffeine protocol recommended by the North American Malignant Hyperthermia Group for clinical diagnosis because of the heterogenic inheritance associated for malignant hyperthermia in humans. [23] 

The probable explanation for the in vitro contracture responses of skeletal muscle, as well as for the in vivo initiation of malignant hyperthermia, following the administration of 4-chloro-m-cresol is likely its known ability to modulate the gating of the calcium-release channel of the sarcoplasmic reticulum (the ryanodine receptor). [1-3]It is described here that high dosages of 4-chloro-m-cresol within the plasma caused transient cardiovascular reactions (e.g., tachycardia, arrhythmias, and severe hypotension) as well as cytotoxic or hemolytic responses. As this agent circulates through the cardiovascular system, it likely causes abnormal calcium regulation in cardiac tissue, vascular smooth muscle, and blood cells. Fortunately, the half-life of this agent is short, so its effects are relatively transient, except for the hemolysis that was noted to accumulate as larger bolus doses were administered.

It is concluded that 4-chloro-m-cresol will trigger episodes of malignant hyperthermia in susceptible swine when serum concentrations are sustained at critical levels that activate skeletal muscle intracellular Ca (⁺²) release. However, the serum levels of 4-chloro-m-cresol needed to initiate such events are far above those likely to be encountered in humans. Consequently, it is unlikely that the clinical use of substances containing these agents will initiate an episode of malignant hyperthermia in a susceptible individual and thus could be considered safe for use.

The authors thank V. Komanduri for her technical assistance; G. Williams for his help with the data analysis; and Nellcor Incorporated for the use of the gas analyzer.