IDENTIFICATION OF SUSCEPTIBILITY TO MALIGNANT HYPERPYREXIA IN SWINE

Br.J. Anaesth. (1979), 51, 171

IDENTIFICATION OF SUSCEPTIBILITY TO MALIGNANT HYPERPYREXIA IN SWINE F.

OKUMURA, B. D.

CROCKER AND M.

A.

DENBOROUGH

SUMMARY

Individuals who are susceptible to malignant hyperpyrexia (MH) have an underlying disease of muscle. The most precise means of identifying susceptibility to MH is pharmacological testing in vitro on muscle from the individual in question. Several different pharmacological tests are used. One examines the contracture response to caffeine (Kalow et al., 1970), another to halothane (Ellis et al., 1972), and a third studies the contracture responsejto a variety of agents including caffeine, halothane, suxamethonium and potassium chloride (Moulds and Denborough, 1974). In the present study an attempt has been made to define the most reliable pharmacological tests for identifying susceptibility to MH, using susceptible swine as an animal model. MATERIALS AND METHODS

Pure Landrace, pure Large White or crossbred Landrace-Large White swine were screened for susceptibility to MH using halothane anaesthesia. Anaesthesia was induced with 3 % halothane in oxygen via a face mask, and maintained with 1-1.5% halothane. Rectal temperature was monitored continuously using a thermistor. MH was diagnosed by a rapid increase in temperature, stiffness of the legs, abnormal respiration and blotchy cyanosis of the skin. F. OKUMURA,* M.D., PH.D.; B. D. CROCKER, B.SC; M. A. DENBOROUGH, M.D., D.PHIL., D.SC, F.R.C.P., F.R.A.C.P.;

Department of Clinical Science, The John Curtin School of Medical Research, The Australian National University, Canberra, A.C.T., Australia. * Present address: Department of Anaesthesia, Shiga Medical College, Shiga, Japan. 0007-0912/79/030171-06 S01.00

As soon as the diagnosis was made, the halothane was discontinued and the animal was cooled by sponging with water or alcohol. Tracheal intubation and artificial ventilation with pure oxygen was carried out and procaine hydrochloride 3-5 mg kg- 1 and sodium bicarbonate were given i.v. if necessary. All the control swine failed to develop abnormal symptoms or signs or an increase in temperature after halothane anaesthesia for 1 h followed by suxamethonium 2 mg kg - 1 i.v. Nineteen of 326 swine were susceptible, of which 10 died during the challenge. One pig which died developed MH only after being given halothane and suxamethonium. The remainder developed the syndrome with halothane alone. The nine survivors consisted of five Landrace, three crossbred Landrace-Large White and one Large White. The mean maximum body temperature during the challenge in the 19 susceptible swine was 40.2 + 1.2 (SD) °C and the mean time to peak temperature from the beginning of halothane administration was 27.3 ± 14.3 min. The mean increase in temperature was 2.0 ± 1.0 °C. Muscle biopsy and pharmacological testing

Muscle biopsy was performed in nine swine susceptible to MH and 16 control swine. Each pig was premedicated with ketamine hydrochloride 4-5 mg kg- 1 i.m. and diazepam 0.5-1 mg kg- 1 i.m. Tracheal intubation was performed after anaesthesia was induced with thiopentone 3-4 mg kg- 1 . Anaesthesia was maintained with nitrous oxide in oxygen, supplemented with thiopentone. © Macmillan Journals Ltd 1979

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In vitro muscle contracture responses in swine susceptible to malignant hyperpyrexia (MH) were similar to those found in muscle from humans susceptible to this anaesthetic complication, confirming the suitability of the pig as an animal model for studying MH. The results suggest that there are different degrees of susceptibility to MH. Whichever drug was used, there was some overlap in the contracture responses between susceptible animals and controls, suggesting that the most accurate way of identifying susceptibility to MH is to use a variety of chemical agents, the best of which seem to be halothane, caffeine, suxamethonium and potassium chloride. Thymol, which is used as a preservative in commercial preparations of halothane, potentiates halothane contractures, but it is not known if this is significant clinically.

172

BRITISH JOURNAL OF ANAESTHESIA

Statistical analysis Rigorous statistical analysis of the data was difficult, as there were large differences in the number of

muscle preparations taken from individual pigs. One-way analysis of variance, of the type used for random effects and unequal sample size, was performed on the results from MH-susceptible pigs where the data allowed. The muscle preparations used for any particular drug test in the control group came from a large number of swine and there were few preparations from any individual pig. For this reason, the data were unsuitable for such an analysis, since the within-pig variance could not be estimated accurately. The variances for the tests in the control group result mainly from between-pig variability. P values were obtained using Student's t test for unequal population variances, there being a greater variability in the MH-susceptible swine than in the control swine. The methods used were those described by Snedecor and Cochran (1967). RESULTS

The results of the analyses of variance are summarized in table I. Although variance ratios (F ratio) were not significant at either the 1 or 2.5% levels, they strongly suggest that heterogeneity exists in the MH-susceptible pig group, in that between-pig variability seemed larger than within-pig variability. Thus it was decided to estimate the components of variance and use these estimates to calculate the standard errors. In a single tension measurement the results show that, for the different drugs, between 77 and 88% results from within-pig differences and only 12-23% is associated with between-pig differences.

TABLE I. Summary of one-way analysis of variance of drug-induced contraction in muscle from MH-susceptible swine

Between animals INUIllLJCr Ul

Drug

Mean muscle preparations square d.f.*

Components of variance of a single measurement

Within animals Mean square d.f.

P'siiniisit'pr1 OalLUldLCL

F ratio

Variance Tabulated estimate F Between Within of grand ratio (2.5% point) animals animals mean

3% Halothane Caffeine 2 mmol litre" 1

58 47

0.45 0.32

7 8

0.22 0.13

50 38

2.0 2.5

2.6 2.6

0.032 0.038

0.222 0.127

0.008 0.008

KC1

34

1.40

8

0.92

25

1.5

2.8

0.133

0.922

0.047

46

0.08

8

0.04

37

1.9

2.6

0.007

0.041

0.002

30

0.44

3

0.14

26

3.1

3.7

0.044

0.141

0.019

80 mmol litre" 1 Suxamethonium 1 mmol litre" 1 Thymol 100 (imol litre" 1

* Between-animal d.f. + 1 = number of animals.

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Biopsy specimens (4 cm long, 1 cm wide and 0.5 cm thick) were removed from the gracilis muscle after it had been clamped, and placed immediately in a muscle buffer at 37 °C bubbled with 5% carbon dioxide in oxygen (carbogen). The methods of preparing the muscle strips for pharmacological testing and for measurement of tension have been described previously (Moulds and Denborough, 1974). The maximum ischaemic time from excision of the muscle to the end of the experiment was 5 h. The initial resting tension for each preparation was 1 g. Before commencing the contracture experiments a single electric stimulus of 30 V was used to confirm the suitability of the muscle preparation. The stock solutions of the drugs used were caffeine 100 mmol litre" 1 in muscle buffer, KC1 4 mol litre" 1 in water, 5% suxamethonium chloride in water and thymol 50 mmol litre" 1 in ethanol. Halothane was administered by passing carbogen through a calibrated Dragewick vaporizer. Dose-response curves for each drug were obtained at 37 °C. For suxamethonium, potassium chloride and thymol, single bolus injections were used. For caffeine, successive doses were added as soon as the maximal contracture plateau induced by the previous dose of caffeine had been reached. Subsequently a dose was selected for each drug which appeared to give the maximal separation between susceptible and control swine, and the efficacy of the different drugs in identifying susceptibility to MH was compared.

SUSCEPTIBILITY TO HYPERPYREXIA IN SWINE

173

Dose-response curves for caffeine, suxamethonium, potassium chloride and thymol are shown in figures 1-4.

o

a MHS pig muscle

x

x Control pig muscle F/73

o 1

o

o MHS pig muscle

x

x Control pig muscle

0 "

20 40 80 120 KCI (mmol litre"1)

MHS pig muscle Ol—u O.S

1

2

4

Control pig muscle

8 1

CAFFEINE (mmol litre' )

FIG. 1. Caffeine dose-response curves for contractures of gracilis muscle from swine susceptible to MH and from controls. The vertical bars represent SEM. *P<0.05; **P<0.01; ***P<0.005; ****P<0.001. 1.0

o

oMHS pig muscle

*

* Control pig muscle

0 0.5 25

50

100

200

THYMOL (pmol litre"1)

11

0 0725 0.5 1.5 Suxamethonium (mmol litre"1) FIG. 2. Suxamethonium dose-response curves for contractures of gracilis muscle from swine susceptible to MH and from controls. *P<0.05; ***P<0.005; ****P<0.001.

Each of the drugs produced greater contractures in muscle from swine susceptible to MH than in controls. The mean concentration of caffeine required to increase the tension of muscle by 1 g was 4.6 + (SEM) 0.7 mmol litre" 1 in the swine susceptible to MH and 9.5 + 0.7 mmol litre- 1 in the controls (P< 0.001). Halothane induced contracture in muscle from susceptible swine at concentrations of 0.5%, 1% and 3%, but induced little or no contracture in the control muscle (fig. 5).

FIG. 4. Thymol dose-response curves for contractures of gracilis muscle from swine susceptible to MH and from controls. ****P<0.001.

The optimal concentration of each drug for separating susceptible from normal swine was decided by inspection of the dose-response curves. The concentrations selected were caffeine 2 mmol litre"1, suxamethonium 1 mmol litre"1, KCI 80 mmol litre"1, thymol 100 (j.mol litre- 1 and 3% halothane. Responses to these concentrations of the different drugs in swine susceptible to MH were compared with the responses in controls; the standard errors in the susceptible swine data being calculated from the components of variance as in Snedecor and Cochran (1967). The results are shown in table II and figure 6. The mean muscle contraction was significantly greater (P< 0.001) in the susceptible swine than in the

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FIG. 3. Potassium chloride (KCI) dose-response curves for contractures of gracilis muscle from swine susceptible to MH and from controls. ****/>< 0.001.

BRITISH JOURNAL OF ANAESTHESIA

174

TABLE II. Drug-induced contraction in MH-susceptible swine (MHS) and in controls. The results expressed represent mean values + SEM Control swine

M H S swine

Muscle contraction

(g)

Number of muscle preparations

(g)

P

0.635 ±0.09 0.549 ±0.09 1.211 ±0.23 0.337 ±0.04 0.757 ±0.14

26 32 31 28 8

0.005 ±0.003 0.032 ±0.012 0.084 ±0.038 0.024 ±0.008 0.162 ±0.059

< 0.001 < 0.001 < 0.001 < 0.001 < 0.001

Muscle contraction

Drug

Number of muscle preparations

3% Halothane Caffeine 2 mmol litre" 1 KC1 80 mmol litre" 1 Suxamethonium 1 mmol litre" 1 Thymol 100 (xmol litre" 1

58 47 34 46 30

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In a single tension measurement on muscle from a randomly chosen MH-susceptible pig, the results show that between 77% and 88% of the variance for different drugs resulted from within-pig differences and only 12-23% was associated with between-pig differences. The within-pig differences reflect experi* o MHS pig muscle x mental factors which include a difference in the size x Control pig muscle of the muscle preparation, the temperature of the muscle bath and the ischaemic time from excision of the muscle to the end of the experiment. Even when the maximum possible care is taken in the preparation and in vitro testing of muscle samples from both humans and swine, there is overlap in the O O.5 -{Si, amount of contracture induced by each of the agents currently used in identifying susceptibility to MH. This implies that the most accurate means of identifying susceptibility to MH is not to use a single predictive test, but to expose the muscle in vitro to a 0 O5 1 3 variety of chemical agents, the most efficient of which, HALOTHANE ( % ) at present, seem to be caffeine 2 mmol litre"1, 3 % 1 mmol litre^1 and KC1 FIG. 5. Halothane dose-response curves for contractures of halothane, suxamethonium 1 gracilis muscle from swine susceptible to MH and from 80 mmol litre" . controls. ****p<0.001. Since thymol induces contracture of skeletal muscle (Ebashi, 1965) and because thymol is used as a DISCUSSION preservative in commercial preparations of halothane, The in vitro behaviour of muscle from swine suscep- the effects of thymol on muscle were examined also in tible to MH was similar to that of muscle from the present investigation. This seemed important as humans who are susceptible to this anaesthetic thymol appears to have a mode of action which is complication (Moulds and Denborough, 1974). This similar to but much more powerful than caffeine confirms the usefulness of the pig as an animal model (Ogawa, 1970). With a low concentration of thymol for studying MH. (100 (xmol litre"1) the mean muscle contracture was The statistical analysis suggests that there is greater in susceptible than in control swine, but there heterogeneity amongst MH swine in that between-pig was more overlap with thymol than with the other variability seemed larger than within-pig variability. drugs used. It is possible that a lower concentration of The between-pig differences are interesting and thymol (such as 75 (xmol litre"1) might identify confirm clinical observations that the degree of susceptible muscle more precisely (fig. 4). susceptibility may differ both in humans and in Although thymol is a potent inducer of skeletal swine. muscle contracture, the contractures induced by controls with each of the drugs (table II), but there was a wide scatter of results in susceptible swine with each of the drugs, and also some overlap in the results with the controls in each case (fig. 6).

SUSCEPTIBILITY TO HYPERPYREXIA IN SWINE CAFFEINE 2mmolliUe"

3*HALOTHANE

SUXAMETH Immol litre"1

175

THYMOL 100 umol litre' 1

KCI 80 mmol lit re' 1

2.0

5.0

1.8

4.5

1.6

4.0

1.4

3.5

0

1.2

3.0

e e

ItNt;ION

1.0

0.8

2.5

e e 2.0

0.6

1.5

0.4

1.0

0.2

0.5

CONTROL MHS

•

0

tHuuut

CONTROL MHS

I ee

J2b-

0

e e e

CONTROL MHS

CONTROL MHS

CONTROL MHS

FIG. 6. Contractions induced by caffeine 2 mmol litre" 1 , 3% halothane, suxamethonium 1 mmol litre" 1 , thymol 100 (imol litre" 1 and KCI 80 mmol litre" 1 in MH-susceptible swine (MHS) and in controls. Each symbol represents the contraction induced in a single muscle preparation. induced in susceptible swine muscle by thymol (fig. 7), and thymol potentiates halothane and potassium contractures (Okumura, Crocker and Denborough, 1978—unpublished observations), but it is not known if the thymol concentration in blood during halothane anaesthesia is sufficient to potentiate the halothane muscle contracture in an individual who is susceptible toMH. ACKNOWLEDGEMENTS 3% Halothane without Thymol

50

100

50

Thymol ((jmol litre"')

FIG. 7. Potentiation of thymol contracture by halothane in muscle from a pig susceptible to MH. The change in tension is shown on the vertical axis with time in minutes on the horizontal axis. Thymol 50 umol litre" 1 induced a contracture of 0.45 g in the presence of 3% halothane, but had no effect on its own. halothane in muscle from swine susceptible to MH did not appear to be related to the presence of thymol as preservative. Special halothane preparations free from thymol also induced contracture in MH muscle. Halothane does potentiate the muscle contraction

We are grateful to Professor P. A. P. Moran for statistical advice. We are also indebted to Dr C. Proctor of ICI Australia Limited for supplying us with thymol-free halothane for our experiments, and to Mrs J. Schmidt for expert technical assistance.

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TENS ION

en

REFERENCES

Ebashi, S. (1965). Molecular Biology of Muscular Contraction, p. 197. Tokyo: Igaku Shoin Ltd; Amsterdam: Elsevier. Ellis, F. R., Keaney, N. P., Harriman, D. G. F., Sumner, D. W., Kyei-Mensah, K., Tyrrell, J. H., Hargreaves, J. B., Parikh, R. K., and Mulrooney, P. L. (1972) Screening for malignant hyperpyrexia. Br. Med.J., 3,559. Kalow, W., Britt, B. A., Terreau, M. E., and Haist, C. (1970). Metabolic error of muscle metabolism after recovery from malignant hyperthermia. Lancet, 2, 895.

BRITISH JOURNAL OF ANAESTHESIA

176 Moulds, R. F. W., and Denborough, M. A. (1974). Biochemical basis of malignant hyperpyrexia. Br. Med.J., 2, 241. Ogawa, Y. (1970). Some properties of fragmented frog sarcoplasmic reticulum with particular reference to its response to caffeine. J. Biochem., 67, 667. Snedecor, G. W., and Cochran, W. G. (1967). Statistical Methods, 6th edn, p. 289. Ames: The Iowa State University Press. IDENTIFICATIONS DE LA SUSCEPTIBILITE A L'HYPERPYREXIE MALIGNE CHEZ LES PORCS RESUME

ZUSAMMENFASSUNG

Die Zusammenziehungsreflexe von Schweinemuskeln in vitro von Tieren die fiir bosartige Hyperpyrexie anfallig waren ahnelten denen von Menschen, die ftir diese Narkosekomplikation anfallig sind, damit wurde bestatigt, dass das Schwein als Versuchstier dienen kann, um bosartige Hyperpyrexie zu studieren. Die Resultate deuten an, dass es verschiedenen Grade der Anfalligkeit fur bosartige Hyperpyrexie gibt. Es kamzu einer Uberschneidung der Ergebnisse der zusammenziehungsreflexe—ganz gleich, welche Droge angewendet wurde—zwischen den anfalligen Tieren und ihrer Kontrollgruppe, was darauf hinweist, dass die genaueste Methode zur Identifikation der Anfalligkeit auf bosartige Hyperpyrexie die ist, die eine Anzahl verschiedener chemischer Mittel benutzt. Die besten scheinen Halothan, Koffein, Suxamethonium und Kaliumchlorid zu sein. Thymol, das als Konservierungsmittel in Halothanpraparationen in Handel angewendet wird, verstarkt die Halothanzusammenziehungen, aber es ist nicht bekannt, ob dies klinisch von Bedeutung ist. IDENTIFICACION DE SUSCEPTIBILIDAD A LA HIPERPIREXIA MALIGNA EN EL CERDO SUMARIO

Las respuestas de contractura muscular in vitro fueron semejantes en los cerdos susceptibles a hiperpirexia maligna (MH) a las producidas en el musculo humano susceptible a esta complicaci6n anestesica, confirmandose la conveniencia del cerdo como animal modelo para estudiar MH. Los resultados sugieren que hay diferentes grados de susceptibilidad a la MH. Con cualquiera de las drogas empleadas, se produjo cierta superposition de respuestas de contractura entre los animales susceptibles y los controles, lo que sugiere que la forma mas precisa de identificar la susceptibilidad a la MH es el empleo de una variedad de agentes quunicos, los mejores de los cuales parecen ser el halotano, cafeina, suxametonio y cloruro potasico. El timol, que se emplea como preservative en preparaciones comerciales de halotano, da potencia a las contracturas de halotano, pero no se sabe si esto tiene significado clinico.

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Les reactions a la contracture du muscle in vitro ont ete similaires, chez les pores susceptibles a l'hyperpyrexie maligne (MH), a celles observees sur les muscles humains susceptibles a cette complication anesthesique, ce qui confirme que le pore convient comme animal type pour etudier la MH. Les resultats laissent penser qu'il y a differents degres de susceptibilite a la MH. Quel que soit le medicament que Ton ait utilise, il y a eu certains chevauchements dans les reactions a la contracture entre les animaux susceptibles et les animaux temoins, ce qui laisse penser que la methode la plus precise d'identifier la susceptibilite a la MH est d'utiliser une variete d'agents chimiques, parmi lesquels les meilleurs semblent etre l'balothane, la cafeine, le suxamethonium et le chlorure de potassium. Le thymol que Ton utilise comme agent de preservation dans les preparations commerciales d'halothane, peut entrainer les contractures par l'halothane, mais on ne sait pas si cela a une importance du point de vue clinique.

IDENTIFIZIERUNG DER ANFALLIGKEIT FUR BOSARTIGE HYPERPYREXIE IN SCHWEINEN