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INFLUENCE OF pH, TEMPERATURE, HALOTHANE AND ITS METABOLITES ON OSMOTIC FRAGILITY OF ERYTHROCYTES OF MALIGNANT HYPERTHERMIA-SUSCEPTIBLE AND RESISTANT PIGS
Br. J. Anaesth. (1981), 53, 499
INFLUENCE OF pH, TEMPERATURE, HALOTHANE AND ITS METABOLITES ON OSMOTIC FRAGILITY OF ERYTHROCYTES OF MALIGNANT HYPERTHERMIA-SUSCEPTIBLE AND RESISTANT PIGS J. J. A. HEFFRON AND G. MITCHELL SUMMARY
There is much to suggest that erythrocyte fragility is increased in individuals and swine susceptible to malignant hyperthermia (MH). Harrison and Verburg (1973) found that the median erythrocyte fragility of MH-susceptible pigs lay between saline 0.094 and 0.086 mol litre" ' whereas that of control pigs lay between saline 0.086 and 0.077 mol litre" 1 . Similarly, King, Ollivier and Basrur, (1976) have shown that erythrocytes from susceptible pigs show an increase in haemolysis. More recently, susceptibility to MH in pigs has been associated with a deficiency of glutathione peroxidase in erythrocytes (Schanus, Glass and Lovrien, 1979). Such a deficiency would greatly increase the possibility of haemolysis. In man, Britt and Kalow (1970) noted the occurrence of haemolysis during an episode of MH and Zsigmond, Penner and Kothary (1978) have reported a MH proband with increased erythrocyte fragility. Reske-Nielsen (1978) found that erythrocyte fragility was increased in 10 members of a family susceptible to MH.
Such an inherent membrane defect is likely to be exacerbated by conditions prevailing under anaesthesia. Seeman and colleagues (1969) reported, for example, that many types of cell and intracellular membranes are stabilized at low concentrations of anaesthetic agents, but are lysed at greater concentrations. Since halothane anaesthesia is the most common cause of MH (Britt and Kalow, 1970) and since it has profound effects on cell membranes (Chang, Dudley and Lee, 1975), it is possible that halothane could precipitate haemolysis. Halothane, however, is not metabolically inert. In the liver it is metabolized to trifiuoroacetic acid (TFA), chloride and bromide (Rehder et al., 1967; Atallah and Geddes, 1973) and these substances could also cause haemolysis. MH is characterized by hyperthermia and lactacidosis, which alone or in combination with halothane or its metabolites may alter fragility. These observations, and the need to find a reliable, non-invasive test of susceptibility to MH have led us to investigate the effects of halothane, its metabolites, pH and temperature on the fraJ. J. A. HEFFRON,* PH.D.; G. MITCHELL^ PH.D., M.R.C.V.S.; gility of erythrocytes of MH-susceptible and resisDepartments of Physiological Chemstry and Physiology, University of the Witwatersrand Medical School, Hospital tant pigs.
Street, Johannesburg, South Africa. Present addresses: * Department of Biochemistry, University College Cork, Cork, Ireland. t Department of Physiology, School of Veterinary Science, Park Row, Bristol, England. Correspondence to J.H., Cork. 0007-0912/81/050499-06 J01.00
METHODS
Twelve pure-bred German Landrace pigs were obtained from a commercial herd with a high frequency of MH. After the experiments to be reported here were completed, a halothane chal© Macmillan Publications Ltd 1981
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Erythrocytc fragility of malignant hyperthermia-susccptible and resistant pigs has been examined as a function of pH and temperature in the absence and presence of halothane and its metabolites. A decrease in pH from 7.40 to 6.95 increased the fragility of both susceptible and resistant pigs, but the increase in susceptible animals was significantly greater than that in resistant animals. An increase in temperature led to a reduction of erythrocyte fragility of both types of pig. The most consistent differences in haemolysis between susceptible and resistant pigs occurred at sodium chloride concentrations of 0.120 and 0.137mollitre" '. At all pH values examined and at 39 and 41 °C, but not at 43°C, erythrocytes from susceptible pigs showed greater haemolysis in sodium chloride 0.120 and 0.137mollitre~' than did erythrocytes from resistant pigs. Halothane or its metabolites, at concentrations occurring during anaesthesia, did not alter erythrocyte fragility.
BRITISH JOURNAL OF ANAESTHESIA
500
brated with 3% halothane in nitrogen. After 15 min of halothane anaesthesia the concentration of blood bromide increases from a preoperative value of 100 umol litre "' to 125 umol litre " ' (Atallah and Geddes, 1973). Consequently, bromide 25 umol litre" 1 was added to each of the group III samples. Similarly TFA 25 umol litre"' was added to the group IV samples. After incubation a 20-ulitre sample from each of the incubated blood samples was added to 4 ml of each of the saline concentrations specified above. Thirty minutes later each saline-blood mixture was centrifuged at 1500^. The amount of haemoglobin in each supernatant was then determined spectrophotometrically by measuring absorbance at 543 nm. The supernatant from the tube containing sodium chloride 0.154mol litre"' was used as the blank for all readings in each series. Each analysis was done in duplicate. In the next series of experiments a second 20-ml sample was obtained from each of the 12 pigs in the same way. This sample was used to assess the effect of temperature, with and without added halothane and its metabolites, on osmotic fragility. In this experiment the samples were incubated at 39, 41 and 43 °C for 30min at a constant pH of 7.40. The
TABLE I. Scheme of procedure for determination of erythrocytc fragility
Heparinized blood 20 ml I Group II
Group I lml
1 ml
I
I
1 ml
I
pH7.40 7.15 6.95 39°Cfor 30 min
lml
lml
I
I
I Group III 1 ml
lml
I
1 ml
pH7.40
I
7.15
I
% Haemolysis measured in each tube
6 95
I
% Haemolysis at each concentration of NaCl averaged
lml
I
pH7.40 7.15 6.95 39°Cfor 30 min
Serial saline concentrations % Haemolysis measured in each tube
lml
TFA 25 umol litre"
39°Cfor 30 min pH7.40 7.15 6.95 39°Cfor 30 min Blood 20ulitre Blood 20nlitre Serial saline concentrations
lml
I
Bromide 25 umol litre"
3% Halothane in nitrogen
I
1 ml
I Group IV
repeated for each treatment, at each pH
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lenge (Heffron and Mitchell, 1975) identified three of the pigs as being MH-susceptible and nine as being MH-resistant. Heparinized blood was drawn from the jugular vein of each pig at the thoracic inlet through a 14gauge needle with minimum suction. Osmotic fragility of erythrocytes was determined by estimating the percentage haemolysis of a standard amount of blood placed in a series of buffered saline concentrations ranging from 0.154 mol litre""1 (0.9%) to 0.051 mol litre" 1 (0.3%; see also table II). The stock solution from which these dilutions were prepared contained sodium chloride 1.54mol litre" 1 buffered with phosphate 0.1 mol litre"', pH 7.4. The first series of experiments was designed to assess the effect of pH, with and without added halothane and halothane metabolites, on osmotic fragility. A 20-ml blood sample was taken from each of the 12 pigs for these experiments. The sample was divided into 12 x 1-ml aliquots which were grouped into four groups of 3 x 1 -ml samples as outlined in table I. Samples were incubated at 39 °C for 30min. The pH of the saline solutions was adjusted to 7.15 and 6.95 with HC1 1 mol litre"'. The samples in group II were equili-
FRAGILITY OF ERYTHROCYTES IN MHS AND RESISTANT PIGS
tant pigs at all saline concentrations in the range 0.077-0.137 mol litre" 1 . The mean osmotic fragility (F 50 ) increased significantly with decreasing pH in both types of pig (table III). The increase in F J 0 of erythrocytes from susceptible pigs at pH 6.95 was significantly greater than the increase in resistant pigs (table III). The effect of increasing temperature from 39 to 43 °C on erythrocyte fragility is shown in table IV. At 39 °C, erythrocyte fragility of susceptible pigs was greater than that of resistant pigs with saline 0.120 and 0.137 mol litre"' only. At 41 °C, significant differences in fragility were found with saline 0.051, 0.068, 0.077, 0.120 and 0.137 mol litre" 1 . However, no significant differences in fragility were found at 43 °C. F 5 0 values of both susceptible and resistant pigs decreased from 0.089 mol litre" 1 +0.0026 to 0.086 mol litre" 1
TABLE II. Mean percentage haemolysis in various concentrations of saline, at various pH, in resistant and susceptible pigs. MHS = susceptible; MHR = resistant; P = statistical significance; n.s. = not significant atP ^0.05. There were three MHS and nine MHRpigs
Sodium chloride (mol litre" ')
pH7.40 units MHS MHR P pH7.15 units MHS MHR P pH6.95 units MHS MHR P
0.051
0.068
0.077
0.086
0.094
0.103
0.120
0.137
92.5±4.6 97.4 ±0.7 n.s.
98.6±4.1 93.6 ±1.6 n.s.
79.6 ±1.0 77.4±2.3 n.s.
58.5±2.6 53.6±1.7 n.s.
40.5 ±4.5 36.2 ±4.3 n.s.
28.3 ±4.0 24.4 ±2.9 n.s.
20.6±1.7 13.6±2.1 0.025
5.0±1.0 1.1 ±0.7 0.01
99.4±0.3 97.3±0.3 0.001
95.3±0.4 98.3±2.5 n.s.
83.7±3.1 84.3±1.0 n.s.
65.8 ±2.4 57.9 ±0.9 0.02
46.0±4.6 43.2 ±2.9 n.s.
37.7 ±5.1 29.4±2.3 n.s.
22.2 ±1.9 14.3 ±1.2 0.005
6.8 ±2.0 1.9 ±0.6 0.05
99.0±0.5 97.4±0.7 n.s.
99.2±1.6 94.2 ±2.3 n.s.
88.9 ±3.0 85.5±2.5 n.s.
74.3±3.0 70.1 ±1.4 n.s.
55.7 ±3.9 52.8 ±1.7 n.s.
40.7 ±6.1 30.9±4.8 n.s.
28.4±2.4 16.0±0.6 0.001
9.4 ±1.9 4.5 ±0.9 0.05
significance for differences were performed using Student's t test. RESULTS
TABLE III. The effect of pH on mean osmotic fragility (Fi0). Temperature was 39°C; other abbreviatiions as in table II
F, 0 (NaCl, mol litre"1)
MH MH Table II shows the percentage haemolysis of susceptible resistant erythrocytes from MH-susceptible and resistant pigs over the range of saline concentrations at three pH (units) pH values, 7.40, 7.15 and 6.95 at a constant 7.40 0.087 ±0.0024 0.084±0.0033 temperature of 39 °C. At each pH, erythrocytes of 7.15 0.091 ±0.0027 0.087 ±0.0029 6.95 0.101 ±0.0022 0.094 ±0.0027 MH-susceptible pigs exhibited significantly greater haemolysis than those of resistant pigs at P 7.40 v. 7.15 n.s. n.s. sodium chloride concentrations of 0.120 and 1 7.15 v. 6.95 n.s. <0.05 0.137 mol litre" . Decreasing pH increased 7.40 v. 6.95 < 0.025 <0.01 erythrocyte fragility of both susceptible and resis-
r MHS v. MHR
n.s. n.s. <0.05
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temperature range chosen covers that commonly found during MH (Mitchell, Heffron and Van Rensburg, 1980). In all other respects the analysis of fragility was performed as described above. The data obtained from estimations of haemolysis at each concentration of saline in each series of dilutions were used to calculate the saline concentration at which 50% haemolysis (F 50 ) occurred. To obtain F 5 0 for each treatment the paired estimations of haemolysis at each saline concentration, for each temperature or pH and for each treatment were averaged. Thereafter the mean, and standard error of the mean, of the haemolysis occurring in all MH-resistant pigs and for all MHsusceptible pigs at each saline concentration and for each treatment were calculated. These data were plotted graphically, a best-fit curve drawn and the F 5 0 determined by inspection. Tests of
501
BRITISH JOURNAL OF ANAESTHESIA
502
TABLE IV. Mean percentage haemolysis at various concentrations of saline, at various temperatures, in resistant and susceptible pigs. For legend, see table II
Sodium chloride (mol litre '') Temp. 39
41
0.068
0.077
0.086
0.099
0.103
0.120
0.137
99.4 ±0.4 97.8±0.7 n.s. 99.5 ±0.2 98.6 ±0.2 0.01 98.4 ±0.5 97.7 ±0.8 n.s.
97.2 ±1.5 93.8 ±1.7 n.s. 96.2±1.5 91.7±1.1 0.05 94.9 ±1.8 92.9 ±0.4 n.s.
79.3 ±2.1 75.4±2.3 n.s. 80.8 ±2.3 74.1 ±0.8 0.02 75.6±3.0 70.6 ±1.5 n.s.
54.6 ±2.0 51.5±1.1 n.s. 57.5 ±3.3 50.6±l.l n.s. 48.4 + 6.0 44.1 ±1.3 n.s.
38.4±4.8 34.9±1.9 n.s. 35.4±2.4 33.4 ±1.3 n.s. 32.2±3.8 29.7±1.6 n.s.
26.8±2.5 23.5 ±1.6 n.s. 23.9±2.2 21.7±1.5 n.s. 21.3±1.6 18.9±4.2 n.s.
19.5±0.8 12.8±0.7 0.001 15.8±0.4 12.5±0.6 0.001 13.9±1.5 13.3±1.0 n.s.
4.4 ±1.2 0.5 ±0.6 0.02 3.7±1.1 1.1 ±0.2 0.05 1.5±0.7 1.4 ±0.4 n.s.
±0.0027 as the temperature increased from 39 to 43 °C and there was no difference between the values for the two types of pig. No significant effect of halothane, TFA or bromide on erythrocyte fragility of susceptible or resistant pigs was observed at any of the temperatures examined (data not shown). In all instances, F 5 0 varied from 0.089 to 0.082mol litre" 1 when temperature increased from 39 to 43 °C. Similar results were obtained when erythrocyte fragility was examined as a function of pH in the presence of halothane, TFA and bromide. DISCUSSION
Several tests are used for the pre-anaesthetic identification of predisposition to MH in man (Isaacs, 1978). The need for a reliable test of predisposition to MH in the pig arises because it is the only useful animal model for the human MH syndrome and because breeders wish to eliminate susceptible animals from their herds. Extrapolation of findings from experiments with susceptible pigs to MH in man is common. In these circumstances, unequivocal identification of susceptible pigs is necessary before proper conclusions can be drawn, especially in those experiments in which pigs are treated with putative therapeutic or preventative drugs before exposure to halothane. A good predictive test should be non-invasive, require the minimum of technical skill and be easy and rapid to carry out. For these reasons haematological tests have been used, the most common of which is measurement of serum creatine phosphokinase (CPK) activity (Isaacs and Barlow, 1970; Woolf et al., 1970). However, there are several constraints which make interpretation of
increases in CPK concentration difficult, and there are many causes of an increase in CPK concentration (Mitchell and Heffron, 1975; Britt et al., 1976). The observations by Harrison and Verburg (1973), King, Ollivier and Basrur (1976) and Cheah and Cheah (1979), that erythrocyte fragility is increased in susceptible pigs, offer an alternative, easy and more specific test. While our results corroborate the previous work in that there was a tendency for MH-susceptible pigs to have a greater erythrocyte fragility than normal, few clear differences emerged. In our experiments, as in those of King, Ollivier and Basrur (1976), significant increases in fragility occurred most consistently at sodium chloride concentrations of 0.120 and 0.137 mol litre" 1 , whereas Harrison and Verburg (1973) showed changes over the range sodium chloride 0.077-0.103 mol litre" 1 . A more serious divergence in the results is that the absolute values of haemolysis differed markedly. Harrison and Verburg (1973) showed 0.7% haemolysis with sodium chloride 0.128 mol litre" 1 , whereas King, Ollivier and Basrur (1976) found 83% with sodium chloride 0.120 mol litre" 1 and our results vary from 20 to 30% with sodium chloride 0.120mol litre" 1 . In these circumstances establishing normal values for haemolysis at particular sodium chloride concentrations against which all pigs could be evaluated, seems impossible. Although at 39 °C and pH 7.4 significant differences in fragility between susceptible and resistant pigs can be shown, differences are more marked at pH6.95. King, Ollivier and Basrur (1976) argue that prolonging the time allowed for haemolysis to occur will uncover more marked differences in
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43
MHS MHR P MHS MHR P MHS MHR P
0.051
FRAGILITY OF ERYTHROCYTES IN MHS AND RESISTANT PIGS
503
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Heffron, J. J. A., and Mitchell, G. (1975). Diagnostic value of serum creatine phosphokinase activity for the porcine malignant hyperthermia syndrome. Anesth. Analg. (Cleve.), 54, 536. Hende, C. v. d., Lister, D., Muyllc, E., Ooms, L., and Oyaert, W. (1976). Malignant hyperthermia in Landrace pigs. Br.J. Anaesth., 48, 821. Isaacs, H. (1978). Comments on predictive tests for malignant hyperthermia; in 2nd Internatl Symp. on Malignant Hyperthermia (eds J. A. Aldrete and B. A. Britt), p. 351. New York: Grune & Stratton. Barlow, M. B. (1970). The genetic background to malignant hyperthermia revealed by serum creatine phosphokinase estimations in asymptomatic relatives. Br. J. Anaesth., 42, 1077. King, W. A., Ollivicr, L., and Basrur, P. K. (1976). Erythrocyte osmotic response test on malignant hyperthermiasusceptible pigs. Arm. Genet. Sel. Anim., 8, 537. Mitchell, G., and Heffron, J. J. A. (1975). Factors affecting serum creatine phosphokinase activity in pigs. J. 5. Afr. Vet. Ass., 46, 145. van Rensburg, A. J. J. (1980). A halothane-induced biochemical defect in muscle of normal and malignant hyperthermia-susceptible Landrace pigs. Anesth. Analg. (Cleve.), 59, 250. Rehder, K., Forbes, J., Alter, H., Hessler, O., and Stier, A. (1967). Halothane biotransformation in man: a quantitative study. Anesthesiology, 28, 711. Reske-Nielsen, E. (1978). Malignant hyperthermia in Denmark: Survey of a family study and investigations into muscular morphology in ten additional cases; in 2nd Internatl Symp. on Malignant Hyperthermia (eds J. A. Aldrete and B. A. Britt), p. 287. New York: Grune & Stratton. Schanus, E., Glass, R., and Lovrien, R. (1979). Molecular basis ACKNOWLEDGEMENTS for malignant hyperthermia involving erythrocyte glutaWe acknowledge the help of M. Bagg. This research was thione peroxidase. Fed. Proc., 38, 571. supported by the Medical Research Council of South Africa Seeman, P., Kwant, W. O., Sauks, T., and Argent, W. (1969). and the Council of the University of the Witwatersrand. Membrane expansion of intact erythrocytes by anaesthetics. Biochim. Biophys. Ada, 183, 490. Woolf, N., Hall, L., Thome, S., Down, M., and Walker, R. REFERENCES (1970). Serum creatine phosphokinase levels in pigs reacting abnormally to halogenated anaesthetics. Br. Med. J., 3, 386. Atallah, M. M., and Geddes, I. C. (1973). Metabolism of halothane during and after anaesthesia in man. Br. J. Zsigmond, E. K., Penner, ]., and Kothary, S. P. (1978). Normal erythrocyte fragility and abnormal platelet aggreAnaesth., 45, 464. gation in MH families: a pilot study; in 2nd Internatl Symp. Bermari, M. C , Harrison, G. G., Bull, A. B., and Kench, J. E. on Malignant Hyperthermia (eds J. A. Aldrete and B. A. (1970). Changes underlying halothane-induced malignant Britt), p. 213. New York: Grune & Stratton. hyperpyrexia in Landrace pigs. Nature (Land.), 225, 653. Britt, B. A., Endrenyi, L., Peters, P. L., Kwong, F. H. F., and Kadjivek, L. (1976). Screening of malignant hyperthermia susceptible families by creatine phosphokinase measurement INFLUENCE DU pH, DE LA TEMPERATURE, DE and other clinical investigations. Can. Anaesth. Soc. J., 23, L'HALOTHANE ET DE SES METABOLITES SUR LA 263. FRAGILITE OSMOTIQUE DES ERYTHROCYTES Kalow, W. (1970). Malignant hyperthermia: aetiology CHEZ LES COCHONS SUSCEPTIBLES OU unknown. Can. Anaesth. Soc.J., 17, 316. RESISTANTS A L'HYPERTHERMIE MALIGNE Chang, L. W., Dudley, A. W., and Lee, Y. K. (1975). Ultrastructural changes in the kidney following chronic RESUME exposure to low levels of halothane. Am.J. Pathol., 78, 225. Cheah, K. S., and Cheah, A. M. (1979). Mitochondrial On a examine la fragilite des erythrocytes chez les cochons calcium, erythrocyte fragility and porcine malignant hyper- susceptibles ou resistants a 1'hyperthermie maligne, en tant que thermia. FEBS Lett., 107, 265. fonction du pH et de la temperature, lorsque l'halothane et ses Harrison, G. G., and Vcrburg, C. (1973). Erythrocyte osmotic metabolites etaient absents et lorsqu'ils etaient presents. La fragility in hyperthermia-susceptiblc swine. Br.J. Anaesth., baisse du pH dc 7,40 a 6,95 a augmente la fragilite des cochons 45, 131. susceptibles et celles des cochons resistants, mais cette
fragility. However, our results suggest that this is unlikely since, in our experiments, haemolysis was allowed to take place over 30min, six times longer than in the study of King, Ollivier and Basrur. We suggest, therefore, that exposing erythrocytes to both an osmotic and pH shock may reveal more marked differences in fragility. Our results provide evidence that halothane and its metabolites at anaesthetic concentrations do not increase erythrocyte fragility. The effects of high temperature and low pH combined with those of halothane and its metabolites are also negligible. The reported haemolysis occurring during MH (Britt and Kalow, 1970) is unlikely to be a result of any effect of halothane or its metabolites. Haemolysis is most likely to occur when a severe acidosis precedes an increase in temperature, since high temperatures tend to stabilize the red cell membrane whereas low pH increases fragility. However, in pigs, these specific circumstances appear to occur rarely, since studies of changes in plasma constituents during episodes of MH have reported no increase in plasma haemoglobin concentration (Berman et al., 1970; Hende et al., 1976).
BRITISH JOURNAL OF ANAESTHESIA
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von 0,120 und 0,137 mol litre '. Bei alien untersuchten pHWerten und bei 39 und 41 °C, aber nicht bei 43 °C, zeigten die Erythrozyten anfalliger Schweine eine grossere Hamolyse bei 0,120 und 0,137 mol litre"' als die Erythrozyten nichtanfalliger Schweine. Halothan und dessen Metaboliten ergaben in wahrend der Narkose vorhandenen Konzentrationen keine Anderung der Fragilitat von Erythrozyten. INFLUENCIA DEL pH, DE LA TEMPERATURA, DEL HALOTANO Y DE SUS METABOLITOS SOBRE LA FRAGILIDAD OSMOTICA DE LOS ERITROCITOS EN CERDOS RESISTENTES Y SUSCEPTIBLES A LA HIPERTERMIA MALIGNA SUMARIO
EINFLUSS VON pH, TEMPERATUR, HALOTHAN UND SEINER METABOLITEN AUF DIE OSMOTISCHE FRAGILITAT DER ERYTHROZYTEN BEI SCHWEINEN, DIE AUF MALIGNE HYPERTHERMIE ANFALLIG UND NICHTANFALLIG SIND ZUSAMMENFASSUNG
Die Erythrozyten-Fragilitat von auf maligne Hyperthermie anfalligen und nicht-anialligen Schweinen wurde als Funktion von pH und Temperatur untersucht, in Abwcsenheit und Anwesenheit von Halothan und seinen Metaboliten. Ein pHAbsticg von 7,40 auf 6,95 erhohte die Fragilitat bei beiden Schweinegruppen, doch war diese Erhohung wesentlich grosser bei anfalligen als bei nich-anfalligen Schweinen. Ein Temperaturanstieg reduziene die Fragilitat bei beiden Gruppen. Die Hauptunterschicde in dcr Hamolyse bei beiden Gruppen ergaben sich bei Natriumchloridkonzentrationen
Se examino la fragilidad de los eritrocitos de cerdos resistentes y susceptibles a la hipertermia maligna cual una funcion del pH y de la temperature, en ausencia y en prcsencia de halotano y dc sus metabolitos. Una disminucion del desdc 7,40 a 6,95 incremento la fragilidad tanto dc los cerdos resistentes como de los susceptibles, pero el incremento en los animales susceptibles fuc significativamente superior al acaecido en los resistentes. Un incremento de la temperature produjo una reduccion de la fragilidad de los eritrocitos en ambos tipos de cerdo. Las diferencias mas consistentes en hemolisis, entre los cerdos susceptibles y los resistente, tuvieron lugar bajo concentraciones de cloruro sodico de 0,120 y de 0,137 mol litro "'. Para todos los valores de pH examinados y a temperatures de 39 y de 41 °C, pero no a 43 °C, los eritrocitos pertenecientes a los cerdos susceptibles mostraron una mayor hemolisis en cloruro sodico al 0,120 y al 0,137 mol litro" ' que los correspondientes a los cerdos resistentes. Ni el halotano ni sus metabolitos llegaron a alterar la fragilidad de los eritrocitos a las concentraciones presentes durante la anestesia.
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augmentation a ete chez les animaux susceptibles beaucoup plus forte que chex les animaux resistants. L'augmentation de la temperature a conduit a une baisse de la fragilite des erythrocytes chez les deux types de cochons. Les differences les plus regulieres dans l'hemolyse, entre les cochons susceptibles et les cochons resistants, ont etc evidentes lorsque les concentrations de chlorure de sodium etaient de 0,120 et de 0,137 mol litre " '. A toutes les valeurs de pH examinees et a 39 ° et a 41 °C, mais pas a 43 °C, les erythrocytes des cochons susceptibles ont montre une plus grande concentration de chlorure de sodium dans l'hemolyse: 0,120 et 0,137 mol litre"', que les erythrocytes des cochons resistants. L'halothane, ou ses metabolites, aux concentrations rencontrees pendant l'anesthesie, n'ont pas modine la fragilite des erythrocytes.
INFLUENCE OF pH, TEMPERATURE, HALOTHANE AND ITS METABOLITES ON OSMOTIC FRAGILITY OF ERYTHROCYTES OF MALIGNANT HYPERTHERMIA-SUSCEPTIBLE AND RESISTANT PIGS J. J. A. HEFFRON AND G. MITCHELL SUMMARY
There is much to suggest that erythrocyte fragility is increased in individuals and swine susceptible to malignant hyperthermia (MH). Harrison and Verburg (1973) found that the median erythrocyte fragility of MH-susceptible pigs lay between saline 0.094 and 0.086 mol litre" ' whereas that of control pigs lay between saline 0.086 and 0.077 mol litre" 1 . Similarly, King, Ollivier and Basrur, (1976) have shown that erythrocytes from susceptible pigs show an increase in haemolysis. More recently, susceptibility to MH in pigs has been associated with a deficiency of glutathione peroxidase in erythrocytes (Schanus, Glass and Lovrien, 1979). Such a deficiency would greatly increase the possibility of haemolysis. In man, Britt and Kalow (1970) noted the occurrence of haemolysis during an episode of MH and Zsigmond, Penner and Kothary (1978) have reported a MH proband with increased erythrocyte fragility. Reske-Nielsen (1978) found that erythrocyte fragility was increased in 10 members of a family susceptible to MH.
Such an inherent membrane defect is likely to be exacerbated by conditions prevailing under anaesthesia. Seeman and colleagues (1969) reported, for example, that many types of cell and intracellular membranes are stabilized at low concentrations of anaesthetic agents, but are lysed at greater concentrations. Since halothane anaesthesia is the most common cause of MH (Britt and Kalow, 1970) and since it has profound effects on cell membranes (Chang, Dudley and Lee, 1975), it is possible that halothane could precipitate haemolysis. Halothane, however, is not metabolically inert. In the liver it is metabolized to trifiuoroacetic acid (TFA), chloride and bromide (Rehder et al., 1967; Atallah and Geddes, 1973) and these substances could also cause haemolysis. MH is characterized by hyperthermia and lactacidosis, which alone or in combination with halothane or its metabolites may alter fragility. These observations, and the need to find a reliable, non-invasive test of susceptibility to MH have led us to investigate the effects of halothane, its metabolites, pH and temperature on the fraJ. J. A. HEFFRON,* PH.D.; G. MITCHELL^ PH.D., M.R.C.V.S.; gility of erythrocytes of MH-susceptible and resisDepartments of Physiological Chemstry and Physiology, University of the Witwatersrand Medical School, Hospital tant pigs.
Street, Johannesburg, South Africa. Present addresses: * Department of Biochemistry, University College Cork, Cork, Ireland. t Department of Physiology, School of Veterinary Science, Park Row, Bristol, England. Correspondence to J.H., Cork. 0007-0912/81/050499-06 J01.00
METHODS
Twelve pure-bred German Landrace pigs were obtained from a commercial herd with a high frequency of MH. After the experiments to be reported here were completed, a halothane chal© Macmillan Publications Ltd 1981
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Erythrocytc fragility of malignant hyperthermia-susccptible and resistant pigs has been examined as a function of pH and temperature in the absence and presence of halothane and its metabolites. A decrease in pH from 7.40 to 6.95 increased the fragility of both susceptible and resistant pigs, but the increase in susceptible animals was significantly greater than that in resistant animals. An increase in temperature led to a reduction of erythrocyte fragility of both types of pig. The most consistent differences in haemolysis between susceptible and resistant pigs occurred at sodium chloride concentrations of 0.120 and 0.137mollitre" '. At all pH values examined and at 39 and 41 °C, but not at 43°C, erythrocytes from susceptible pigs showed greater haemolysis in sodium chloride 0.120 and 0.137mollitre~' than did erythrocytes from resistant pigs. Halothane or its metabolites, at concentrations occurring during anaesthesia, did not alter erythrocyte fragility.
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brated with 3% halothane in nitrogen. After 15 min of halothane anaesthesia the concentration of blood bromide increases from a preoperative value of 100 umol litre "' to 125 umol litre " ' (Atallah and Geddes, 1973). Consequently, bromide 25 umol litre" 1 was added to each of the group III samples. Similarly TFA 25 umol litre"' was added to the group IV samples. After incubation a 20-ulitre sample from each of the incubated blood samples was added to 4 ml of each of the saline concentrations specified above. Thirty minutes later each saline-blood mixture was centrifuged at 1500^. The amount of haemoglobin in each supernatant was then determined spectrophotometrically by measuring absorbance at 543 nm. The supernatant from the tube containing sodium chloride 0.154mol litre"' was used as the blank for all readings in each series. Each analysis was done in duplicate. In the next series of experiments a second 20-ml sample was obtained from each of the 12 pigs in the same way. This sample was used to assess the effect of temperature, with and without added halothane and its metabolites, on osmotic fragility. In this experiment the samples were incubated at 39, 41 and 43 °C for 30min at a constant pH of 7.40. The
TABLE I. Scheme of procedure for determination of erythrocytc fragility
Heparinized blood 20 ml I Group II
Group I lml
1 ml
I
I
1 ml
I
pH7.40 7.15 6.95 39°Cfor 30 min
lml
lml
I
I
I Group III 1 ml
lml
I
1 ml
pH7.40
I
7.15
I
% Haemolysis measured in each tube
6 95
I
% Haemolysis at each concentration of NaCl averaged
lml
I
pH7.40 7.15 6.95 39°Cfor 30 min
Serial saline concentrations % Haemolysis measured in each tube
lml
TFA 25 umol litre"
39°Cfor 30 min pH7.40 7.15 6.95 39°Cfor 30 min Blood 20ulitre Blood 20nlitre Serial saline concentrations
lml
I
Bromide 25 umol litre"
3% Halothane in nitrogen
I
1 ml
I Group IV
repeated for each treatment, at each pH
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lenge (Heffron and Mitchell, 1975) identified three of the pigs as being MH-susceptible and nine as being MH-resistant. Heparinized blood was drawn from the jugular vein of each pig at the thoracic inlet through a 14gauge needle with minimum suction. Osmotic fragility of erythrocytes was determined by estimating the percentage haemolysis of a standard amount of blood placed in a series of buffered saline concentrations ranging from 0.154 mol litre""1 (0.9%) to 0.051 mol litre" 1 (0.3%; see also table II). The stock solution from which these dilutions were prepared contained sodium chloride 1.54mol litre" 1 buffered with phosphate 0.1 mol litre"', pH 7.4. The first series of experiments was designed to assess the effect of pH, with and without added halothane and halothane metabolites, on osmotic fragility. A 20-ml blood sample was taken from each of the 12 pigs for these experiments. The sample was divided into 12 x 1-ml aliquots which were grouped into four groups of 3 x 1 -ml samples as outlined in table I. Samples were incubated at 39 °C for 30min. The pH of the saline solutions was adjusted to 7.15 and 6.95 with HC1 1 mol litre"'. The samples in group II were equili-
FRAGILITY OF ERYTHROCYTES IN MHS AND RESISTANT PIGS
tant pigs at all saline concentrations in the range 0.077-0.137 mol litre" 1 . The mean osmotic fragility (F 50 ) increased significantly with decreasing pH in both types of pig (table III). The increase in F J 0 of erythrocytes from susceptible pigs at pH 6.95 was significantly greater than the increase in resistant pigs (table III). The effect of increasing temperature from 39 to 43 °C on erythrocyte fragility is shown in table IV. At 39 °C, erythrocyte fragility of susceptible pigs was greater than that of resistant pigs with saline 0.120 and 0.137 mol litre"' only. At 41 °C, significant differences in fragility were found with saline 0.051, 0.068, 0.077, 0.120 and 0.137 mol litre" 1 . However, no significant differences in fragility were found at 43 °C. F 5 0 values of both susceptible and resistant pigs decreased from 0.089 mol litre" 1 +0.0026 to 0.086 mol litre" 1
TABLE II. Mean percentage haemolysis in various concentrations of saline, at various pH, in resistant and susceptible pigs. MHS = susceptible; MHR = resistant; P = statistical significance; n.s. = not significant atP ^0.05. There were three MHS and nine MHRpigs
Sodium chloride (mol litre" ')
pH7.40 units MHS MHR P pH7.15 units MHS MHR P pH6.95 units MHS MHR P
0.051
0.068
0.077
0.086
0.094
0.103
0.120
0.137
92.5±4.6 97.4 ±0.7 n.s.
98.6±4.1 93.6 ±1.6 n.s.
79.6 ±1.0 77.4±2.3 n.s.
58.5±2.6 53.6±1.7 n.s.
40.5 ±4.5 36.2 ±4.3 n.s.
28.3 ±4.0 24.4 ±2.9 n.s.
20.6±1.7 13.6±2.1 0.025
5.0±1.0 1.1 ±0.7 0.01
99.4±0.3 97.3±0.3 0.001
95.3±0.4 98.3±2.5 n.s.
83.7±3.1 84.3±1.0 n.s.
65.8 ±2.4 57.9 ±0.9 0.02
46.0±4.6 43.2 ±2.9 n.s.
37.7 ±5.1 29.4±2.3 n.s.
22.2 ±1.9 14.3 ±1.2 0.005
6.8 ±2.0 1.9 ±0.6 0.05
99.0±0.5 97.4±0.7 n.s.
99.2±1.6 94.2 ±2.3 n.s.
88.9 ±3.0 85.5±2.5 n.s.
74.3±3.0 70.1 ±1.4 n.s.
55.7 ±3.9 52.8 ±1.7 n.s.
40.7 ±6.1 30.9±4.8 n.s.
28.4±2.4 16.0±0.6 0.001
9.4 ±1.9 4.5 ±0.9 0.05
significance for differences were performed using Student's t test. RESULTS
TABLE III. The effect of pH on mean osmotic fragility (Fi0). Temperature was 39°C; other abbreviatiions as in table II
F, 0 (NaCl, mol litre"1)
MH MH Table II shows the percentage haemolysis of susceptible resistant erythrocytes from MH-susceptible and resistant pigs over the range of saline concentrations at three pH (units) pH values, 7.40, 7.15 and 6.95 at a constant 7.40 0.087 ±0.0024 0.084±0.0033 temperature of 39 °C. At each pH, erythrocytes of 7.15 0.091 ±0.0027 0.087 ±0.0029 6.95 0.101 ±0.0022 0.094 ±0.0027 MH-susceptible pigs exhibited significantly greater haemolysis than those of resistant pigs at P 7.40 v. 7.15 n.s. n.s. sodium chloride concentrations of 0.120 and 1 7.15 v. 6.95 n.s. <0.05 0.137 mol litre" . Decreasing pH increased 7.40 v. 6.95 < 0.025 <0.01 erythrocyte fragility of both susceptible and resis-
r MHS v. MHR
n.s. n.s. <0.05
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temperature range chosen covers that commonly found during MH (Mitchell, Heffron and Van Rensburg, 1980). In all other respects the analysis of fragility was performed as described above. The data obtained from estimations of haemolysis at each concentration of saline in each series of dilutions were used to calculate the saline concentration at which 50% haemolysis (F 50 ) occurred. To obtain F 5 0 for each treatment the paired estimations of haemolysis at each saline concentration, for each temperature or pH and for each treatment were averaged. Thereafter the mean, and standard error of the mean, of the haemolysis occurring in all MH-resistant pigs and for all MHsusceptible pigs at each saline concentration and for each treatment were calculated. These data were plotted graphically, a best-fit curve drawn and the F 5 0 determined by inspection. Tests of
501
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502
TABLE IV. Mean percentage haemolysis at various concentrations of saline, at various temperatures, in resistant and susceptible pigs. For legend, see table II
Sodium chloride (mol litre '') Temp. 39
41
0.068
0.077
0.086
0.099
0.103
0.120
0.137
99.4 ±0.4 97.8±0.7 n.s. 99.5 ±0.2 98.6 ±0.2 0.01 98.4 ±0.5 97.7 ±0.8 n.s.
97.2 ±1.5 93.8 ±1.7 n.s. 96.2±1.5 91.7±1.1 0.05 94.9 ±1.8 92.9 ±0.4 n.s.
79.3 ±2.1 75.4±2.3 n.s. 80.8 ±2.3 74.1 ±0.8 0.02 75.6±3.0 70.6 ±1.5 n.s.
54.6 ±2.0 51.5±1.1 n.s. 57.5 ±3.3 50.6±l.l n.s. 48.4 + 6.0 44.1 ±1.3 n.s.
38.4±4.8 34.9±1.9 n.s. 35.4±2.4 33.4 ±1.3 n.s. 32.2±3.8 29.7±1.6 n.s.
26.8±2.5 23.5 ±1.6 n.s. 23.9±2.2 21.7±1.5 n.s. 21.3±1.6 18.9±4.2 n.s.
19.5±0.8 12.8±0.7 0.001 15.8±0.4 12.5±0.6 0.001 13.9±1.5 13.3±1.0 n.s.
4.4 ±1.2 0.5 ±0.6 0.02 3.7±1.1 1.1 ±0.2 0.05 1.5±0.7 1.4 ±0.4 n.s.
±0.0027 as the temperature increased from 39 to 43 °C and there was no difference between the values for the two types of pig. No significant effect of halothane, TFA or bromide on erythrocyte fragility of susceptible or resistant pigs was observed at any of the temperatures examined (data not shown). In all instances, F 5 0 varied from 0.089 to 0.082mol litre" 1 when temperature increased from 39 to 43 °C. Similar results were obtained when erythrocyte fragility was examined as a function of pH in the presence of halothane, TFA and bromide. DISCUSSION
Several tests are used for the pre-anaesthetic identification of predisposition to MH in man (Isaacs, 1978). The need for a reliable test of predisposition to MH in the pig arises because it is the only useful animal model for the human MH syndrome and because breeders wish to eliminate susceptible animals from their herds. Extrapolation of findings from experiments with susceptible pigs to MH in man is common. In these circumstances, unequivocal identification of susceptible pigs is necessary before proper conclusions can be drawn, especially in those experiments in which pigs are treated with putative therapeutic or preventative drugs before exposure to halothane. A good predictive test should be non-invasive, require the minimum of technical skill and be easy and rapid to carry out. For these reasons haematological tests have been used, the most common of which is measurement of serum creatine phosphokinase (CPK) activity (Isaacs and Barlow, 1970; Woolf et al., 1970). However, there are several constraints which make interpretation of
increases in CPK concentration difficult, and there are many causes of an increase in CPK concentration (Mitchell and Heffron, 1975; Britt et al., 1976). The observations by Harrison and Verburg (1973), King, Ollivier and Basrur (1976) and Cheah and Cheah (1979), that erythrocyte fragility is increased in susceptible pigs, offer an alternative, easy and more specific test. While our results corroborate the previous work in that there was a tendency for MH-susceptible pigs to have a greater erythrocyte fragility than normal, few clear differences emerged. In our experiments, as in those of King, Ollivier and Basrur (1976), significant increases in fragility occurred most consistently at sodium chloride concentrations of 0.120 and 0.137 mol litre" 1 , whereas Harrison and Verburg (1973) showed changes over the range sodium chloride 0.077-0.103 mol litre" 1 . A more serious divergence in the results is that the absolute values of haemolysis differed markedly. Harrison and Verburg (1973) showed 0.7% haemolysis with sodium chloride 0.128 mol litre" 1 , whereas King, Ollivier and Basrur (1976) found 83% with sodium chloride 0.120 mol litre" 1 and our results vary from 20 to 30% with sodium chloride 0.120mol litre" 1 . In these circumstances establishing normal values for haemolysis at particular sodium chloride concentrations against which all pigs could be evaluated, seems impossible. Although at 39 °C and pH 7.4 significant differences in fragility between susceptible and resistant pigs can be shown, differences are more marked at pH6.95. King, Ollivier and Basrur (1976) argue that prolonging the time allowed for haemolysis to occur will uncover more marked differences in
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43
MHS MHR P MHS MHR P MHS MHR P
0.051
FRAGILITY OF ERYTHROCYTES IN MHS AND RESISTANT PIGS
503
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Heffron, J. J. A., and Mitchell, G. (1975). Diagnostic value of serum creatine phosphokinase activity for the porcine malignant hyperthermia syndrome. Anesth. Analg. (Cleve.), 54, 536. Hende, C. v. d., Lister, D., Muyllc, E., Ooms, L., and Oyaert, W. (1976). Malignant hyperthermia in Landrace pigs. Br.J. Anaesth., 48, 821. Isaacs, H. (1978). Comments on predictive tests for malignant hyperthermia; in 2nd Internatl Symp. on Malignant Hyperthermia (eds J. A. Aldrete and B. A. Britt), p. 351. New York: Grune & Stratton. Barlow, M. B. (1970). The genetic background to malignant hyperthermia revealed by serum creatine phosphokinase estimations in asymptomatic relatives. Br. J. Anaesth., 42, 1077. King, W. A., Ollivicr, L., and Basrur, P. K. (1976). Erythrocyte osmotic response test on malignant hyperthermiasusceptible pigs. Arm. Genet. Sel. Anim., 8, 537. Mitchell, G., and Heffron, J. J. A. (1975). Factors affecting serum creatine phosphokinase activity in pigs. J. 5. Afr. Vet. Ass., 46, 145. van Rensburg, A. J. J. (1980). A halothane-induced biochemical defect in muscle of normal and malignant hyperthermia-susceptible Landrace pigs. Anesth. Analg. (Cleve.), 59, 250. Rehder, K., Forbes, J., Alter, H., Hessler, O., and Stier, A. (1967). Halothane biotransformation in man: a quantitative study. Anesthesiology, 28, 711. Reske-Nielsen, E. (1978). Malignant hyperthermia in Denmark: Survey of a family study and investigations into muscular morphology in ten additional cases; in 2nd Internatl Symp. on Malignant Hyperthermia (eds J. A. Aldrete and B. A. Britt), p. 287. New York: Grune & Stratton. Schanus, E., Glass, R., and Lovrien, R. (1979). Molecular basis ACKNOWLEDGEMENTS for malignant hyperthermia involving erythrocyte glutaWe acknowledge the help of M. Bagg. This research was thione peroxidase. Fed. Proc., 38, 571. supported by the Medical Research Council of South Africa Seeman, P., Kwant, W. O., Sauks, T., and Argent, W. (1969). and the Council of the University of the Witwatersrand. Membrane expansion of intact erythrocytes by anaesthetics. Biochim. Biophys. Ada, 183, 490. Woolf, N., Hall, L., Thome, S., Down, M., and Walker, R. REFERENCES (1970). Serum creatine phosphokinase levels in pigs reacting abnormally to halogenated anaesthetics. Br. Med. J., 3, 386. Atallah, M. M., and Geddes, I. C. (1973). Metabolism of halothane during and after anaesthesia in man. Br. J. Zsigmond, E. K., Penner, ]., and Kothary, S. P. (1978). Normal erythrocyte fragility and abnormal platelet aggreAnaesth., 45, 464. gation in MH families: a pilot study; in 2nd Internatl Symp. Bermari, M. C , Harrison, G. G., Bull, A. B., and Kench, J. E. on Malignant Hyperthermia (eds J. A. Aldrete and B. A. (1970). Changes underlying halothane-induced malignant Britt), p. 213. New York: Grune & Stratton. hyperpyrexia in Landrace pigs. Nature (Land.), 225, 653. Britt, B. A., Endrenyi, L., Peters, P. L., Kwong, F. H. F., and Kadjivek, L. (1976). Screening of malignant hyperthermia susceptible families by creatine phosphokinase measurement INFLUENCE DU pH, DE LA TEMPERATURE, DE and other clinical investigations. Can. Anaesth. Soc. J., 23, L'HALOTHANE ET DE SES METABOLITES SUR LA 263. FRAGILITE OSMOTIQUE DES ERYTHROCYTES Kalow, W. (1970). Malignant hyperthermia: aetiology CHEZ LES COCHONS SUSCEPTIBLES OU unknown. Can. Anaesth. Soc.J., 17, 316. RESISTANTS A L'HYPERTHERMIE MALIGNE Chang, L. W., Dudley, A. W., and Lee, Y. K. (1975). Ultrastructural changes in the kidney following chronic RESUME exposure to low levels of halothane. Am.J. Pathol., 78, 225. Cheah, K. S., and Cheah, A. M. (1979). Mitochondrial On a examine la fragilite des erythrocytes chez les cochons calcium, erythrocyte fragility and porcine malignant hyper- susceptibles ou resistants a 1'hyperthermie maligne, en tant que thermia. FEBS Lett., 107, 265. fonction du pH et de la temperature, lorsque l'halothane et ses Harrison, G. G., and Vcrburg, C. (1973). Erythrocyte osmotic metabolites etaient absents et lorsqu'ils etaient presents. La fragility in hyperthermia-susceptiblc swine. Br.J. Anaesth., baisse du pH dc 7,40 a 6,95 a augmente la fragilite des cochons 45, 131. susceptibles et celles des cochons resistants, mais cette
fragility. However, our results suggest that this is unlikely since, in our experiments, haemolysis was allowed to take place over 30min, six times longer than in the study of King, Ollivier and Basrur. We suggest, therefore, that exposing erythrocytes to both an osmotic and pH shock may reveal more marked differences in fragility. Our results provide evidence that halothane and its metabolites at anaesthetic concentrations do not increase erythrocyte fragility. The effects of high temperature and low pH combined with those of halothane and its metabolites are also negligible. The reported haemolysis occurring during MH (Britt and Kalow, 1970) is unlikely to be a result of any effect of halothane or its metabolites. Haemolysis is most likely to occur when a severe acidosis precedes an increase in temperature, since high temperatures tend to stabilize the red cell membrane whereas low pH increases fragility. However, in pigs, these specific circumstances appear to occur rarely, since studies of changes in plasma constituents during episodes of MH have reported no increase in plasma haemoglobin concentration (Berman et al., 1970; Hende et al., 1976).
BRITISH JOURNAL OF ANAESTHESIA
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von 0,120 und 0,137 mol litre '. Bei alien untersuchten pHWerten und bei 39 und 41 °C, aber nicht bei 43 °C, zeigten die Erythrozyten anfalliger Schweine eine grossere Hamolyse bei 0,120 und 0,137 mol litre"' als die Erythrozyten nichtanfalliger Schweine. Halothan und dessen Metaboliten ergaben in wahrend der Narkose vorhandenen Konzentrationen keine Anderung der Fragilitat von Erythrozyten. INFLUENCIA DEL pH, DE LA TEMPERATURA, DEL HALOTANO Y DE SUS METABOLITOS SOBRE LA FRAGILIDAD OSMOTICA DE LOS ERITROCITOS EN CERDOS RESISTENTES Y SUSCEPTIBLES A LA HIPERTERMIA MALIGNA SUMARIO
EINFLUSS VON pH, TEMPERATUR, HALOTHAN UND SEINER METABOLITEN AUF DIE OSMOTISCHE FRAGILITAT DER ERYTHROZYTEN BEI SCHWEINEN, DIE AUF MALIGNE HYPERTHERMIE ANFALLIG UND NICHTANFALLIG SIND ZUSAMMENFASSUNG
Die Erythrozyten-Fragilitat von auf maligne Hyperthermie anfalligen und nicht-anialligen Schweinen wurde als Funktion von pH und Temperatur untersucht, in Abwcsenheit und Anwesenheit von Halothan und seinen Metaboliten. Ein pHAbsticg von 7,40 auf 6,95 erhohte die Fragilitat bei beiden Schweinegruppen, doch war diese Erhohung wesentlich grosser bei anfalligen als bei nich-anfalligen Schweinen. Ein Temperaturanstieg reduziene die Fragilitat bei beiden Gruppen. Die Hauptunterschicde in dcr Hamolyse bei beiden Gruppen ergaben sich bei Natriumchloridkonzentrationen
Se examino la fragilidad de los eritrocitos de cerdos resistentes y susceptibles a la hipertermia maligna cual una funcion del pH y de la temperature, en ausencia y en prcsencia de halotano y dc sus metabolitos. Una disminucion del desdc 7,40 a 6,95 incremento la fragilidad tanto dc los cerdos resistentes como de los susceptibles, pero el incremento en los animales susceptibles fuc significativamente superior al acaecido en los resistentes. Un incremento de la temperature produjo una reduccion de la fragilidad de los eritrocitos en ambos tipos de cerdo. Las diferencias mas consistentes en hemolisis, entre los cerdos susceptibles y los resistente, tuvieron lugar bajo concentraciones de cloruro sodico de 0,120 y de 0,137 mol litro "'. Para todos los valores de pH examinados y a temperatures de 39 y de 41 °C, pero no a 43 °C, los eritrocitos pertenecientes a los cerdos susceptibles mostraron una mayor hemolisis en cloruro sodico al 0,120 y al 0,137 mol litro" ' que los correspondientes a los cerdos resistentes. Ni el halotano ni sus metabolitos llegaron a alterar la fragilidad de los eritrocitos a las concentraciones presentes durante la anestesia.
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augmentation a ete chez les animaux susceptibles beaucoup plus forte que chex les animaux resistants. L'augmentation de la temperature a conduit a une baisse de la fragilite des erythrocytes chez les deux types de cochons. Les differences les plus regulieres dans l'hemolyse, entre les cochons susceptibles et les cochons resistants, ont etc evidentes lorsque les concentrations de chlorure de sodium etaient de 0,120 et de 0,137 mol litre " '. A toutes les valeurs de pH examinees et a 39 ° et a 41 °C, mais pas a 43 °C, les erythrocytes des cochons susceptibles ont montre une plus grande concentration de chlorure de sodium dans l'hemolyse: 0,120 et 0,137 mol litre"', que les erythrocytes des cochons resistants. L'halothane, ou ses metabolites, aux concentrations rencontrees pendant l'anesthesie, n'ont pas modine la fragilite des erythrocytes.