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ERYTHROCYTE OSMOTIC FRACILITY IN HYPERTHERMIA-SUSCEPTIBLE SWINE
Brit. J. Anaesth. (1973), 45, 131
ERYTHROCYTE OSMOTIC FRAGILITY IN HYPERTHERMIA-SUSCEPTIBLE SWINE G.
G.
HARRISON AND C. VERBURG SUMMARY
The observation is reported that hyperthermia-susceptible swine manifest an increased red cell osmotic fragility. The importance of this observation is discussed. Anaesthetic-induced malignant hyperpyrexia in humans appears to result from some intrinsic abnormality of muscle (Satnick, 1969; Harrison et aL, 1970; King, Denborough and Zapf, 1972). Evidence points to this defect being in the sarcoplasmic reticulum (Kalow et al., 1970). A certain strain of Landrace pigs manifests a similar condition and has been used as an animal experimental model for the investigation of various aspects of the disease and trials of therapeutic measures (Berman et al., 1970; Harrison, 1971). While taking blood specimens for various investigations from such hyperthermia-susceptible pigs, we observed that more often than not these specimens were markedly haemolysed. This observation prompted us to investigate the erythrocyte osmotic fragility of these pigs. METHODS
Hyperthermia-susceptible swine were initially chosen by observing the reaction of all pigs acquired for the Surgical Research Laboratory to a screening challenge of halothane inhalation (Harrison et al., 1969). Hyperthermia-susceptible swine react in an obvious and highly characteristic fashion by manifesting extreme muscle rigor within about 10 min of exposure to halothane and recover if the halothane is immediately discontinued. Susceptibility to hyperthermia was confirmed in positive reactors by estimation of serum c.p.k. levels, which in hyperthermiasusceptible swine remain persistently raised (Woolf et al., 1970).
The red cell fragility of three such pigs, aged 9-14 weeks, was investigated over a time some weeks after the original test exposure to halothane. Three specimens of blood—a week elapsing beween successive specimens—were taken from each pig under brief thiopentone anaesthesia. This blood was drawn from one of the large veins in the thoracic inlet through a wide-bore needle (15 s.w.g.), care being taken to exert the minimum of suction. The fragility of blood drawn under similar conditions from nine non-reactor pigs, non-reactivity being confirmed by low serum c.p.k. levels, served as a control. Osmotic fragility was tested by the standard method (Dacie and Lewis, 1968) of estimating colorometrically the degree of haemolysis achieved in serial dilutions of saline solutions. Differences between groups of mean percentage haemolysis at each concentration of saline were tested statistically by a standard Student (-test. RESULTS
The results (table I and figs. 1 and 2) reveal that these hyperthermia-susceptible pigs manifest a greater erythrocyte osmotic fragility than normal. The median corpuscular fragility of the hyperthermia-susceptible swine lay between 0.55 and 0.50% saline, while that of the control pigs was between 0.50 and 0.45% saline. (The human value G. G. HARRISON, MX>., F.F.A.R.C.S.; C. VERBURG; Depart-
ment of Anaesthetics, Groote Schuur Hospital and University of Cape Town, South Africa.
TABLE L Mean (and SE) percentage haemolysis at % concentration of saline. Type of Pig
I.I
Hyperth.- susceptible pigs 0 (9 samples) 0 Control pigs C9 samples) P value Significance of difference
0.95 0 0
0.85 0-2
0.75 0.7
±0.1
±0.1
0
P<0.10
0.2 ±0.1 P<0.05 s.
0.65
0.60
0.55
0.50
0.45
0.40
0.30
0^0
0.K
7.8
20.7 ±2J 10.0 ±1.0 P< 0.001
38.9 ±2.2 21.5 ±2.1 P< 0.001
68.2 ±2.6 37.9 ±Z5 P< 0.001 s.
83.2 ±1.9 72.0 ±23 P<0.01 s.
84.4 ±1.7 88.8 ±2.4
94.6 ±1.1 94J ±2.8
97.1
100
99.3
100
nj.
nj.
ax
±1.1
5.1 ±1.1 P
s. "•significant difference
3.
*•
—not significant difference
BRITISH JOURNAL OF ANAESTHESIA
132
90
PIGS HYPERTXERMIA
SUSCEPTBLE PICS
H*£MCOSIS SO
Ql__
O85
OB
O7S
O7
OtS
%
O*
O 5S
O.5
O
4S
O4
Q3S
O.3
O!i
O.2
CONCENTRATION SACWE SOLUTION
FIG. 1. Red cell fragility curve (mean values with SE).
for comparison lies between 0.445 and 0.40% saline; Dacie and Lewis, 1968.) The increment haemolysis curve (fig. 2) demonstrates a difference of 0.05% saline between peaks of maximal haemolysis.
tently included hyperthermia-susceptible swine (Harrison, 1972), comparisons of fragility in this paper are made only with the screened controls described. DISCUSSION
5O|
3O % HAEMOLYSIS 2O
OB
O7
O4 %
OS
O4
O3
CONCENTRATION SALI>C
O3
Ol
SOLUTOM
FIG. 2. Increment haemolysis curve. Hyperthermiasusceptible pigs. Control pigs.
There is quite marked disparity in the few reported estimations of porcine red cell fragility (Schalm, 1965). As the pig populations on which these reported estimations were performed may well have inadver-
Abnormalities in red cell fragility depend to a large extent on the functional state of its surface membrane (Dacie and Lewis, 1968). The marked morphological similarity between this cell membrane and the membranes of cell organelles such as mitochondria, endoplasmic reticulum and Golgi apparatus led to Robertson's concept of these as "unit" membrane (Robertson, 1959). Though there are important functional differences between membranes which doubtless reside in the protein enzymes associated with the phospholipid skeleton, there is indeed a structural organization that is common to most cellular membranes (Fawcett, 1962; Giese, 1968). It is this that confers relevance to the finding of increased red cell fragility in hyperthermia-susceptible swine reported here. There is evidence that anaesthetic-induced malignant hyperpyrexia results from some intrinsic abnormality in muscle, more specifi-
133
ERYTHROCITE OSMOTIC FRAGILITY IN SWINE cally of the sarcoplasmic reticulum. Here is evidence that another "unit" membrane in animals susceptible to anaesthetic induced malignant hyperpyrexia is also defective. ACKNOWLEDGEMENTS
We wish to thank Brian Sassman for the initial screening and subsequent anaesthesia of the pigs used and Dr J. Smith for technical assistance in obtaining blood samples. We wish to thank the Liver Research Group and the J. S. Marais Surgical Research Laboratory of the University of Cape Town for access to and supply of pigs. This project was finanriaiiy supported by the AngloAmerican and De Beers Anaesthetic Research Fund and the Joseph Stone Anaesthetic Research Foundation. REFERENCES
Berman, M. C , Harrison, G. G., Bull, A. B., and Kench, J. E. (1970). Biochemical changes associated with malignant hyperpyrexia induced by halothane in susceptible Landrace pigs. Nature (Lond.), 225, 653. Dacde, J. V., and Lewis, S. M. (1968). Practical Haematology, 4th edn., p. 166. London: Churchill. Fawcett, D. W. (1962). Physiologically significant specialization of the cell surface. Circulation, 26, 1105. Giese, A. C. (1968). Cell Physiology, 3rd edn., p. 118. Philadelphia, London, Toronto: Saunders. Harrison, G. G. (1971). Anaesthetic induced malignant hyperpyrexia—a suggested method of treatment. Brit, med. J.. 3. 454. (1972). Pale, soft, exudative pork, porcine stress syndrome and malignant hyperpyrexia—an identity? J. S. Afr. vet. med. Ats., 43, 57. Berman, M. C , Hickman, R., Bull, A. B., Terblanche, J., and Kench, J. E. (1970). Anaesthetic induced malignant hyperpyrexia—some observations of the syndrome in Landrace pigs. Proceedings of the 3rd Asian and Australasian Congress of Anaesthesiology (eds. Shea, L., and Dwyer, B.), p. 158. Sydney: Butterworth. Saunders, S. J., Biebuyck, J. F., Hickman, R., Dent, D. M., Weaver, V., and Terblanche, J. (1969). Anaesthetic-induced malignant hyperpyrexia and a method for its prediction. Brit. J. Anaesth., 41, 844. 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.
King, J. O., Denborough, M. A., and Zapf, P. W. (1972). The inheritance of malignant hyperpyrexia. Lancet, I, 365. Robertson, J. D. (1959). The ultrastructure of cell membranes and their derivatives. Biochem. Soc. Symposia, Cambridge, England, 16, 3. Satnick, J. H. (1969). Hyperthermia under anesthesia with muscle flaccidity. Anesthesiology, 30, 472. Schalm, O. W. (1965). Veterinary Hematology, 2nd edn., p. 279. Philadelphia: Lea & Febiger. Woolf, N., Hall, L., Thorpe, C , Down, M., and Walker, R. (1970). Serum creatinephosphokinase levels in pigs reacting abnormally to halogenated anaesthetics. Bnt. med. J., 3. 386. FRAGILITE OSMOTIQUE ERYTHROCYTAIRE CHEZ LE PORC SUSCEPTIBLE A L"HYPERTHERMIE SOMMAIRE
Les auteurs rapportent avoir observi que des pores susceptibles a rhyperthermie manifestent une fragilite' osmotique accrue des globules rouges et discutent rimportance de cette observation. OSMOTISCHE FRAGILITAT DER ERYTHROCYTEN BEI HYPERTHERMIEEMPFINDLICHEN SCHWEINEN ZUSAMMENFASSUNG
Es wird fiber die Beobachtung berichtet, da8 Hyperthermieempfindliche Schweine eine gesteigerte osmotische Fragilitat ihrer roten Zellen aufweisen. Die Bedeutung dieser Beobachtung wird diskutiert. FRAGILIDAD OSMOTICA ERITROCITARIA EN CERDOS SUSCEPTIBLES A LA HIPERTERMINA RESUMEN
Se informa sobre la observation de que los cerdos susceptibles a la hipertermia presentan un incremento en la fragilidad osmdtica de sus gl6bulos rojos. Se discute la importancia de esta observaaon.
ERYTHROCYTE OSMOTIC FRAGILITY IN HYPERTHERMIA-SUSCEPTIBLE SWINE G.
G.
HARRISON AND C. VERBURG SUMMARY
The observation is reported that hyperthermia-susceptible swine manifest an increased red cell osmotic fragility. The importance of this observation is discussed. Anaesthetic-induced malignant hyperpyrexia in humans appears to result from some intrinsic abnormality of muscle (Satnick, 1969; Harrison et aL, 1970; King, Denborough and Zapf, 1972). Evidence points to this defect being in the sarcoplasmic reticulum (Kalow et al., 1970). A certain strain of Landrace pigs manifests a similar condition and has been used as an animal experimental model for the investigation of various aspects of the disease and trials of therapeutic measures (Berman et al., 1970; Harrison, 1971). While taking blood specimens for various investigations from such hyperthermia-susceptible pigs, we observed that more often than not these specimens were markedly haemolysed. This observation prompted us to investigate the erythrocyte osmotic fragility of these pigs. METHODS
Hyperthermia-susceptible swine were initially chosen by observing the reaction of all pigs acquired for the Surgical Research Laboratory to a screening challenge of halothane inhalation (Harrison et al., 1969). Hyperthermia-susceptible swine react in an obvious and highly characteristic fashion by manifesting extreme muscle rigor within about 10 min of exposure to halothane and recover if the halothane is immediately discontinued. Susceptibility to hyperthermia was confirmed in positive reactors by estimation of serum c.p.k. levels, which in hyperthermiasusceptible swine remain persistently raised (Woolf et al., 1970).
The red cell fragility of three such pigs, aged 9-14 weeks, was investigated over a time some weeks after the original test exposure to halothane. Three specimens of blood—a week elapsing beween successive specimens—were taken from each pig under brief thiopentone anaesthesia. This blood was drawn from one of the large veins in the thoracic inlet through a wide-bore needle (15 s.w.g.), care being taken to exert the minimum of suction. The fragility of blood drawn under similar conditions from nine non-reactor pigs, non-reactivity being confirmed by low serum c.p.k. levels, served as a control. Osmotic fragility was tested by the standard method (Dacie and Lewis, 1968) of estimating colorometrically the degree of haemolysis achieved in serial dilutions of saline solutions. Differences between groups of mean percentage haemolysis at each concentration of saline were tested statistically by a standard Student (-test. RESULTS
The results (table I and figs. 1 and 2) reveal that these hyperthermia-susceptible pigs manifest a greater erythrocyte osmotic fragility than normal. The median corpuscular fragility of the hyperthermia-susceptible swine lay between 0.55 and 0.50% saline, while that of the control pigs was between 0.50 and 0.45% saline. (The human value G. G. HARRISON, MX>., F.F.A.R.C.S.; C. VERBURG; Depart-
ment of Anaesthetics, Groote Schuur Hospital and University of Cape Town, South Africa.
TABLE L Mean (and SE) percentage haemolysis at % concentration of saline. Type of Pig
I.I
Hyperth.- susceptible pigs 0 (9 samples) 0 Control pigs C9 samples) P value Significance of difference
0.95 0 0
0.85 0-2
0.75 0.7
±0.1
±0.1
0
P<0.10
0.2 ±0.1 P<0.05 s.
0.65
0.60
0.55
0.50
0.45
0.40
0.30
0^0
0.K
7.8
20.7 ±2J 10.0 ±1.0 P< 0.001
38.9 ±2.2 21.5 ±2.1 P< 0.001
68.2 ±2.6 37.9 ±Z5 P< 0.001 s.
83.2 ±1.9 72.0 ±23 P<0.01 s.
84.4 ±1.7 88.8 ±2.4
94.6 ±1.1 94J ±2.8
97.1
100
99.3
100
nj.
nj.
ax
±1.1
5.1 ±1.1 P
s. "•significant difference
3.
*•
—not significant difference
BRITISH JOURNAL OF ANAESTHESIA
132
90
PIGS HYPERTXERMIA
SUSCEPTBLE PICS
H*£MCOSIS SO
Ql__
O85
OB
O7S
O7
OtS
%
O*
O 5S
O.5
O
4S
O4
Q3S
O.3
O!i
O.2
CONCENTRATION SACWE SOLUTION
FIG. 1. Red cell fragility curve (mean values with SE).
for comparison lies between 0.445 and 0.40% saline; Dacie and Lewis, 1968.) The increment haemolysis curve (fig. 2) demonstrates a difference of 0.05% saline between peaks of maximal haemolysis.
tently included hyperthermia-susceptible swine (Harrison, 1972), comparisons of fragility in this paper are made only with the screened controls described. DISCUSSION
5O|
3O % HAEMOLYSIS 2O
OB
O7
O4 %
OS
O4
O3
CONCENTRATION SALI>C
O3
Ol
SOLUTOM
FIG. 2. Increment haemolysis curve. Hyperthermiasusceptible pigs. Control pigs.
There is quite marked disparity in the few reported estimations of porcine red cell fragility (Schalm, 1965). As the pig populations on which these reported estimations were performed may well have inadver-
Abnormalities in red cell fragility depend to a large extent on the functional state of its surface membrane (Dacie and Lewis, 1968). The marked morphological similarity between this cell membrane and the membranes of cell organelles such as mitochondria, endoplasmic reticulum and Golgi apparatus led to Robertson's concept of these as "unit" membrane (Robertson, 1959). Though there are important functional differences between membranes which doubtless reside in the protein enzymes associated with the phospholipid skeleton, there is indeed a structural organization that is common to most cellular membranes (Fawcett, 1962; Giese, 1968). It is this that confers relevance to the finding of increased red cell fragility in hyperthermia-susceptible swine reported here. There is evidence that anaesthetic-induced malignant hyperpyrexia results from some intrinsic abnormality in muscle, more specifi-
133
ERYTHROCITE OSMOTIC FRAGILITY IN SWINE cally of the sarcoplasmic reticulum. Here is evidence that another "unit" membrane in animals susceptible to anaesthetic induced malignant hyperpyrexia is also defective. ACKNOWLEDGEMENTS
We wish to thank Brian Sassman for the initial screening and subsequent anaesthesia of the pigs used and Dr J. Smith for technical assistance in obtaining blood samples. We wish to thank the Liver Research Group and the J. S. Marais Surgical Research Laboratory of the University of Cape Town for access to and supply of pigs. This project was finanriaiiy supported by the AngloAmerican and De Beers Anaesthetic Research Fund and the Joseph Stone Anaesthetic Research Foundation. REFERENCES
Berman, M. C , Harrison, G. G., Bull, A. B., and Kench, J. E. (1970). Biochemical changes associated with malignant hyperpyrexia induced by halothane in susceptible Landrace pigs. Nature (Lond.), 225, 653. Dacde, J. V., and Lewis, S. M. (1968). Practical Haematology, 4th edn., p. 166. London: Churchill. Fawcett, D. W. (1962). Physiologically significant specialization of the cell surface. Circulation, 26, 1105. Giese, A. C. (1968). Cell Physiology, 3rd edn., p. 118. Philadelphia, London, Toronto: Saunders. Harrison, G. G. (1971). Anaesthetic induced malignant hyperpyrexia—a suggested method of treatment. Brit, med. J.. 3. 454. (1972). Pale, soft, exudative pork, porcine stress syndrome and malignant hyperpyrexia—an identity? J. S. Afr. vet. med. Ats., 43, 57. Berman, M. C , Hickman, R., Bull, A. B., Terblanche, J., and Kench, J. E. (1970). Anaesthetic induced malignant hyperpyrexia—some observations of the syndrome in Landrace pigs. Proceedings of the 3rd Asian and Australasian Congress of Anaesthesiology (eds. Shea, L., and Dwyer, B.), p. 158. Sydney: Butterworth. Saunders, S. J., Biebuyck, J. F., Hickman, R., Dent, D. M., Weaver, V., and Terblanche, J. (1969). Anaesthetic-induced malignant hyperpyrexia and a method for its prediction. Brit. J. Anaesth., 41, 844. 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.
King, J. O., Denborough, M. A., and Zapf, P. W. (1972). The inheritance of malignant hyperpyrexia. Lancet, I, 365. Robertson, J. D. (1959). The ultrastructure of cell membranes and their derivatives. Biochem. Soc. Symposia, Cambridge, England, 16, 3. Satnick, J. H. (1969). Hyperthermia under anesthesia with muscle flaccidity. Anesthesiology, 30, 472. Schalm, O. W. (1965). Veterinary Hematology, 2nd edn., p. 279. Philadelphia: Lea & Febiger. Woolf, N., Hall, L., Thorpe, C , Down, M., and Walker, R. (1970). Serum creatinephosphokinase levels in pigs reacting abnormally to halogenated anaesthetics. Bnt. med. J., 3. 386. FRAGILITE OSMOTIQUE ERYTHROCYTAIRE CHEZ LE PORC SUSCEPTIBLE A L"HYPERTHERMIE SOMMAIRE
Les auteurs rapportent avoir observi que des pores susceptibles a rhyperthermie manifestent une fragilite' osmotique accrue des globules rouges et discutent rimportance de cette observation. OSMOTISCHE FRAGILITAT DER ERYTHROCYTEN BEI HYPERTHERMIEEMPFINDLICHEN SCHWEINEN ZUSAMMENFASSUNG
Es wird fiber die Beobachtung berichtet, da8 Hyperthermieempfindliche Schweine eine gesteigerte osmotische Fragilitat ihrer roten Zellen aufweisen. Die Bedeutung dieser Beobachtung wird diskutiert. FRAGILIDAD OSMOTICA ERITROCITARIA EN CERDOS SUSCEPTIBLES A LA HIPERTERMINA RESUMEN
Se informa sobre la observation de que los cerdos susceptibles a la hipertermia presentan un incremento en la fragilidad osmdtica de sus gl6bulos rojos. Se discute la importancia de esta observaaon.