J. Physiol. (I958) I41,

Transcription

1 377 J. Physiol. (I958) I41, THE RELATION OF POTASSIUM DEFICIENCY TO MUSCULAR PARALYSIS BY INSULIN BY F. G. J. OFFERIJNS,* D. WESTERINK AND A. F. WILLEBRANDS From the University Department of Physiology, the Second Medical Service. Wilhelmina-Gasthuis, and the Institute of Human Nutrition, Amsterdam, the Netherlands (Received 21 August 1957) It is known that patients treated for diabetic coma with large amounts of insulin and replacement of fluid may develop generalized paralysis of the extremities and of the respiratory muscles. During the paralysis the potassium level of the serum is extremely low, but when the serum potassium is raised by administration of potassium the paralysis disappears (Holler, 1946; Frenkel, Groen & Willebrands, 1947 a, b; Nicholson & Brauning, 1947; Logsdon & McGavack, 1948; Tuynman & Wilhelm, 1948; Stephens, 1949; Danowski, Peters, Rathburn, Quashnock & Greenman, 1949). It seemed possible that the paralysis might be explained by a combination of potassium depletion with an effect of insulin on the potassium distribution in the muscles, and further that the paralysis might be related to a diminution of the extra- or intracellular potassium or both. To test these possibilities we examined the effect of insulin on the contractions of the isolated rat diaphragm suspended in a potassium-free medium. The diaphragm preparations were obtained from normal rats and from rats fed on a potassium-free diet to produce a low intracellular potassium content of the muscle. METHODS Male wbite rats of Wistar strain were used from the time they weighed g. Some were fed on a potassium-free diet, others, the control rats, received the same diet with additional potassium in the form of K.HPO g/kg mixed food. Gardner's diet slightly modified to the following composition was used. One kilogram of mixed food consisted of (g): sugar 725, casein 180, arachin oil 50, cod-liver oil 5 and salt mixture 40. The composition of the salt mixture was as follows (g): CaCo3 670, CaHPO4.2H.O 119, NaCl 234, Na2HPO4 795, MgSO4.7H2O 508, NaI 0-15, CuSO.5HH20 22, ZnCl 0-4, MnSO4.4H , and FeNH4.citrate 148. In addition, the following amounts (mg) of B vitamins were added to each kilogram of mixed food: thiamine 3, riboflavin 3, * Correspondence address: Second Medical Service, pav. 7, Wilhelmina-Gasthuis, Amsterdam, the Netherlands. 24 PHYSIO. CXLI

2 378 F. G. J. OFFERIJNS AND OTHERS niacin 20, paraaminobenzoic acid 250, Ca-pantothenic acid 20, pyridoxine 2, inositol 100 and choline 1 g. Each rat received further 0-2 ml. of wheat germ oil per week. After being fed on these diets for at least 21 days the rats were killed by decapitation. Blood was collected and allowed to clot and then centrifuged. The hind leg muscles were removed. The serum and the hind leg muscles were then analysed for water content, sodium, potassium and chloride concentrations. In the muscles 'neutral' fat was determined as well. The water content was determined by estimating the loss of weight after drying at 1050 C during hr. Sodium and potassium were determined by flame photometry; chloride was determined as described by Schales & Schales (1941) and 'neutral' fat by the method of Cotlove, Holliday, Schwartz & Wallace (1951). In the calculations of the distribution of the electrolytes in the intracellular and extracellular spaces of the muscle, the chloride space was assumed to be identical with the extracellular space. Thus, by dividing the quantity of chloride per kg fresh fat-free muscle by the chloride concentration in the extracellular fluid, the quantity of extracellular water per kg fat-free muscle was obtained. A correction was made for the fat content of the tissue. For the calculation of the extracellular concentrations of chloride, sodium and potassium from the serum values a Donnan factor of 0-96 was used. By subtracting the extracellular quantity of water (in 1./kg fresh fat-free muscle) from the determined water content of the whole muscle, the amount of intracellular water per kilogram of fat-free muscle was obtained. By multiplying the extracellular concentration of sodium or potassium, by the amount of extracellular water, the quantity of extracellular sodium or potassium per kilogram fat-free muscle was found. By subtracting this figure from the total quantity of sodium or potassium per kilogram of fat-free muscle, the intracellular amounts of these electrolytes were obtained. The concentration of sodium or potassium in the intracellular water was then computed by dividing the total quantity of sodium or potassium by the quantity of intracellular water per kilogram of fatfree tissue (Westerink, Offerijns & WiJlebrands., 1955). Rat diaphragm preparation. A phrenic nerve with the corresponding half of the diaphragm was suspended according to the technique described by Builbring (1946) in a Tyrode solution in which the potassium concentration was varied. The bath volume was 100 ml., the temperature was 370 C, and a mixture of 95 % 0O and 5% C02 was bubbled through the solution. The nerve was stimulated with regular condenser discharges at a frequency of 12-16/min. The contractions of the diaphragm were recorded on a smoked kymograph. RESULTS Ion and water content of serum and muscle in rats fed on potassium-free diet In the serum of rats fed on the potassium-deficient diet low blood levels of both potassium and chloride were found. In the muscles the intracellular sodium concentration was considerably increased and the intracellular potassium concentration was decreased (Table 1). For every 3 m-equiv intracellular potassium lost there was an increase in intracellular sodium of about 2 m- equiv. The chloride concentration in the muscles was not changed significantly. The amount of extracellular water in the muscles of the potassium-deficient animals was increased, whereas the amount of intracellular water was decreased by about the same amount. Effects on the rat diaphragm The diaphragms of the control rats contracted in Tyrode solution for hours without decrease in the height of the contractions. The potassium in the bath

3 MUSCULAR PARALYSIS BY INSULIN 379 fluid could be diminished without greatly affecting the contractions. Even when potassium was entirely omitted from the Tyrode solution the diaphragms contracted for hours with only a small decrease in the height of the contraction (Fig. 1). The addition of 40 i.u. insulin to the bath fluid had no effect on the TABLE 1. Electrolyte changes brought about in rats fed on potassium-free diet. The values are means ( + S.E.) obtained from 40 potassium-deficient and 12 control rats Potassium-deficient rats Controls P Serum (m-equiv/kg H20) Na 150 i i 3-1 >0-1 K 3.95± ± 0-6 <0-001 C1 94 ± ± 3.5 -<0-001 Muscle (m-equiv/kg fresh fat-free tissue) Total 50 ± <0-001 Na Extracellular 26-5 i ± 2-6 <0-001 Intracellular i 1-9 <0-001 Total 68 i <0-001 K Extracellular i 0-14 <0-001 Intracellular ± 7-2 <0-001 C1 17 ± ±2-4 >0-05 <0-1 Na Intracellular) *q k H20) 41-5 ± ± 3-1 <0-001 K Intracellulari (m-equiv/kgh ± ±12-1 <0-001 Muscle water (ml./kg fresh fat-free tissue) Total 771 ± ± 4-4 <0-05 Extracellular 182 ± ±18-7 <0-001 Intracellular 590 ± ± 18-5 <0-001 Fig. 1. Contractions of rat diaphragm suspended in 100 ml. potassium-free Tyrode solution to stimulation of the phrenic nerve. Contractions after stimulation for 30 min (at A) and for- 150 min (at B). A few minutes later (at C) 40 i.u. insulin added to the bath (at the arrow). Time in minutes. contractions of the diaphragms of the control rats, independent of whether they contracted in normal Tyrode solution (potassium content 2 m-equiv/l.) or in potassium-free solution (Fig. 1). The diaphragms of potassium-deficient rats contracted in ordinary and potassium-free Tyrode solution in the same way as the diaphragms of the control rats. Insulin had no influence on the contractions of these diaphragms 24-2

4 380 F. G. J. OFFERIJNS AND OTHERS when they were suspended in normal Tyrode solution, but it impaired their contractility when they were suspended in potassium-free Tyrode solution. In thirty-three experiments of this kind, 40 i.u. insulin was added to the potassium-free fluid when the diaphragms of the potassium-deficient rats had contracted to a constant height for 30 min. In eleven of these experiments the insulin caused complete paralysis within a few minutes (Fig. 2); in nineteen it caused a great reduction in the height of the contractions (Fig. 3), and in three there was no effect. During the paralysis the diaphragms were relaxed. Fig. 2. Contractions of rat's diaphragm suspended in 100 ml. Tyrode solution. Rat fed on potassium-free diet. At A paralysis after adding 40 i.u. insulin at arrow; at B return of muscular contractions after adding 20 mg KC1; C, 5 min later. Time in minutes. In this condition direct stimulation of the diaphragm caused no contraction. The effect of insulin was usually tested by adding 40 i.u. (2 mg) to the 100 ml. bath, but the addition of i.u. (10-100,tg) was sufficient to produce a reduction in the height of the contractions. The effect of insulin could be abolished by adding KCI to the bath fluid (Fig. 2B and Fig. 3B). Similarly the addition of KCl to the bath fluid before the addition of insulin prevented the paralysing effect. The addition of purified human serum albumin to the potassium-free solution did not influence the contractions of the diaphragms from rats fed on

5 MUSCULAR PARALYSIS BY INSULIN 381 Fig. 3. Contractions of rat's diaphragm suspended in 100 ml. Tyrode solution. Rat fed on potassium-free diet. A, effec t of 2 mg albumin and of 40 i.u. insulin; B, effect of 20 mg KCI. Time in minutes. TABLE 2. Mean concentrations of Na+, K+ and Cl- in 28 potassium-deficient rats and in 12 normal rats, arranged according to the effect of insulin on the contractions of the diaphragm in vitro Intracellular concentrations (muscle) Group I II III Normal (controls) Effect of insulin Paralysis Contraction height diminished No effect No effect No. of animals 8 17 Extracellular fluid (m-equiv/kg fresh fat-free (m-equiv/kg H20) tissue) (m-equiv/kg H20) Na+ K+ Cl- Na+ K+ Na+ K * *

6 382 F. G. J. OFFERIJNS AND OTHERS a potassium-free diet, nor did it abolish the paralysing effect of insulin (Fig. 3). The mean concentrations were determined of Na+, K+ and Cl- in the serum and in the muscles from twenty-eight potassium-deficient rats. In Table 2 these values have been arranged in three groups. It is evident that insulin produced full paralysis in those rats which were most deficient in potassium. In those rats which were somewhat less deficient in potassium, insulin produced only a decrease in the height of the contraction, and in the three animals in which potassium deficiency was least pronounced insulin had no influence on the contractions. There was no correlation of the insulin effect with the sodium content of the muscles. DISCUSSION The electrolyte concentrations which we found in the normal and in the potassium-deficient rats are of the same order as those obtained by previous workers (Miller & Darrow, 1940; Darrow, Schwartz, Iannucci & Corille, 1948; Gardner, Talbot, Cook, Berman & Uribe, 1950; Cotlove et al. 1951). Only the values for the extracellular potassium concentration of the control animals were somewhat higher (7 m-equiv/kg) than those of most investigators (5 m- equiv K+/kg) probably owing to the fact that we did not heparinize the rats before sampling the blood and that during coagulation the erythrocytes may lose considerable amounts of potassium. Our findings on the rat diaphragm preparation, which showed that neither extracellular nor intracellular potassium deficiency per se causes paralysis of the muscle, agree with those of Miller & Darrow (1940) who could find no abnormality in the contractility of the gastrocnemius muscles from potassiumdeficient rats. In man also a potassium-free diet does not cause paralysis (Black & Milne, 1952; Fourman, 1954; McArdle, 1954) nor does a low concentration of extracellular and intracellular potassium caused by pathological loss of potassium. Thus potassium deficiency alone is insufficient to produce paralysis in vivo or in vitro. On the other hand, our finding that insulin produces paralysis of the diaphragm obtained from rats fed on a potassiumdeficient diet and suspended in potassium-free Tyrode solution shows that insulin has a paralysing effect on the potassium-deficient muscle and this action of insulin may well explain the paralysis which can occur when patients with diabetic coma are treated with insulin. This effect of insulin does not necessarily depend on the extracellular or intracellular potassium concentration alone; other factors of the 'potassium deficiency complex', like the altered sodium and chloride concentrations, the changes in water content, ph, etc., may contribute to it. The paralysing effect insulin exerts on the potassium-deficient diaphragm illustrates that even a hormone which has been studied as extensively as

7 MUSCULAR PARALYSIS BY INSULIN 383 insulin reveals, when tested during a disturbed electrolyte balance, unexpected ' new ' properties. The same may apply to other drugs; for instance, it is known that the action of digitalis is counteracted by high concentrations of potassium in serum and accentuated by low concentrations (Cohen, 1952). Since many drugs are used in patients with disturbed electrolyte metabolism, it seems necessary that they should be investigated not only in normal animals, as is customary, but also in animals with disturbances in their electrolyte equilibrium similar to those that occur in human patients. SUMMARY 1. Insulin exerts a paralysing effect on the isolated diaphragm preparation of rats fed on a potassium-free diet and suspended in potassium-free Tyrode solution. 2. This action of insulin may explain the paralysis which may develop in patients with diabetic coma treated with large doses of insulin. The authors are greatly indebted to Professor Dr J. ten Cate, Dr J. Groen and Professor Dr B. C. P. Jansen for their advice and encouragement. This research was supported by a grant from the Professor Dr D. A. de Jong Foundation. REFERENCES BLACK, D. A. K. & M1LNE, M. D. (1952). Experimental potassium depletion in man. Lancet, 262, BiLJBRING, E. (1946). Observations on the isolated phrenic nerve diaphragm preparation of the rat. Brit. J. Pharmacol. 1, COHEN, B. M. (1952). Digitalis poisoning and its treatment. New Engl. J. Med. 246, COTLOVE, E. M., HOLLIDAY, M. A., SCHWARTZ, R. & WALLAcE, W. M. (1951). Effects of electrolyte depletion and acid-base disturbance on muscle cations. Amer. J. Physiol. 167, DANOWSKi, T. S., PETERS, J. H., RATHBURN, J. C., QUASHNOCK, J. M. & GREENMAN, L. (1949). Studies in diabetic acidosis and coma with particular emphasis on the retention of administered potassium. J. clin. Inve8t. 28, 1-9. DARRow, D. C., SCHWARTZ, R., IANNUCCI, J. E. & CORILLE, F. (1948). The relation of serum bicarbonate concentration to muscle composition. J. clin. Invest. 27, FOURMAN, P. (1954). Depletion of potassium induced in man with an exchange resin. Clin. Sci. 13, FRENKEL, M., GROEN, J. & WILLEBRANDS, A. F. (1947a). Low serum potassium level during recovery from diabetic coma. Arch. intern. Med. 80, FRENKEL, M., GROEN, J. & WILLEBRANDS, A. F. (1947b). Daling van het serumkaliumgehalte met verschijnselen van gegeneraliseerde spierzwakte en een kenmerkend cardiovasculair syndroom tijdens de behandeling van het coma diabeticum. Ned. Tijd8chr. Geneesk. 91, GARDNER, L. I. TALBOT, N. B., COOK, C. D., BERMAN, H. & URIBE, C. R. (1950). The effect of potassium deficiency on carbohydrate metabolism. J. Lab. clin. Med. 35, HOLLER, J. W. (1946). Potassium deficiency occurring during the treatment of diabetic acidosis. J. Amer. med. As8. 131, LOGSDON, C. S. & McGAvAcK, T. H. (1948). Death, probably due to potassium deficiency, following control of diabetic coma. J. clin. Endocrin. 8, MCARDLE, B. (1954). Le role du potassium dans la paralysie periodique familiale: in Physiopathologie du Potassium. Paris: Centre National de la Recherche Scientifique. MILLER, H. C. & DARROW, D. C. (1940). The effect of changes in muscle electrolyte on the response of skeletal muscle to tetanic stimulation with particular reference to potassium. Amer. J. Physiol. 129,

8 384 F. G. J. OFFERIJNS AND OTHERS NICHOLSON, W. M. & BIAUNING, W. S. (1947). Potassium deficiency in diabetic acidosis. J. Amer. med. A , OFFERIJNS, F. G. J., WESTERINK, D. & WILLEBRANDS, A. F. (1953). Effect of insulin in vitro upon the contractility of the isolated rat diaphragm in potassium deficiency. XIiXnt. physiol. Congr. Montreal, pp SCHALES, 0. & SCHALES, S. S. (1941). A simple and accurate method for the determination of chloride in biological fluids. J. biol. Chem. 140, STEPHENS, F. J. (1949). Paralysis due to reduced serum potassium concentration during treatment of diabetic acidosis. Ann. int. Med. 30, TuYNMAN, P. E. & WILHELM, S. K. (1948). Potassium deficiency associated with diabetic acidosis. Ann. int. Med. 29, WESTERINK, D., OFFERIJNS, F. G. J. & WILLEBRANDS, A. F. (1955). De invloed van kalium. deficientie op het gehalte aan water en electrolyten in serum en spierweefsel bij de rat. Ned. Tijd8chr. Genee8k. 99,