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Gossypol and hypokalaemia
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GOSSYPOL AND HYPOKALAEMIA* Christina Wang** and Rosie T.T. Yeung Department of Medicine, University of Hong Kong Queen Mary Hospital, Hong Kong ABSTRACT One major side effect of administration of gossypol as a male fertility regulating agent is the occurrence of hypokalaemic paralysis. We have reviewed the common causes of hypokalaemia in clinical practice and previous studies of gossypol-induced hypokalaemia in animals and man. The available evidences suggest that gossypol induced renal leakage of potassium. The most likely mechanism is a direct toxic effect of gossypol on the renal tubules.
~ossypol has been used by Chinese scientists since 1970 as a male method for fertility regulation (l-5). The antifertility efficacy as determined by sperm concentration (below 4 million/ml) was over 99%. Full recovery of spermatogenesis occurred in 73.7% of the volunteers. The common side effects reported include fatigue, gastrointestinal symptoms, decreased libido and potency, dizziness, and dryness of mouth. However the most serious side effect of administration of gossypol is hypokalaemic paralysis reported in 66 out of 8806 volunteers,i.e. 0.75% (3-5). The clinical syndrome is that of hypokalaemia: fatigue, muscle weakness followed by flaccid paralysis. Recovery is prompt and complete after potassium (K) repletion. However, 2 patients have been reported to have chronic persistent hypokalaemia after cessation of gossypol treatment (5,6). In one small-scale study of 12 Brazilian men, Coutinho (7) reported similar antifertility efficacy but serum K and biochemistry remained normal throughout the 12-month study. In this brief review, we will discuss some of the common causes of hypokalaemia in clinical practice, in particular hypokalaemic periodic paralysis and previous studies of gossypol-induced hypokalaemia in Chinese men. Such an inspection may shed light as to the basis for the gossypol-induced hypokalaemia.
*
Part of a review submitted in July, 1984 to the Steering Committee of the Task Force on Male Methods of Fertility Regulation of the WHO Special Programme on Research, Development and Research Training in Human Reproduction. ** To whom correspondence should be addressed. Submitted for publication July 2, 1985 Accepted for publication August 26, 1985
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HYPOKALAEMIA
IN CLINICAL PRACTICE
The principal causes of hypokalaemia encountered in clinical practice are listed in the Table (8). Dietary deprivation of K alone can lead to moderate hypokalaemia. However hypokalaemia seen in clinical practice is most frequently due to excessive loss of K either through the gastrointestinal tract or through the kidneys. K deficiency occurs commonly in gastrointestinal disorders in which vomiting, diarrhoea or loss of gastrointestinal secretions are prominent. Metabolic alkalosis which accompanies the loss of gastric secretions further enhances renal K loss. Gastrointestinal losses with vomiting or diarrhoea together with a compromised K intake in chronic alcoholics have been suggested to be the possible causes (9). Table.
Causes of Hypokalaemia in Men
1.
Gastrointestinal a. deficient intake starvation malnutrition b. excessive loss vomiting (including surreptitious vomiting) diarrhoea (including laxative abuse) nasogastric aspiration fistulas villous adenoma alcohol (?I
2.
Renal a. diuretics diuretic abuse osmotic diuresis (e.g. diabetic ketoacidosis) b. excessive mineralocorticoid effects primary hyperaldosteronism secondary hyperaldosteronism glucocorticoid excess licorice ingestion renal tubular diseases C. renal tubular acidosis renal proximal tubulopathy with alkalosis Liddle's syndrome monocytic/myelocytic leukaemia metabolic alkalosis nephrotoxins (e.g. amphotericin, aminoglycosides, heavy metals)
3.
Shift of K into cells a. familial hypokalaemic periodic paralysis b. thyrotoxic periodic paralysis barium poisoning C. a. insulin with glucose
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With the exception of spironolactone, triamterene and amiloride, all diuretics increase renal K loss and significant hypokalaemia occurs in patients with a low K dietary intake. Osmotic diuresis due to glycosuria and ketonuria regularly causes hypokalaemia in diabetic ketoacidosis. Mineralocorticoids and mineralocorticoid-like substances (e.g. licorice) produce renal K loss by increasing secretion of K and hydrogen ion by the distal and collecting tubules. In renal tubular acidosis, alkalosis which normally accompanies K depletion cannot occur because of either defective hydrogen ion secretion, inability to establish a pH gradient across the renal tubular lumen or bicarbonate loss in the proximal renal tubules. In Bartter's syndrome, inappropriate renal K loss is accompanied by the combined presence of hypokalaemia, alkalosis, hyperaldosteronism, hyperreninaemia, overproduction of renal postaglandins (PG), normal blood pressure, relative resistance to the pressor effects of angiotension II and norepinephrine and hyperplasia of the juxta-glomerular apparatus (10-12). The most recent pathogenetic clue lies in the demonstration of a defective reabsorption of chloride in the loop of Henle (10,111. However, the classic clinical and biochemical features of Bartter's syndrome are also encountered in surreptitious vomiting, diuretics and laxative abuse and measurement of urinary chloride distinguishes Bartter's syndrome from these conditions (10,13-15). Gullner et al. (16) recently described a familial hypokalaemiculopathy associated with alkalosis which is distinct from Bartter's syndrome because firstly, pathological changes occur in the proximal tubules and secondly, renal chloride reabsorption is normal. In Liddle's syndrome, a familial disease with hypertension, renal K wasting is probably due to an intrinsic renal tubular abnormality (10). Renal toxins, e.g. amphotericin, outdated tetracyclines, aminoglycosides and heavy metals, can cause renal tubular dysfunction and K leakage (8). Hypokalaemia due to redistribution of K between intracellular and extracellular space leads to muscle weakness and periodic paralysis. HYPOKALAEMIC PERIODIC PARALYSIS Although the detailed pathogenesis of hypokalaemic periodic paralysis is still unknown, during acute attacks, a large amount of extracellular K shifts into the muscle. Muscle paresis occurs because of depolarization of the sarcolemmal membrane. Active transport of K and sodium (Na) in and out of cells is mediated by Na-K-adenosine triphosphatase (Na-K-ATPase) in the cell membrane. It has been suggested that alterations in the muscle permeability to K or Na and the activity of the Na-K-pump (Na-K-ATPase
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activity) results in shift of extracellular K into the muscle, until the Na-K-pump turns off leading to depolarization and muscle paralysis in all types of hypokalaemic paralysis (17). In periodic paralysis caused by the shift of extracellular K into the muscle compartment, considerably greater muscle weakness occurs than expected from the change in serum K. In fact, some patients have paralytic attacks with little change in serum K. Familial periodic paralysis, thyrotoxic periodic paralysis and paralysis associated with barium poisoning have common clinical features. The attacks are precipitated by rest after exercise, ingestion of sodium chloride, large carbohydrate diet and epinephrine or insulin administration. During the onset of weakness, gentle exercise improves strength and the patient "walks" off the attack. During the attack, there is flaccid paralysis of the limb muscles associated with hyporeflexia. The paralyzed muscles are refractory to electrical stimulation. Respiratory and cardiac muscles are rarely involved. Although the detailed pathophysiology is not clarified, during attacks, a large amount of K moves into the muscles leading to extracellular hypokalaemia. In-between attacks, serum K is normal (18). Familial hypokalaemic periodic paralysis has been reported from all parts of the world and from different ethnic groups, e.g. Caucasians (18), Orientals (19, 20), Negroes (21). The disease affects males predominantly with a male-to-female ratio of about 4-to-l. The attacks begin in the second decade of life. Familial hypokalaemic periodic paralysis is inherited as an autosomal dominant trait although sporadic cases are present (18, 22). At present there is no animal model for this condition. During attacks, the body K is normal and urinary K excretion is decreased (18, 22). The primary abnormality in familial hypokalaemic periodic paralysis is not known. Acute attacks are treated with K supplement and long-term prophylaxis consists of K salts and azetazolamide. In a few patients chronic progressive interattack muscle weakness and myopathy develop usually a few years after the onset of the acute attacks (22-24). Thyrotoxic periodic paralysis occurs in males of oriental ancestry. Consequently over 90% of the case reports before 1961 came from Japan. This complication of thyrotoxicosis is rare in Caucasians (25). In Japan, periodic paralysis has been reported to occur in around 2% of all thyrotoxic patients (26). The overall incidence (1.8%) of periodic paralysis was similar in Chinese thyrotoxic patients (27), but the incidence was 25.6% in the Chinese male thyrotoxic subjects (28). During the attacks serum K falls below the normal range in about 60 to 70% of
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the patients. Urinary K excretion decreases in proportion to the fall in serum K (27). Treatment of the Familial thyrotoxicosis cures the periodic paralysis. occurrence of thyrotoxic periodic paralysis was first reported by McFadzean and Yeung (29). HLA typing in Chinese patients with thyrotoxic periodic paralysis in Singapore also suggests the association of certain HLA antigens with thyrotoxic periodic paralysis (30). The pathophysiology of thyrotoxic periodic paralysis is also unclear. In animals and in man, thyroid hormones increase the Na-K-ATPase activity in a number of tissues including the skeletal muscle (31-33). There is no animal model for thyrotoxic periodic paralysis and little information is available on the muscle Na-K-ATPase activity in patients with thyrotoxic periodic paralysis. Thyrotoxicosis produces a state of 8-adrenergic mechanisms hypersensitivity to catecholamines. may also play a role in thyrotoxic periodic paralysis since propanolol has been shown to completely or partially suppress the attacks in most patients (28, 34). However, the familial occurrence of thyrotoxic periodic paralysis and the rarity of this syndrome in non-Orientals indicate that an additional factor, e.g. genetic susceptibility, is required to act in concert with thyroid hormones or catecholamine hypersensitivity. Accidental contamination of table salt with soluble barium salts resulting in an endemic form of periodic paralysis 'P'a Ping" or soft disease was first reported in Sichuan province of China (35-37). Subsequent reports confirmed that accidental or suicidal ingestion of soluble barium salts produced hypokalaemic paralysis. Severe intoxication is accompanied by profound hypokalaemia sometimes resulting in respiratory paralysis and cardiac arrest. Administration of K reverses the paralysis and permits full recovery (38,391. Infusion of barium chloride into dogs produced paralysis associated with prompt decrease in serum K which was due to the transfer of K into the muscles (40). In vitro studies of skeletal muscles showed that the events occurring in the barium-treated muscle closely simulate that in-familial hypokalaemic periodic paralysis (41). This suggests that chronic barium poisoning in dogs might be a possible animal model to examine the pathogenesis of hypokalaemic periodic paralysis in man (17). Transient hypokalaemic paralysis occasionally occurs in patients with chronic K deficiency associated with primary hyperaldosteronism (42), renal tubular acidosis (43) and in Bartter's syndrome (10-12). Plasma K tends to be low in-between attacks and falls to lower values during The paralysis can be reversed by K paralysis. administration. In dogs, as in the humans, chronic K depletion leads to muscle weakness and paralysis occurs either spontaneously or after provocation by insulin or
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epinephrine (44,451. Epinephrine or insulin activates the Na-K-pump in muscles (46, 47). The increased activity of the Na-K-pump then causes a sustained influx of K into the muscle, lowering the extracellular K further, until the Na-K-pump is turned off (48) and the membrane potential falls to an unexcitable level leading to muscle paralysis (17). GOSSYPOL-RELATED
HYPOKALAEMIA
Clinical Studies The hypokalaemic paralysis associated with gossypol was reported in 66 out of 8806 volunteers in China (3-6). Coutinho did not find any changes in serum K levels or paralysis in Brazilian men treated with similar doses of gossypol (7). The incidence of hypokalaemia showed marked regional differences in China, e.g. in Nanjing (Central China) the incidence was 4.7% and in Taian (North China) not a single subject had paralysis (4-6). The dietary K intake in Nanjing was below the recommended nutritional requirements in China (49) and the mean serum K was significantly lower in healthy men from Nanjing (6). This suggests that dietary K intake may be one of the factors accounting for the regional difference in gossypol-related hypokalaemia observed in China. The attacks of paralysis can be prevented by the administration of K at the onset of symptoms. The incidence of gossypol-related hypokalaemia in China was reduced from 5% before 1977 to zero after 1977 when K-chloride was administered routinely with gossypol (50). In a recent controlled study reported by Liu et it was clearly demonstrated that oral G.(51), administration of gossypol caused a gradual and steady decline of serum K which levelled at the lower limit of the normal range after one year. Mean serum K was 4.29 5 0.03 mmol/l (mean + SE, 152 subjects) at the beginning of the study and fell significantly to 3.75 + 0.04 mmol/l (86 subjects) over a 12-month period. Of the 152 subjects studied at the beginning of the trial, 6 (3.9%) had K levels below 3.0 mmol/l and amongst these 6 subjects only one (0.6%) developed hypokalaemic paralysis. In a subsequent study in progress, gossypol was administered alone or together with K supplements and a K-sparing diuretic (triameterene). In the groups of men treated, serum K values declined over seven months of observation. However no clinical symptoms of hypokalaemia were reported (52). The hypokalaemic paralysis associated with gossypol occurs usually in the summer months in Nanjing (Central China) when people sweat a great deal (5, 6). This seasonal variation is similar to that of thyrotoxic periodic
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paralysis reported in Chinese in Hong Kong (South China)(271. The clinical picture of gossypol-related hypokalaemic paralysis is similar to that of other origin, During e.g. familial, thyrotoxicosis and barium poisoning. the attack there is flaccid paralysis starting in the lower limbs and gradually spreading upwards but usually sparing the respiratory muscles. The biochemical findings are that of moderate hypokalaemia associated with increased urinary K In the 21 patients reported by Qian et a1.(50), excretion. 14 gossypol-treated subjects had serum K between 3.0 to 3.5 mmol/l and 24-hour urinary K excretion was over 30 mmol/l in 11. In the-remaining seven subjects, the serum K was less than 3.0 mmol/l, and the 24-hour urinary K still exceeded 30 mmol/l in six. This is in contrast to the findings in periodic paralysis due to redistribution of K where urinary Similarly Bi et al-(531 studied 3 K is normal or decreased. subjects from Beijing (North China) with gossypol-related hypokalaemic paralysis admitted to hospital. During the attack the serum K fell and the intracellular (erythrocyte) K decreased, but the urinary K excretion remained high. When serum K was measured in 38 patients treated with gossypol for 1 to 2 years and compared with a group of 73 controls, the serum K was similar in the 2 groups, but the intraerythrocytic K was significantly lower in the gossypol-treated group (53). Recent in vitro studies using human red blood cells showed that gossypol lowered intracellular K concentration without inhibiting the Na-K-ATPase activity. The results indicated that K loss from the red blood cells could be the consequence of the damage of the cell membrane by gossypol leading to increased K permeability and K loss (54). Although urinary or plasma aldosterone and plasma renin activity were not measured in these studies, salivary ratio of Na to K were normal in these subjects with hypokalaemic paralysis suggesting that there was no hyperaldosteronism (501. Other laboratory investigations including blood pH, serum Na, chloride, renal concentrating and diluting ability, renal acidifying power, renograms, and thyroid functions were reported to be normal in subjects given gossypol (3-6, 55). Most patients with gossypol-related hypokalaemia recover promptly after K repletion (5, 6). A few patients remained hypokalaemic a long time after cessation of gossypol treatment. Qian (6) reported 2 patients with chronic persistent hypokalaemia after gossypol. The clinical status of the patients before gossypol treatment was not described. These 2 patients had increase in urinary prostaglandin E2-like substance. K repletion did not correct the hypokalaemia or ameliorate the symptoms. Indomethacin, a prostaglandin synthetase inhibitor, together with K supplement led to normal K levels and disappearance
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of symptoms. From these observations, Qian proposed that prostaglandin is an important mediator of gossypol action in the development of renal K loss leading to hypokalaemia (5, 6). Studies in Animals Gossypol does not produce hypokalaemia or paralysis in animals, although different animals show different degree of tolerance to gossypol (5). In the dog, where experimental K depletion can lead to paralysis (20, 21), gossypol has been shown to be very toxic. Low doses of gossypol damage the heart and cause sudden death (5). In the rat, gossypol does not significantly affect thg2urine excretion of K (56, 571, but urinary excretion of K was reported to be increased after intravenous administration of 42K in gossypol-treated rats (58). Qian (6) showed in studies using isolated rabbit heart that gossypol reduced the myocardial K content. This could be prevented by the addition of magnesium to the perfusate. He suggested that gossypol may inhibit Na-K-ATPase or other magnesium-dependent enzyme systems thus interfering with K transport (6). Similarly, in rats fed a low K diet, gossypol reduced the intracellular K and magnesium This concentration although the serum K remained unchanged. effect of gossypol was not observed in rats fed a normal diet (59). Prompted by these observations, in vitro studies on the Na-K-ATPase activity of rat, guinea pig and human fetal renal cortex slices showed that gossypol markedly inhibited the Na-K-ATPase (60, 61). At the same time gossypol also decreased the oxygen consumption, magnesium-ATPase activty and intracellular K. This suggests that gossypol inhibits Na-K-ATPase due to non-specific binding of gossypol to the enzyme molecule (61). However, in rats, administration of the usual antifertility doses of gossypol in vivo did not cause a suppression of the renal Na-K-ATPase activity (60, 62, 631. In guinea pigs treated with gossypol for 5-9 weeks, Na-K-ATPase activity was lowered. This effect was diminished by giving the guinea pigs a K-rich diet (60). When large doses of gossypol were administered to rats and guinea pigs, the renal Na-K-ATPase activity was decreased (64, 65). In guinea pigs, the Na-K-ATPase activity in skeletal muscle was also decreased (64). Based on these studies in men and animals, Qian (5, 6) suggested the following mechanisms of action of gossypol especially in relation to the production of hypokalaemia: Gossypol may enhance renal prostaglandin biosynthesis (5, 6). Gossypol may also decrease renal Na-K-ATPase (57-63). These two factors led to leakage of K from intracellular to extracellular space, renal loss of K and K depletion. Hypokalaemia would further increase the renal biosynthesis of prostaglandin E2 (66) leading to further loss of K from
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the kidneys. Prostaglandin E2 (67) and hypokalaemia (48) are both inhibitory to Na-K-ATPase in some tissues. All these relationships lead to a vicious cycle resulting in further renal loss and hypokalaemia. The extent of problem and possible mechanisms It is evident from the recent clinical studies in China (50, 51, 52) that during gossypol administration mean serum K levels tend to fall and level at the lower limit of the normal range. It should be noted in the single clinical study done outside China (7), gossypol administration did not lower serum K levels in the 12 subjects. However, moderate hypokalaemia (K level below 3.0 mmol/l) occurs in about 4 to 5% of the subjects (50, 51). In most instances hypokalaemia has a gradual onset over several months and may express as the clinical symptom of fatique. From the studies of Qian et a1.(5,-6; 501, dietary K intake probably plays a role in the gossypol-induced hypokalaemia as hypokalaemia occurs more frequently in provinces where the average K intake is low (Jiangsu). The clinical studies in men suggest renal loss of K Although is the most likely cause of the hypokalaemia. absence of gastrointestinal loss of K has not been documented, vomiting and diarrhoea have not been reported as prominent side effects of gossypol administration. Furthermore, the urinary excretion of K remained high in the presence of low serum K levels in gossypol-induced hypokalaemia (50). Mineralcorticoid excess is unlikely as gossypol-related hypokalaemia is not associated with other features of hypernatraemia, alkalosis and hypertension. Absence of hyperaldosteronism is evidenced by the normal Na/K ratios in saliva and serum aldostexone levels (50, 55). The most likely cause of the hypokalaemia is direct toxic effect of gossypol on the renal tubules. Gossypol has been shown to cause leakage of K from human red blood cells (54) in vitro without inhibiting Na-K-ATPase activity suggesti'ng a direct effect of gossypol on cell membrane permeability. In vivo animal studies have also shown that renal Na-K-ATPase activity was not affected with the usual antifertility doses of gossypol (60, 62, 63). The direct toxic effects of gossypol on renal tubules could be similar to other renal toxins like heavy metals, amphotericin and aminoglycosides. The specific mechanism of the direct action of gossypol on renal tubules has not been elucidated. Although the incidence of gossypol-induced hypokalaemia is about 4 to 5% in China, that of hypokalaemic paralysis varied from zero or very low in north China (e.g. Taian, Beijing) to higher incidences in cental and south The differences in China (e.g. Nanjing, Shanghai). incidence is at least partly dependent on the dietary intake of K (4-6). It is also possible that the prevalence of
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paralysis in Chinese is at least dependent on genetic This genetic susceptibility is most evident susceptibility. in periodic paralysis of thyrotoxicosis which occurs exclusively in the Orientals (26-30). Our own studies showed periodic paralysis was a presenting feature in over 40% of Chinese patients with primary hyperaldosteronism (68). The hypokalaemic periodic paralysis caused by gossypol is most likely the result of chronic depletion of K resulting from renal leakage, but a direct toxic effect of gossypol on muscle membrane permeability has not been excluded. To conclude, the available clinical evidences suggest that gossypol increased K excretion by the renal tubules. The most likely mechanism is a direct toxic effect In most subjects given of gossypol on the renal tubules. gossypol, a very mild degree of hypokalaemia is present. However, in susceptible subjects, moderate or severe hypokalaemia ensues leading to muscle weakness and paralysis. This susceptibility to hypokalaemia is related to dietary K intake and probably also to genetic constitution. Acknowledgment The authors thank Drs. C. van Ypserle, T. Clausen, N. Skakkebaeh and C.A. Paulsen for their helpful suggestions and MS. S. Yim for the preparation of the manuscript. REFERENCES 1.
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Liu GZ, Chiu Lyle K, Gao J. Clinical trial of gossypol as a male contraceptive agent for man. In: Endocrinology teds. Labrie F, Proulix Lf. Excerpta Medica ICS 655, Amderstam, 1984, p. 227 to 230.
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Liu GZ, Chiu Lyle K, Gao J. Alleviation of side effects produced by gossypol. Proc. III International Congress of Andrology, Boston (1985).
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Bi XF, Ye YX, Yang HF, Zhang ZR. Preliminary study on gossypol in causing hypokalemia. Acta Acad Med Sinica 3:175-178 (1981).
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Yao KQ, Song MZ, Lei HP. Effect of gossypol on K metabolism of human red blood cells. Proc. Symposium on Fertility Regulation in Male, Nanjing (1984).
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55.
The long-term follow-up study of 22 cases You Gc. with hypokalaemic paralysis after administration of Proc Symposium on Male Fertility gossypol. Regulation in Male. Nanjing (1984).
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Sun YB, Chen QQ, Wang Y, Su M. Effect of gossypol on K excretion of male rat. Acta Acad Med Sinica 4:126-127 (1982).
57.
Yu TH, Zhang XD, Wang ZH. Gossypol did not affect the hypokalemia by DOCA in the rat. Reprod Contracep China 2:39-41 (1981).
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Xue SP, Liang DC, Shao TS, Wu YW, Liu Y, Zhow ZH, K Wang NY. Studies on the effect of gossypol on metabolism of rat. Acta Anat Sinica 10:78-87 (1979).
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Chen ZC, Gao LM, Sun SG, Qian SZ, Xu Y, Tang XC, Wang YE, Shen LY, Zhu MK. The influence of gossypol on the potassium metabolism of rats and the effect of some possible contributing factors. Acta Pharm Sinica 14:513-520 (1979).
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Bi XF, Zheng YJ, Yang HF, Zhang ZR. Effect of gossypol on ATPase activity of the kidney. Sci Sinica 24:573-580 (1981).
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Hong SK, Haspel HC, Sonenberg M, Goldriger JM. Effects of gossypol on PAH transport in the rabbit kidney slice.. Toxicology & Applied Pharmacology 71: 430-435 (1983).
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Fu YF, Zhao RS, Liu JG. Effect of gossypol acetic acid on renal Na-K-ATPase activity of rabbit and rat. Nature China 2:724-725 (1979).
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Wu YW, Wang NY, Tong DS, Shieh SP. Ultracytochemical observations on the effect of gossypol on Na-K-ATPase of the renal cell membrane of rats and guinea pigs. Acta Anat Sinica 12:289-292 (1981).
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Fei RR, Liang DC, Gao Y, Liu Y, Guo XR, Zhow ZH, Xue SP. Gossypol effect on the activity of renal cell membrane Na-K-ATPase of rats and guinea pigs. Reprod Contracep China 1:42-45 (1982).
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Su SY, Liu Y, Zhow ZH, Shieh SP, Zhao XJ, Xu MY, Zhuang YZ. Relationship between gossypol administration and activity of Na-K-ATPase in animal. Acta Anat Sinica 13:85-75 (1982).
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66.
Dusing R, Attallah AA, Presyna AP, Lee JB. Renal biosynthesis of PGE2 and F2, dependence on extracellular potassium. J Lab Clin Med 92:669-672 (1978).
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Mozsik G, Kutas J, Magy L, Nemeth G. Inhibition of Na-K-ATPase system from human gastric mucosa by PGEl and PGE2. Eur J Pharmacol 29:133-137 (1974).
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Ma JTC, Wang C, Lam KSL, Yeung RTT, Chan FL, Boey J,_ Cheung PSY, Coghlan JP, Scoggins BA, Stockigt JR. Studies of 50 consecutive patients with primary hyperaldosteronism in Hong Kong Chinese (submitted for publication).
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GOSSYPOL AND HYPOKALAEMIA* Christina Wang** and Rosie T.T. Yeung Department of Medicine, University of Hong Kong Queen Mary Hospital, Hong Kong ABSTRACT One major side effect of administration of gossypol as a male fertility regulating agent is the occurrence of hypokalaemic paralysis. We have reviewed the common causes of hypokalaemia in clinical practice and previous studies of gossypol-induced hypokalaemia in animals and man. The available evidences suggest that gossypol induced renal leakage of potassium. The most likely mechanism is a direct toxic effect of gossypol on the renal tubules.
~ossypol has been used by Chinese scientists since 1970 as a male method for fertility regulation (l-5). The antifertility efficacy as determined by sperm concentration (below 4 million/ml) was over 99%. Full recovery of spermatogenesis occurred in 73.7% of the volunteers. The common side effects reported include fatigue, gastrointestinal symptoms, decreased libido and potency, dizziness, and dryness of mouth. However the most serious side effect of administration of gossypol is hypokalaemic paralysis reported in 66 out of 8806 volunteers,i.e. 0.75% (3-5). The clinical syndrome is that of hypokalaemia: fatigue, muscle weakness followed by flaccid paralysis. Recovery is prompt and complete after potassium (K) repletion. However, 2 patients have been reported to have chronic persistent hypokalaemia after cessation of gossypol treatment (5,6). In one small-scale study of 12 Brazilian men, Coutinho (7) reported similar antifertility efficacy but serum K and biochemistry remained normal throughout the 12-month study. In this brief review, we will discuss some of the common causes of hypokalaemia in clinical practice, in particular hypokalaemic periodic paralysis and previous studies of gossypol-induced hypokalaemia in Chinese men. Such an inspection may shed light as to the basis for the gossypol-induced hypokalaemia.
*
Part of a review submitted in July, 1984 to the Steering Committee of the Task Force on Male Methods of Fertility Regulation of the WHO Special Programme on Research, Development and Research Training in Human Reproduction. ** To whom correspondence should be addressed. Submitted for publication July 2, 1985 Accepted for publication August 26, 1985
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HYPOKALAEMIA
IN CLINICAL PRACTICE
The principal causes of hypokalaemia encountered in clinical practice are listed in the Table (8). Dietary deprivation of K alone can lead to moderate hypokalaemia. However hypokalaemia seen in clinical practice is most frequently due to excessive loss of K either through the gastrointestinal tract or through the kidneys. K deficiency occurs commonly in gastrointestinal disorders in which vomiting, diarrhoea or loss of gastrointestinal secretions are prominent. Metabolic alkalosis which accompanies the loss of gastric secretions further enhances renal K loss. Gastrointestinal losses with vomiting or diarrhoea together with a compromised K intake in chronic alcoholics have been suggested to be the possible causes (9). Table.
Causes of Hypokalaemia in Men
1.
Gastrointestinal a. deficient intake starvation malnutrition b. excessive loss vomiting (including surreptitious vomiting) diarrhoea (including laxative abuse) nasogastric aspiration fistulas villous adenoma alcohol (?I
2.
Renal a. diuretics diuretic abuse osmotic diuresis (e.g. diabetic ketoacidosis) b. excessive mineralocorticoid effects primary hyperaldosteronism secondary hyperaldosteronism glucocorticoid excess licorice ingestion renal tubular diseases C. renal tubular acidosis renal proximal tubulopathy with alkalosis Liddle's syndrome monocytic/myelocytic leukaemia metabolic alkalosis nephrotoxins (e.g. amphotericin, aminoglycosides, heavy metals)
3.
Shift of K into cells a. familial hypokalaemic periodic paralysis b. thyrotoxic periodic paralysis barium poisoning C. a. insulin with glucose
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With the exception of spironolactone, triamterene and amiloride, all diuretics increase renal K loss and significant hypokalaemia occurs in patients with a low K dietary intake. Osmotic diuresis due to glycosuria and ketonuria regularly causes hypokalaemia in diabetic ketoacidosis. Mineralocorticoids and mineralocorticoid-like substances (e.g. licorice) produce renal K loss by increasing secretion of K and hydrogen ion by the distal and collecting tubules. In renal tubular acidosis, alkalosis which normally accompanies K depletion cannot occur because of either defective hydrogen ion secretion, inability to establish a pH gradient across the renal tubular lumen or bicarbonate loss in the proximal renal tubules. In Bartter's syndrome, inappropriate renal K loss is accompanied by the combined presence of hypokalaemia, alkalosis, hyperaldosteronism, hyperreninaemia, overproduction of renal postaglandins (PG), normal blood pressure, relative resistance to the pressor effects of angiotension II and norepinephrine and hyperplasia of the juxta-glomerular apparatus (10-12). The most recent pathogenetic clue lies in the demonstration of a defective reabsorption of chloride in the loop of Henle (10,111. However, the classic clinical and biochemical features of Bartter's syndrome are also encountered in surreptitious vomiting, diuretics and laxative abuse and measurement of urinary chloride distinguishes Bartter's syndrome from these conditions (10,13-15). Gullner et al. (16) recently described a familial hypokalaemiculopathy associated with alkalosis which is distinct from Bartter's syndrome because firstly, pathological changes occur in the proximal tubules and secondly, renal chloride reabsorption is normal. In Liddle's syndrome, a familial disease with hypertension, renal K wasting is probably due to an intrinsic renal tubular abnormality (10). Renal toxins, e.g. amphotericin, outdated tetracyclines, aminoglycosides and heavy metals, can cause renal tubular dysfunction and K leakage (8). Hypokalaemia due to redistribution of K between intracellular and extracellular space leads to muscle weakness and periodic paralysis. HYPOKALAEMIC PERIODIC PARALYSIS Although the detailed pathogenesis of hypokalaemic periodic paralysis is still unknown, during acute attacks, a large amount of extracellular K shifts into the muscle. Muscle paresis occurs because of depolarization of the sarcolemmal membrane. Active transport of K and sodium (Na) in and out of cells is mediated by Na-K-adenosine triphosphatase (Na-K-ATPase) in the cell membrane. It has been suggested that alterations in the muscle permeability to K or Na and the activity of the Na-K-pump (Na-K-ATPase
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activity) results in shift of extracellular K into the muscle, until the Na-K-pump turns off leading to depolarization and muscle paralysis in all types of hypokalaemic paralysis (17). In periodic paralysis caused by the shift of extracellular K into the muscle compartment, considerably greater muscle weakness occurs than expected from the change in serum K. In fact, some patients have paralytic attacks with little change in serum K. Familial periodic paralysis, thyrotoxic periodic paralysis and paralysis associated with barium poisoning have common clinical features. The attacks are precipitated by rest after exercise, ingestion of sodium chloride, large carbohydrate diet and epinephrine or insulin administration. During the onset of weakness, gentle exercise improves strength and the patient "walks" off the attack. During the attack, there is flaccid paralysis of the limb muscles associated with hyporeflexia. The paralyzed muscles are refractory to electrical stimulation. Respiratory and cardiac muscles are rarely involved. Although the detailed pathophysiology is not clarified, during attacks, a large amount of K moves into the muscles leading to extracellular hypokalaemia. In-between attacks, serum K is normal (18). Familial hypokalaemic periodic paralysis has been reported from all parts of the world and from different ethnic groups, e.g. Caucasians (18), Orientals (19, 20), Negroes (21). The disease affects males predominantly with a male-to-female ratio of about 4-to-l. The attacks begin in the second decade of life. Familial hypokalaemic periodic paralysis is inherited as an autosomal dominant trait although sporadic cases are present (18, 22). At present there is no animal model for this condition. During attacks, the body K is normal and urinary K excretion is decreased (18, 22). The primary abnormality in familial hypokalaemic periodic paralysis is not known. Acute attacks are treated with K supplement and long-term prophylaxis consists of K salts and azetazolamide. In a few patients chronic progressive interattack muscle weakness and myopathy develop usually a few years after the onset of the acute attacks (22-24). Thyrotoxic periodic paralysis occurs in males of oriental ancestry. Consequently over 90% of the case reports before 1961 came from Japan. This complication of thyrotoxicosis is rare in Caucasians (25). In Japan, periodic paralysis has been reported to occur in around 2% of all thyrotoxic patients (26). The overall incidence (1.8%) of periodic paralysis was similar in Chinese thyrotoxic patients (27), but the incidence was 25.6% in the Chinese male thyrotoxic subjects (28). During the attacks serum K falls below the normal range in about 60 to 70% of
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the patients. Urinary K excretion decreases in proportion to the fall in serum K (27). Treatment of the Familial thyrotoxicosis cures the periodic paralysis. occurrence of thyrotoxic periodic paralysis was first reported by McFadzean and Yeung (29). HLA typing in Chinese patients with thyrotoxic periodic paralysis in Singapore also suggests the association of certain HLA antigens with thyrotoxic periodic paralysis (30). The pathophysiology of thyrotoxic periodic paralysis is also unclear. In animals and in man, thyroid hormones increase the Na-K-ATPase activity in a number of tissues including the skeletal muscle (31-33). There is no animal model for thyrotoxic periodic paralysis and little information is available on the muscle Na-K-ATPase activity in patients with thyrotoxic periodic paralysis. Thyrotoxicosis produces a state of 8-adrenergic mechanisms hypersensitivity to catecholamines. may also play a role in thyrotoxic periodic paralysis since propanolol has been shown to completely or partially suppress the attacks in most patients (28, 34). However, the familial occurrence of thyrotoxic periodic paralysis and the rarity of this syndrome in non-Orientals indicate that an additional factor, e.g. genetic susceptibility, is required to act in concert with thyroid hormones or catecholamine hypersensitivity. Accidental contamination of table salt with soluble barium salts resulting in an endemic form of periodic paralysis 'P'a Ping" or soft disease was first reported in Sichuan province of China (35-37). Subsequent reports confirmed that accidental or suicidal ingestion of soluble barium salts produced hypokalaemic paralysis. Severe intoxication is accompanied by profound hypokalaemia sometimes resulting in respiratory paralysis and cardiac arrest. Administration of K reverses the paralysis and permits full recovery (38,391. Infusion of barium chloride into dogs produced paralysis associated with prompt decrease in serum K which was due to the transfer of K into the muscles (40). In vitro studies of skeletal muscles showed that the events occurring in the barium-treated muscle closely simulate that in-familial hypokalaemic periodic paralysis (41). This suggests that chronic barium poisoning in dogs might be a possible animal model to examine the pathogenesis of hypokalaemic periodic paralysis in man (17). Transient hypokalaemic paralysis occasionally occurs in patients with chronic K deficiency associated with primary hyperaldosteronism (42), renal tubular acidosis (43) and in Bartter's syndrome (10-12). Plasma K tends to be low in-between attacks and falls to lower values during The paralysis can be reversed by K paralysis. administration. In dogs, as in the humans, chronic K depletion leads to muscle weakness and paralysis occurs either spontaneously or after provocation by insulin or
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epinephrine (44,451. Epinephrine or insulin activates the Na-K-pump in muscles (46, 47). The increased activity of the Na-K-pump then causes a sustained influx of K into the muscle, lowering the extracellular K further, until the Na-K-pump is turned off (48) and the membrane potential falls to an unexcitable level leading to muscle paralysis (17). GOSSYPOL-RELATED
HYPOKALAEMIA
Clinical Studies The hypokalaemic paralysis associated with gossypol was reported in 66 out of 8806 volunteers in China (3-6). Coutinho did not find any changes in serum K levels or paralysis in Brazilian men treated with similar doses of gossypol (7). The incidence of hypokalaemia showed marked regional differences in China, e.g. in Nanjing (Central China) the incidence was 4.7% and in Taian (North China) not a single subject had paralysis (4-6). The dietary K intake in Nanjing was below the recommended nutritional requirements in China (49) and the mean serum K was significantly lower in healthy men from Nanjing (6). This suggests that dietary K intake may be one of the factors accounting for the regional difference in gossypol-related hypokalaemia observed in China. The attacks of paralysis can be prevented by the administration of K at the onset of symptoms. The incidence of gossypol-related hypokalaemia in China was reduced from 5% before 1977 to zero after 1977 when K-chloride was administered routinely with gossypol (50). In a recent controlled study reported by Liu et it was clearly demonstrated that oral G.(51), administration of gossypol caused a gradual and steady decline of serum K which levelled at the lower limit of the normal range after one year. Mean serum K was 4.29 5 0.03 mmol/l (mean + SE, 152 subjects) at the beginning of the study and fell significantly to 3.75 + 0.04 mmol/l (86 subjects) over a 12-month period. Of the 152 subjects studied at the beginning of the trial, 6 (3.9%) had K levels below 3.0 mmol/l and amongst these 6 subjects only one (0.6%) developed hypokalaemic paralysis. In a subsequent study in progress, gossypol was administered alone or together with K supplements and a K-sparing diuretic (triameterene). In the groups of men treated, serum K values declined over seven months of observation. However no clinical symptoms of hypokalaemia were reported (52). The hypokalaemic paralysis associated with gossypol occurs usually in the summer months in Nanjing (Central China) when people sweat a great deal (5, 6). This seasonal variation is similar to that of thyrotoxic periodic
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paralysis reported in Chinese in Hong Kong (South China)(271. The clinical picture of gossypol-related hypokalaemic paralysis is similar to that of other origin, During e.g. familial, thyrotoxicosis and barium poisoning. the attack there is flaccid paralysis starting in the lower limbs and gradually spreading upwards but usually sparing the respiratory muscles. The biochemical findings are that of moderate hypokalaemia associated with increased urinary K In the 21 patients reported by Qian et a1.(50), excretion. 14 gossypol-treated subjects had serum K between 3.0 to 3.5 mmol/l and 24-hour urinary K excretion was over 30 mmol/l in 11. In the-remaining seven subjects, the serum K was less than 3.0 mmol/l, and the 24-hour urinary K still exceeded 30 mmol/l in six. This is in contrast to the findings in periodic paralysis due to redistribution of K where urinary Similarly Bi et al-(531 studied 3 K is normal or decreased. subjects from Beijing (North China) with gossypol-related hypokalaemic paralysis admitted to hospital. During the attack the serum K fell and the intracellular (erythrocyte) K decreased, but the urinary K excretion remained high. When serum K was measured in 38 patients treated with gossypol for 1 to 2 years and compared with a group of 73 controls, the serum K was similar in the 2 groups, but the intraerythrocytic K was significantly lower in the gossypol-treated group (53). Recent in vitro studies using human red blood cells showed that gossypol lowered intracellular K concentration without inhibiting the Na-K-ATPase activity. The results indicated that K loss from the red blood cells could be the consequence of the damage of the cell membrane by gossypol leading to increased K permeability and K loss (54). Although urinary or plasma aldosterone and plasma renin activity were not measured in these studies, salivary ratio of Na to K were normal in these subjects with hypokalaemic paralysis suggesting that there was no hyperaldosteronism (501. Other laboratory investigations including blood pH, serum Na, chloride, renal concentrating and diluting ability, renal acidifying power, renograms, and thyroid functions were reported to be normal in subjects given gossypol (3-6, 55). Most patients with gossypol-related hypokalaemia recover promptly after K repletion (5, 6). A few patients remained hypokalaemic a long time after cessation of gossypol treatment. Qian (6) reported 2 patients with chronic persistent hypokalaemia after gossypol. The clinical status of the patients before gossypol treatment was not described. These 2 patients had increase in urinary prostaglandin E2-like substance. K repletion did not correct the hypokalaemia or ameliorate the symptoms. Indomethacin, a prostaglandin synthetase inhibitor, together with K supplement led to normal K levels and disappearance
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of symptoms. From these observations, Qian proposed that prostaglandin is an important mediator of gossypol action in the development of renal K loss leading to hypokalaemia (5, 6). Studies in Animals Gossypol does not produce hypokalaemia or paralysis in animals, although different animals show different degree of tolerance to gossypol (5). In the dog, where experimental K depletion can lead to paralysis (20, 21), gossypol has been shown to be very toxic. Low doses of gossypol damage the heart and cause sudden death (5). In the rat, gossypol does not significantly affect thg2urine excretion of K (56, 571, but urinary excretion of K was reported to be increased after intravenous administration of 42K in gossypol-treated rats (58). Qian (6) showed in studies using isolated rabbit heart that gossypol reduced the myocardial K content. This could be prevented by the addition of magnesium to the perfusate. He suggested that gossypol may inhibit Na-K-ATPase or other magnesium-dependent enzyme systems thus interfering with K transport (6). Similarly, in rats fed a low K diet, gossypol reduced the intracellular K and magnesium This concentration although the serum K remained unchanged. effect of gossypol was not observed in rats fed a normal diet (59). Prompted by these observations, in vitro studies on the Na-K-ATPase activity of rat, guinea pig and human fetal renal cortex slices showed that gossypol markedly inhibited the Na-K-ATPase (60, 61). At the same time gossypol also decreased the oxygen consumption, magnesium-ATPase activty and intracellular K. This suggests that gossypol inhibits Na-K-ATPase due to non-specific binding of gossypol to the enzyme molecule (61). However, in rats, administration of the usual antifertility doses of gossypol in vivo did not cause a suppression of the renal Na-K-ATPase activity (60, 62, 631. In guinea pigs treated with gossypol for 5-9 weeks, Na-K-ATPase activity was lowered. This effect was diminished by giving the guinea pigs a K-rich diet (60). When large doses of gossypol were administered to rats and guinea pigs, the renal Na-K-ATPase activity was decreased (64, 65). In guinea pigs, the Na-K-ATPase activity in skeletal muscle was also decreased (64). Based on these studies in men and animals, Qian (5, 6) suggested the following mechanisms of action of gossypol especially in relation to the production of hypokalaemia: Gossypol may enhance renal prostaglandin biosynthesis (5, 6). Gossypol may also decrease renal Na-K-ATPase (57-63). These two factors led to leakage of K from intracellular to extracellular space, renal loss of K and K depletion. Hypokalaemia would further increase the renal biosynthesis of prostaglandin E2 (66) leading to further loss of K from
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the kidneys. Prostaglandin E2 (67) and hypokalaemia (48) are both inhibitory to Na-K-ATPase in some tissues. All these relationships lead to a vicious cycle resulting in further renal loss and hypokalaemia. The extent of problem and possible mechanisms It is evident from the recent clinical studies in China (50, 51, 52) that during gossypol administration mean serum K levels tend to fall and level at the lower limit of the normal range. It should be noted in the single clinical study done outside China (7), gossypol administration did not lower serum K levels in the 12 subjects. However, moderate hypokalaemia (K level below 3.0 mmol/l) occurs in about 4 to 5% of the subjects (50, 51). In most instances hypokalaemia has a gradual onset over several months and may express as the clinical symptom of fatique. From the studies of Qian et a1.(5,-6; 501, dietary K intake probably plays a role in the gossypol-induced hypokalaemia as hypokalaemia occurs more frequently in provinces where the average K intake is low (Jiangsu). The clinical studies in men suggest renal loss of K Although is the most likely cause of the hypokalaemia. absence of gastrointestinal loss of K has not been documented, vomiting and diarrhoea have not been reported as prominent side effects of gossypol administration. Furthermore, the urinary excretion of K remained high in the presence of low serum K levels in gossypol-induced hypokalaemia (50). Mineralcorticoid excess is unlikely as gossypol-related hypokalaemia is not associated with other features of hypernatraemia, alkalosis and hypertension. Absence of hyperaldosteronism is evidenced by the normal Na/K ratios in saliva and serum aldostexone levels (50, 55). The most likely cause of the hypokalaemia is direct toxic effect of gossypol on the renal tubules. Gossypol has been shown to cause leakage of K from human red blood cells (54) in vitro without inhibiting Na-K-ATPase activity suggesti'ng a direct effect of gossypol on cell membrane permeability. In vivo animal studies have also shown that renal Na-K-ATPase activity was not affected with the usual antifertility doses of gossypol (60, 62, 63). The direct toxic effects of gossypol on renal tubules could be similar to other renal toxins like heavy metals, amphotericin and aminoglycosides. The specific mechanism of the direct action of gossypol on renal tubules has not been elucidated. Although the incidence of gossypol-induced hypokalaemia is about 4 to 5% in China, that of hypokalaemic paralysis varied from zero or very low in north China (e.g. Taian, Beijing) to higher incidences in cental and south The differences in China (e.g. Nanjing, Shanghai). incidence is at least partly dependent on the dietary intake of K (4-6). It is also possible that the prevalence of
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paralysis in Chinese is at least dependent on genetic This genetic susceptibility is most evident susceptibility. in periodic paralysis of thyrotoxicosis which occurs exclusively in the Orientals (26-30). Our own studies showed periodic paralysis was a presenting feature in over 40% of Chinese patients with primary hyperaldosteronism (68). The hypokalaemic periodic paralysis caused by gossypol is most likely the result of chronic depletion of K resulting from renal leakage, but a direct toxic effect of gossypol on muscle membrane permeability has not been excluded. To conclude, the available clinical evidences suggest that gossypol increased K excretion by the renal tubules. The most likely mechanism is a direct toxic effect In most subjects given of gossypol on the renal tubules. gossypol, a very mild degree of hypokalaemia is present. However, in susceptible subjects, moderate or severe hypokalaemia ensues leading to muscle weakness and paralysis. This susceptibility to hypokalaemia is related to dietary K intake and probably also to genetic constitution. Acknowledgment The authors thank Drs. C. van Ypserle, T. Clausen, N. Skakkebaeh and C.A. Paulsen for their helpful suggestions and MS. S. Yim for the preparation of the manuscript. REFERENCES 1.
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