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Cardiovascular and respiratory effects of Gaboon viper venom
Gen. Pharmac., 1975, Vol. 6, pp. 35 to 41. Pergamon Press. Printed in Great Britain
C A R D I O V A S C U L A R A N D RESPIRATORY EFFECTS OF GABOON VIPER VENOM B. C. WaAL~R Department of Physiology, Queen Elizabeth College, Campden Hill Road, London W8 7AH, England (Received 20 May 1974) Abstract--1. Doses of venom (rabbit 0.7-2.7 mg/kg; monkey 0.204).33 mg/kg) given intravenously and killing in 20-200 rain produce a marked and maintained fall in blood pressure. 2. There is a progressive reduction in O2 consumption accompanied in rabbits with hyperventilation and terminal pulmonary oedema. 3. The heart is directly affected. 4. Blood becomes incoagulable and there is also extensive internal haemorrhage, particularly in visceral tissues. INTRODUCTION THE GABOON viper, Bitis gabonica, is the largest of the African vipers, producing considerable quantities of a venom (Whaler, 1971) which is believed to be both haemotoxic and neurotoxic (Grasset, 1946; Christensen, 1955). Few fatalities have been directly attributable to this snake; however, where it has been known to strike at man or large animals, the outcome is either a fairly early death or extremely severe symptoms (Staley, 1929; Pitman, 1938; Grasset, 1946; Editorial, 1954; Mangili, 1959; Doucet, 1963; Lane, 1963). Interest in the venom has centred mainly on its action upon blood; here both pro-coagulant and anti-coagulant properties, as well as haemolytic activity have been reported (Christensen, 1955; Forbes et aL, 1969; Marsh & Whaler, 1974). In addition to these actions (and probably separate from the anti-coagulant activity) internal haemorrhage and bleeding from external orifices takes place This picture readily explains the "haemotoxic" reputation of the venom. The neurotoxic component is less well documented and perhaps derives from the dyspnoeic condition following venom intoxication; for example, Grasset (1946) comments upon this respiratory effect in the rabbit. However, experiments with the isolated phrenic nerve-diaphragm preparation of the rat have demonstrated quite clearly the absence of a peripheral neurotoxic activity (Ndiku, J. & Whaler, B. C., 1969, unpublished observations) even with doses of venom up to 400 ~tg/ml. In common with work on most venomous snakes the major stimulus for an understanding of Gaboon viper venom activity has been derived from its medical importance to snake-bite victims, and in the closely related factor, anti-venom production. Partly as a result of this and secondarily because of the rarity of bites from this species, we remain relatively
ignorant of the detailed mechanism of the envenomation. It was to remedy this that the present investigations were begun, using rabbits and monkeys as the main experimental animals. A preliminary report of the results has already appeared (Whaler, 1972). METHODS AND MATERIALS (a) Venom This was taken from animals caged in the laboratory (Whaler, 1971) and stored at approx. --23°C until required. It was diluted ( × 30) with 0.9 ~ NaCI prior to use. Venom dose was expressed only in terms of its protein content as measured using Folin-Ciocalteau reagent with bovine serum albumin (Sigma fraction V) as standard. Fresh whole venom usually contained 180260 mg/ml protein; dilutions gave 6-9 mg/ml and after intravenous injection, doses of 1.0-0.1 ml killed in 3-200 min. (b) Animal preparations Rabbits were anaesthetized with i.v. chloraloseurethane, supplemented with Nembutal (Abbott Laboratories Ltd.) and ether where necessary; for monkeys, i.m. phencyclidine (Parke-Davies & Co.) (2 mg/kg) was used. Carotid blood pressure was recorded using either a mercury manometer or a suitable transducer with its output displayed on a Devices 2-channel recorder. In a few experiments fight ventricular pressure was recorded from a catheter passed into the heart through the right jugular vein; the position of the cannula was checked post mortem. Heparin was used as an anticoagulant. The trachea was always cannulated and a one-way valve of small dead-space and low opening impedance (2-3 mm water, inlet; 3-4 mm water, outlet) attached. Expired air passed through silica-gel to a Servomex oxygen meter, and thence to a Kipps-Zonen Capnograph for CO2 analysis. The dead-space of this post-valve system was about 200 ml, and served to eliminate fluctuations in oxygen and CO2 content at the samplers; hence mean values for O~ and CO2 content of expired air were available. Although this whole system added to the ventilation work load, the animal came to steady-state 35
36
B. C. WHALER
values for I;'E, l?Eo2 and l?Eco~ before venom was injected The input side of the respiratory valve was usually open to the atmosphere but could be connected without increase in the air flow resistance to a balanced spirometer of about 1 1. capacity for measuring ventilation, when the time taken to withdraw some arbitrary volume (8001000 ml) was measured. From the respiration rate during this period it was possible to calculate minute and mean tidal volumes, as well as obtaining the data necessary for calculating oxygen consumption and carbon dioxide output of the animal. The overall accuracy of these methods was within -+-1.4 ml/min of COs and O2. A cannula into a small branch of the right jugular vein served for the administration of anaesthetic, heparin, saline and venom. Suitable taps on the arterial side enabled blood samples (1-2 ml each) to be withdrawn at intervals during the course of an experiment.
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RESULTS The effect o f venom upon the rabbit Figure 1 shows the effect of a small dose o f v e n o m upon blood pressure, respiration rate, minute v o l u m e and O ~ uptake for an experiment fully representative o f eight carried out with v e n o m doses o f 0.84.1mg/kg. Table 1 gives data for five other experiments.
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(c) Assay methods Packed cell volume was measured in capillary tubes; plasma glucose was determined using a glucose-oxidase method. Inspection of post-venom plasma samples showed that no marked haemolysis occurred; however, in spite of beparin, some formed weak clots upon standing. Hence, a semi-quantitative measure of fibrinogen content was made by precipitating the fibrinogen with 25 % sodium sulphate, redissolving in salicylate and measuring the turbidity of the solution. In most experiments the erythrocytes were re-suspended in saline and returned to the animal at convenient times. (d) In vitro tests Using simple systems, the effect of venom upon rat, monkey, rabbit and guinea pig plasma was tested. Doubling dilutions of venom were made and 0.05-0-1 ml added to 0.25 ml of citrated, oxalated or heparinized plasma; calcium or protamine was added when required but venom alone was sufficient to cause clotting except in the weakest dilutions. No tests were included for anticoagulant activity in vitro. Following early suggestions of a cardiotoxic action, Langendorff preparations were made, mostly using rabbit or monkey hearts. These were perfused with Krebs' bicarbonate saline, using a Watson-Marlow roller pump, and contractions recorded on a smoked drum. Those from monkeys were taken from phencyclidineanaesthetized animals used to provide tissue for another experiment; rabbit hearts were taken from animals killed by a blow on the head. In both cases the hearts were transferred immediately to ice-cold saline, trimmed of excess tissue, set up and perfused with saline at room temperature (22-25°C) until free of blood, before warming to 36-37°C. Venom was added to the reservoir from which the pump drew its supply and usually ran to waste after leaving the heart; occasionally it was re-cycled.
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Fig. 1. The effect of an injection of venom (at V2.3 mg) upon respiration rate, respiratory minute volume, Oa uptake and systemic blood pressure in the rabbit. Following the injection there was a marked fall in arterial pressure, beginning about 8 see after the v e n o m was introduced, and fully developed within 14 see. After small doses this recovered in minutes to near-normal levels and could be re-elicited, but to a progressively reduced extent, by further doses. In other cases recovery was absent, or only partial, and followed by a slow decline until the experiment terminated suddenly by respiratory and heart failure. In general, heart rate was not m u c h affected, in spite o f the hypotension; diastolic pressure fell considerably m o r e than systolic and pulse pressure was thus invariably increased. Right ventricular pressure did not fall immediately after v e n o m ; if anything, it increased over 5-10 min before declining gradually. However, it was always maintained better than systemic pressure. The maintained systolic values suggest that, at least initially, cardiac muscle performance itself was fairly normal. Ventilation increased considerably both in the period associated with the initial hypotension, and subsequently even when b l o o d pressure recovered. D u r i n g this latter period there was a progressive decrease in both oxygen uptake and C O z output although the latter fell rather m o r e slowly than the former, and R.Q. therefore reached values of 1.5 o r
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more. Blood Os levels were not measured but the animal appeared to become progressively hypoxic during this period, and arterial blood samples drawn for glucose and haematocrit analysis had a marked venous appearance. These conditions were associated with both tachypnoea and apparent dyspnoea. Respiration failed with extreme suddennness, often within the one minute or so required for a ventilation measurement, and at O~ uptake values much less than normal (Table 1). Left ventricular activity, as measured by the systemic blood pressure, failed more or less at the same time, and the moment of these events was usually marked by massive exudation of pulmonary oedema fluid into the tracheal cannula. Blood sugar levels always increased 2-3-fold after venom and, although declining somewhat before death, never returned to pre-venom values. Haematocrit tended to fall only slightly, and perhaps not significantly, by about 4 ~o from values of 42-37 Yo. In two experiments blood taken 10 and 25 min after venom was virtually free of fibrinogen as measured by sodium sulphite precipitation. Venom injection was frequently associated with vigorous defaecation; in addition, expulsion of blood-stained faecal pellets commonly occurred some minutes before death and was often preceded or accompanied by bladder emptying; the urine contained erythrocytes, but no free haemoglobin was present. Examination of animals which died 20-70 rain after venom injection showed extensive haemorrhagic areas in the external muscle and submucosal layers of the alimentary canal (rectum, colon, caecum and small intestine), ureters and bladder. Mesenteric tissue as well as fatty deposits and the pancreas were also haemorrhagic. The abdominal muscle wall and the diaphragm showed similar extensive damage although no distinction between haemorrhage and myoglobin release was made, and the latter could have been responsible for the "bloody" condition of these muscle areas. Superficial lung damage was visible and the heart and its basal structures (connective tissues, thymus and fatty tissue) showed extensive haemorrhagic patches; in some cases these had extended to the apex of the heart. In general the extent o f this haemorrhagic picture depended upon the survival time of the animal; after multiple small doses of venom, killing only after 30-170 min, this damage was severe, whereas it was virtually absent in cases where death occurred within less than 20 min. In addition to the haemorrhage, the left ventricle was strongly contracted, yet the right ventricle was relaxed, and often beating rather ineffectually. Stimulation of the phrenic nerve and a response from the diaphragm confirmed absence of neuromuscular paralysis as a primary cause of respiratory failure. Effect o f venom on the monkey Four similar experiments were carried out (see
38
B. C. WSALEg
Table 1 and Fig. 2); the sensitivity to venom was much greater than in the rabbit, and the hypotension showed little tendency to recover. Respiratory changes were much less than in the rabbit and only transient increases in ventilation occurred, although reduction of oxygen uptake and the very rapid terminal failure of ventilation were exactly as described above. Pulmonary oedema of the kind observed in the rabbit never occurred. v 1.3mg
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The isolated heart Langendorff-type preparations of rabbit, monkey and two cat hearts were perfused with venom. In most cases the venom solution was passed once through the heart with no re-cycling, and was followed by normal Krebs saline. Results are shown in Table 2. Failure in every case was irreversible and no improvement occurred in partially affected preparations perfused with normal saline. As the table shows, failure was usually rapid and, although the venom doses were high, absence of re-cycling presumably means that only a small proportion of the full dose came into contact with the heart.
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DISCUSSION AND CONCLUSIONS
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doses, no proper clot formation whatsoever. In these experiments the clots, when formed, were loose in texture and showed no retraction. Venom added to washed erythrocytes in saline and left for 12-16 hr at r o o m temperature (24°C) produced little evidence of haemolysis, and other in vitro tests have confirmed an almost total absence of phospholipase A, with no direct lytic factor and virtually no indirect lytic factor. The finding of unhaemolysed plasma in rabbits and monkeys dying after venom injection is thus corroborated by these observations. In one experiment a small haemolytic effect was noted with washed cat erythrocytes left at 4°C for 18 hr in the presence of venom (600 lag/tube) but a tenfold reduction in venom concentration abolished this.
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Fig. 2. The effect of an injection of venom (at V 1"3 mg) upon respiration rate, minute volume, O= uptake and blood pressure in the monkey. Upon post-mortem examination there was the impression of more intensive haemorrhage in all areas, in spite o f the fact that the dose of venom used was distinctly less than in the rabbits. As in the rabbit, plasma from blood drawn post-venom either showed a tendency to clot, or in samples drawn later, had a reduced fibrinogen content. In vitro effects on blood and plasma The addition of venom (0.33-29 lag venom protein per tube) to heparinised or oxalated plasma taken from guinea pig, rat and monkey produced clotting in the absence of both protamine and calcium. However, larger doses of venom (up to 780 lag/tube) resulted in delayed clotting, and with the higher
The results presented here fail to confirm fully the usually accepted effects of G a b o o n viper venom. N o evidence has been obtained for a neurotoxic effect upon the phrenic-nerve diaphragm preparation in vitro, and in the present experiments a vigorous response of the rabbit diaphragm to stimulation of the phrenic nerve immediately following death confirms this finding. In addition, the increased ventilation which so often occurred after venom injection and which declined only slowly until shortly before "death" is evidence against a developing peripheral neurotoxicity. However, the apparent difficulty of respiration (as demonstrated by the pronounced hyperpnoea) could be readily interpreted as an effort to maintain an otherwise failing respiratory system, and it is probably this aspect of the envenomation response which has led to suggestions of neurotoxic action. Rejection of this concept leaves a need for some explanation of the respiratory effects, and three factors are relevant. Hypotension itself is a respiratory stimulant and is probably involved in the initial stages. However in many experiments the increased ventilation continued after the hypotension reversed, suggesting a more generalized central stimulation. Thirdly, and contributary for perhaps the whole of the post-venom phase, the progressive hypoxia
Effects of Gaboon viper venom
39
Table 2. The effect of venom upon isolated perfused hearts Preparation No. 1 2a b 3a b 4 5 6 7 8 9 10
Species Monkey Monkey Monkey Monkey Monkey Rabbit Rabbit Rabbit Rabbit Rabbit Cat Cat
Volume* (ml) 110 50 50 50 50 50 30 28 66 36 52 98
Venom cone. (~tg/ml) 30 250 547 220 440 200 200 277 146 146 292 345
Time of venom contact (min) 5.5 4"5 4.5 2'0 4"5 2-3 3"5 ca 4 6"0 4.0 4"5 7.5
Response 80% fail in 25 min Ineffective Fail 18 min Ineffective Fail 15 min Fail 2-3 min Fail 7 min Fail 4 min Fail 8 min Fail 18 min Fail 4-5 min Fail 6-7 min
* Volume represents that containingvenom which passed through the heart. In experiments 1 and 3 it was re-cycled twice; in all others no re-cycling occurred. Failure time calculated from the start of venom perfusion. would provide a powerful stimulus to respiration from peripheral receptors. Only when blood oxygen levels were very low would this reflex response to chemoreceptor drive fail; this would be expected to occur very suddenly after such a period of hypoxia, and indeed does so. The fall in oxygen consumption and venous appearance of the animal suggests effects of venom at a pulmonary site rather than an overall diminution of cell oxygen requirements. Qualitatively, blood oxygenation was depressed during this phase, whereas if tissue oxygen usage was declining, blood would retain its normal oxygenated appearance. Apart from the possibility of phospholipase, which, if present, could seriously damage pulmonary epithelium, other enzymes are probably involved, as well as left heart failure which is itself conducive to pulmonary oedema. Cardiac failure was demonstrated by the BP fall, change in ECG record, beating right but quiescent and contracted left ventricle at death as well as by the Langendorff preparations mentioned. These latter results suggest that a powerful cardiotoxin is present in this venom, as in that from many other snake venoms (Jiminez Porras, 1970). The widespread formation of haemorrhagic petechiae following the administration of venom would also be likely to cause structural damage to lung alveoli and pulmonary capillaries. Whilst specific enzymes of a protease nature have not yet been clearly identified in venom of B. gabonica, they are widespread in snake venoms generally, including those of the Viperidae (see Jiminez Porras, 1970) and our own unpublished .experiments have demonstrated proteolytic activity in several fractions isolated using Sephadex and CM columns: so too has Mebs (1969). Blood sugar changes have been described before
as a result of Bitis spp. envenomation (Grasset & Goldstein, 1947) but no analysis of the occurrence has been attempted. If a specific hyperglycaemic factor was liberated, an early peak and slower decline might be expected. Alternatively perhaps, catecholamine release would occur and contribute to the hyperglycaemic response; however the apparent vasodilation and unchanged heart rate is not entirely consonant with this and the phenomenon requires further study. Blood coagulation changes appear to depend upon venom concentration and upon whether in rive or in vitro conditions are used. In vitro, low doses stimulate and larger amounts prevent clotting. Grasset & Zoutendyke (1938) observed a coagulant effect and Christensen (1955) obtained accelerated clotting with large doses and a reduced clotting with small doses. Forbes et aL (1969) reported only an anti-coagulant effect in vitro. More recent experiments (Marsh & Whaler, 1972, 1974) have shown that both pro- and anti-coagulant factors can be demonstrated in vitro; under in vivo conditions whole venom seems to produce only defibrination. The mechanisms of these responses are being investigated. A substantial clinical report (Staley, 1929) comments upon some of the matters raised above. This report discussed the clinical findings in terms of physiological effects, and many of the responses described are found in the present results, even though species were different. The nature and cause of the haemorrhagic areas needs further investigation. I n appearance this presents itself as mild to gross reddening of many tissues, particularly the diaphragm and gut, an effect which increases roughly with the time during which the venom has acted prior to death. Large doses, killing rapidly, show no effect, whereas those animals
B. C. WHALER in which death takes 20--200 min to occur show extensive haemorrhage. At an early stage in development, these appear as small petechiae in body wall, diaphragm and alimentary canal, but later the induration can be so extensive---and this is seen very clearly in mice used for assay purposes after i.p. venom--as to mask virtually all appearance o f normal structure as seen by the naked eye. The extensive release of myoglobin (as described by Reid (1961) after sea snake bites) could account for this, but the invasion o f tissues by erythrocytes from damaged capillaries is an equally satisfactory explanation, and one which is supported indirectly by two further pieces o f evidence. One is that little or no haemoglobin is present in plasma after venom, and at a time when haemorrhage is extensive; secondly, the haematuria which so frequently occurred was never accompanied by free haemoglobin or myoglobin. These considerations make it unlikely that myoglobin release on an extensive scale was occurring; moreover the haematuria which probably originated from glomerular damage indicates that gross capillary damage elsewhere could occur. However further investigations on these points are continuing. If the findings here can be extrapolated to man, it would seem that the major problems associated with envenomation, and which should require primary clinical attention are: (a) cardiac damage and failure, and (b) changes in the lungs, whereby oxygenation of blood is prevented. If these can be alleviated then other factors are not so crucial. In principle, defibrination per se does not cause haemorrhage, as shown by Reid & Chart (1968) for the Malayan Pit vipers. The evidence of survival of mice used for assay purposes, in spite of extensive tissue haemorrhage, suggests that this factor too may not be crucial in causing death. As far as man is concerned Doucet (1963) published a list of ten cases of G a b o o n viper bite in W. Africa; there were no fatalities. In view of the warnings expressed by both Ionides (quoted by Lane, 1963) and by Pitman (1938 and personal communication) about the deadly nature of this species, Doucet's findings are surprising, and one suspects two sub-species, the East African one having a more lethal venom than that from West Africa. The sensitivity of the monkey (200-300 ~tg/kg is lethal) and even more so the baboon (50-100 ~tg/kg, Whaler, unpublished) would suggest that man might be equally affected by the venom, yet no deaths have been corroborated, and indeed no eases even documented from East Africa. Route of administration of the venom is clearly important. A snake would inject intra-venously only by chance; however they could inject subcutaneously or intra-muscularly some tens or hundreds of milligrams at a strike---a dose which would be expected to cause extremely rapid intoxication; the absence of human fatalities thus remains puzzling
and may, as Pitman suggests, reflect the extraordinary placidity of the animal.
SUMMARY
G a b o o n viper venom produces hypotension which may or may not reverse. Irrespective of this the animal eventually dies in a markedly hypotensive state associated with cardiac failure. The cardiac effect occurs also in vitro. Oxygen consumption declines, often to levels only 10-20yo of the prevenom value; in rabbits (but not monkeys) this change is accompanied by hyperventilation terminating abruptly with. the appearance of pulmonary oedema fluid. Plasma fibrinogen levels fall and blood is eventually incoagulable. Extensive haemorrhage occurs, initially in visceral tissues and later spreading to skeletal muscle, especially the diaphragm. The significance of the results is discussed particularly with respect to treatment of humans bitten by this snake. Surprisingly there are no formal reports o f deaths, in spite of the widespread fear of this snake and its reputedly deadly nature expressed by East Africans. Acknowledgements--A research grant from Makerere University, Uganda, made this work possible; in addition, the author is indebted to Professor P. G. Wright and his staff for their hospitality, and to Mr. B. S. Ssebbuggwaawo for excellent technical assistance.
REFERENCES CHRISTENSENP. A. (1955) South African Snake Venoms and Antivenins. South African Institute for Medical Research, Johannesburg, S.A. DOUCET J. (1963) Les serpents de la Republique de Cote d'Ivoire. Acta Tropica XX, 201-259. EDITORIAL(1954) 30,000--40,000 snake bite deaths every year. S. Aft. Med. J. 28, 654--655. FORBES C. D., TURPIE A. G. G., FERGUSON J. C., McNxCOL G. P. & DOUOLAS A. S. (1969) Effect of Gaboon viper, Bitis gabonica, venom on blood coagulation, platelets and the fibrinolytic enzyme system. J. clin. Path. 22, 312-316. GRASS~T E. (1946) La vipere du Gabon. Acta Tropica. 11I, 97-115. GRASSET E. & GOLDSTEIN L. (1947) Hyperglycaemic action of snake venoms in relation to their toxic and antigenic properties. Trans. R. Soc. Trop. Med. Hyg. 40, 771-788. GRASSET E. & ZOUTENDYK A, (1938) Studies on the Gaboon viper (Bitis gabonica) and the preparation of a specific therapeutic antivenene. Trans. R. Soc. Trop. Med. Hyg. 31, 445--450. J~MINEZ PORRAS J. M. (1970) Biochemistry of snake venoms. Clin. Toxicol. 3, 389--431. LANE M. (1963) In Life with lonides, Chap. III. Hamish Hamilton, London. MANGILIG. (1959) I velami ofidici e la coagulazione del sangue. Arch. Zool. Ital. XLIV, 165-214.
Effects of Gaboon viper venom MARSH N. A. & WHALERB. C. (1972) Separation from Gaboon viper venom of two fractions affecting haemostasis. XIV Internat. Congress of Haematology; Abstracts. S~to Paulo, Brazil. MARSH N. A. & WHALER B. C. (1974) Separation and partial characterisation of a coagulant enzyme from Bitis gabonica venom. Br. J. Haemat. 26, 295-306. MEns D. (1969) Ober Schlangengift-Kallikreine: heinigung und Eigenschaften eines Kinin-freisetzenden Enzyms ans dem Gift der Viper Bitis gabonica. Hoppe-Seyler's Z. PhysioL Chem. 350, 1563-1569. PITMAN C. R. S. (1938) A Guide to the Snakes of Uganda. The Uganda Soc., Kampala, Uganda. REID H. A. (1961) Myoglobinuria and sea snake-bite poisoning. Br. Med. 3". I, 1284.
41
REID H. A. & C'nAR K. E. (1968) The paradox in therapeutic defibrination. Lancet i, 485. STALEY F. H. (1929) A case report of Gaboon viper poisoning with recovery. Bull. Antivenin Inst. Am. HI, 31-39. WrtALER B. C. (1971) Venom yields from captive Gaboon vipers. The Uganda Journal, Kampala 35, 195-206. WHALERB. C. (1972) Gaboon viper venom and its effects. J. PhysioL, Lond. 222, 61P.
Key Word Index Venom; snake venom; vipers; cardiovascular; Bitis gabonica.
C A R D I O V A S C U L A R A N D RESPIRATORY EFFECTS OF GABOON VIPER VENOM B. C. WaAL~R Department of Physiology, Queen Elizabeth College, Campden Hill Road, London W8 7AH, England (Received 20 May 1974) Abstract--1. Doses of venom (rabbit 0.7-2.7 mg/kg; monkey 0.204).33 mg/kg) given intravenously and killing in 20-200 rain produce a marked and maintained fall in blood pressure. 2. There is a progressive reduction in O2 consumption accompanied in rabbits with hyperventilation and terminal pulmonary oedema. 3. The heart is directly affected. 4. Blood becomes incoagulable and there is also extensive internal haemorrhage, particularly in visceral tissues. INTRODUCTION THE GABOON viper, Bitis gabonica, is the largest of the African vipers, producing considerable quantities of a venom (Whaler, 1971) which is believed to be both haemotoxic and neurotoxic (Grasset, 1946; Christensen, 1955). Few fatalities have been directly attributable to this snake; however, where it has been known to strike at man or large animals, the outcome is either a fairly early death or extremely severe symptoms (Staley, 1929; Pitman, 1938; Grasset, 1946; Editorial, 1954; Mangili, 1959; Doucet, 1963; Lane, 1963). Interest in the venom has centred mainly on its action upon blood; here both pro-coagulant and anti-coagulant properties, as well as haemolytic activity have been reported (Christensen, 1955; Forbes et aL, 1969; Marsh & Whaler, 1974). In addition to these actions (and probably separate from the anti-coagulant activity) internal haemorrhage and bleeding from external orifices takes place This picture readily explains the "haemotoxic" reputation of the venom. The neurotoxic component is less well documented and perhaps derives from the dyspnoeic condition following venom intoxication; for example, Grasset (1946) comments upon this respiratory effect in the rabbit. However, experiments with the isolated phrenic nerve-diaphragm preparation of the rat have demonstrated quite clearly the absence of a peripheral neurotoxic activity (Ndiku, J. & Whaler, B. C., 1969, unpublished observations) even with doses of venom up to 400 ~tg/ml. In common with work on most venomous snakes the major stimulus for an understanding of Gaboon viper venom activity has been derived from its medical importance to snake-bite victims, and in the closely related factor, anti-venom production. Partly as a result of this and secondarily because of the rarity of bites from this species, we remain relatively
ignorant of the detailed mechanism of the envenomation. It was to remedy this that the present investigations were begun, using rabbits and monkeys as the main experimental animals. A preliminary report of the results has already appeared (Whaler, 1972). METHODS AND MATERIALS (a) Venom This was taken from animals caged in the laboratory (Whaler, 1971) and stored at approx. --23°C until required. It was diluted ( × 30) with 0.9 ~ NaCI prior to use. Venom dose was expressed only in terms of its protein content as measured using Folin-Ciocalteau reagent with bovine serum albumin (Sigma fraction V) as standard. Fresh whole venom usually contained 180260 mg/ml protein; dilutions gave 6-9 mg/ml and after intravenous injection, doses of 1.0-0.1 ml killed in 3-200 min. (b) Animal preparations Rabbits were anaesthetized with i.v. chloraloseurethane, supplemented with Nembutal (Abbott Laboratories Ltd.) and ether where necessary; for monkeys, i.m. phencyclidine (Parke-Davies & Co.) (2 mg/kg) was used. Carotid blood pressure was recorded using either a mercury manometer or a suitable transducer with its output displayed on a Devices 2-channel recorder. In a few experiments fight ventricular pressure was recorded from a catheter passed into the heart through the right jugular vein; the position of the cannula was checked post mortem. Heparin was used as an anticoagulant. The trachea was always cannulated and a one-way valve of small dead-space and low opening impedance (2-3 mm water, inlet; 3-4 mm water, outlet) attached. Expired air passed through silica-gel to a Servomex oxygen meter, and thence to a Kipps-Zonen Capnograph for CO2 analysis. The dead-space of this post-valve system was about 200 ml, and served to eliminate fluctuations in oxygen and CO2 content at the samplers; hence mean values for O~ and CO2 content of expired air were available. Although this whole system added to the ventilation work load, the animal came to steady-state 35
36
B. C. WHALER
values for I;'E, l?Eo2 and l?Eco~ before venom was injected The input side of the respiratory valve was usually open to the atmosphere but could be connected without increase in the air flow resistance to a balanced spirometer of about 1 1. capacity for measuring ventilation, when the time taken to withdraw some arbitrary volume (8001000 ml) was measured. From the respiration rate during this period it was possible to calculate minute and mean tidal volumes, as well as obtaining the data necessary for calculating oxygen consumption and carbon dioxide output of the animal. The overall accuracy of these methods was within -+-1.4 ml/min of COs and O2. A cannula into a small branch of the right jugular vein served for the administration of anaesthetic, heparin, saline and venom. Suitable taps on the arterial side enabled blood samples (1-2 ml each) to be withdrawn at intervals during the course of an experiment.
._E
60[- Resp. rate
~"6
2o
,'n
RESULTS The effect o f venom upon the rabbit Figure 1 shows the effect of a small dose o f v e n o m upon blood pressure, respiration rate, minute v o l u m e and O ~ uptake for an experiment fully representative o f eight carried out with v e n o m doses o f 0.84.1mg/kg. Table 1 gives data for five other experiments.
°
0
•
• 00 )0000•0
•
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(c) Assay methods Packed cell volume was measured in capillary tubes; plasma glucose was determined using a glucose-oxidase method. Inspection of post-venom plasma samples showed that no marked haemolysis occurred; however, in spite of beparin, some formed weak clots upon standing. Hence, a semi-quantitative measure of fibrinogen content was made by precipitating the fibrinogen with 25 % sodium sulphate, redissolving in salicylate and measuring the turbidity of the solution. In most experiments the erythrocytes were re-suspended in saline and returned to the animal at convenient times. (d) In vitro tests Using simple systems, the effect of venom upon rat, monkey, rabbit and guinea pig plasma was tested. Doubling dilutions of venom were made and 0.05-0-1 ml added to 0.25 ml of citrated, oxalated or heparinized plasma; calcium or protamine was added when required but venom alone was sufficient to cause clotting except in the weakest dilutions. No tests were included for anticoagulant activity in vitro. Following early suggestions of a cardiotoxic action, Langendorff preparations were made, mostly using rabbit or monkey hearts. These were perfused with Krebs' bicarbonate saline, using a Watson-Marlow roller pump, and contractions recorded on a smoked drum. Those from monkeys were taken from phencyclidineanaesthetized animals used to provide tissue for another experiment; rabbit hearts were taken from animals killed by a blow on the head. In both cases the hearts were transferred immediately to ice-cold saline, trimmed of excess tissue, set up and perfused with saline at room temperature (22-25°C) until free of blood, before warming to 36-37°C. Venom was added to the reservoir from which the pump drew its supply and usually ran to waste after leaving the heart; occasionally it was re-cycled.
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Fig. 1. The effect of an injection of venom (at V2.3 mg) upon respiration rate, respiratory minute volume, Oa uptake and systemic blood pressure in the rabbit. Following the injection there was a marked fall in arterial pressure, beginning about 8 see after the v e n o m was introduced, and fully developed within 14 see. After small doses this recovered in minutes to near-normal levels and could be re-elicited, but to a progressively reduced extent, by further doses. In other cases recovery was absent, or only partial, and followed by a slow decline until the experiment terminated suddenly by respiratory and heart failure. In general, heart rate was not m u c h affected, in spite o f the hypotension; diastolic pressure fell considerably m o r e than systolic and pulse pressure was thus invariably increased. Right ventricular pressure did not fall immediately after v e n o m ; if anything, it increased over 5-10 min before declining gradually. However, it was always maintained better than systemic pressure. The maintained systolic values suggest that, at least initially, cardiac muscle performance itself was fairly normal. Ventilation increased considerably both in the period associated with the initial hypotension, and subsequently even when b l o o d pressure recovered. D u r i n g this latter period there was a progressive decrease in both oxygen uptake and C O z output although the latter fell rather m o r e slowly than the former, and R.Q. therefore reached values of 1.5 o r
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more. Blood Os levels were not measured but the animal appeared to become progressively hypoxic during this period, and arterial blood samples drawn for glucose and haematocrit analysis had a marked venous appearance. These conditions were associated with both tachypnoea and apparent dyspnoea. Respiration failed with extreme suddennness, often within the one minute or so required for a ventilation measurement, and at O~ uptake values much less than normal (Table 1). Left ventricular activity, as measured by the systemic blood pressure, failed more or less at the same time, and the moment of these events was usually marked by massive exudation of pulmonary oedema fluid into the tracheal cannula. Blood sugar levels always increased 2-3-fold after venom and, although declining somewhat before death, never returned to pre-venom values. Haematocrit tended to fall only slightly, and perhaps not significantly, by about 4 ~o from values of 42-37 Yo. In two experiments blood taken 10 and 25 min after venom was virtually free of fibrinogen as measured by sodium sulphite precipitation. Venom injection was frequently associated with vigorous defaecation; in addition, expulsion of blood-stained faecal pellets commonly occurred some minutes before death and was often preceded or accompanied by bladder emptying; the urine contained erythrocytes, but no free haemoglobin was present. Examination of animals which died 20-70 rain after venom injection showed extensive haemorrhagic areas in the external muscle and submucosal layers of the alimentary canal (rectum, colon, caecum and small intestine), ureters and bladder. Mesenteric tissue as well as fatty deposits and the pancreas were also haemorrhagic. The abdominal muscle wall and the diaphragm showed similar extensive damage although no distinction between haemorrhage and myoglobin release was made, and the latter could have been responsible for the "bloody" condition of these muscle areas. Superficial lung damage was visible and the heart and its basal structures (connective tissues, thymus and fatty tissue) showed extensive haemorrhagic patches; in some cases these had extended to the apex of the heart. In general the extent o f this haemorrhagic picture depended upon the survival time of the animal; after multiple small doses of venom, killing only after 30-170 min, this damage was severe, whereas it was virtually absent in cases where death occurred within less than 20 min. In addition to the haemorrhage, the left ventricle was strongly contracted, yet the right ventricle was relaxed, and often beating rather ineffectually. Stimulation of the phrenic nerve and a response from the diaphragm confirmed absence of neuromuscular paralysis as a primary cause of respiratory failure. Effect o f venom on the monkey Four similar experiments were carried out (see
38
B. C. WSALEg
Table 1 and Fig. 2); the sensitivity to venom was much greater than in the rabbit, and the hypotension showed little tendency to recover. Respiratory changes were much less than in the rabbit and only transient increases in ventilation occurred, although reduction of oxygen uptake and the very rapid terminal failure of ventilation were exactly as described above. Pulmonary oedema of the kind observed in the rabbit never occurred. v 1.3mg
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The isolated heart Langendorff-type preparations of rabbit, monkey and two cat hearts were perfused with venom. In most cases the venom solution was passed once through the heart with no re-cycling, and was followed by normal Krebs saline. Results are shown in Table 2. Failure in every case was irreversible and no improvement occurred in partially affected preparations perfused with normal saline. As the table shows, failure was usually rapid and, although the venom doses were high, absence of re-cycling presumably means that only a small proportion of the full dose came into contact with the heart.
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DISCUSSION AND CONCLUSIONS
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doses, no proper clot formation whatsoever. In these experiments the clots, when formed, were loose in texture and showed no retraction. Venom added to washed erythrocytes in saline and left for 12-16 hr at r o o m temperature (24°C) produced little evidence of haemolysis, and other in vitro tests have confirmed an almost total absence of phospholipase A, with no direct lytic factor and virtually no indirect lytic factor. The finding of unhaemolysed plasma in rabbits and monkeys dying after venom injection is thus corroborated by these observations. In one experiment a small haemolytic effect was noted with washed cat erythrocytes left at 4°C for 18 hr in the presence of venom (600 lag/tube) but a tenfold reduction in venom concentration abolished this.
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Fig. 2. The effect of an injection of venom (at V 1"3 mg) upon respiration rate, minute volume, O= uptake and blood pressure in the monkey. Upon post-mortem examination there was the impression of more intensive haemorrhage in all areas, in spite o f the fact that the dose of venom used was distinctly less than in the rabbits. As in the rabbit, plasma from blood drawn post-venom either showed a tendency to clot, or in samples drawn later, had a reduced fibrinogen content. In vitro effects on blood and plasma The addition of venom (0.33-29 lag venom protein per tube) to heparinised or oxalated plasma taken from guinea pig, rat and monkey produced clotting in the absence of both protamine and calcium. However, larger doses of venom (up to 780 lag/tube) resulted in delayed clotting, and with the higher
The results presented here fail to confirm fully the usually accepted effects of G a b o o n viper venom. N o evidence has been obtained for a neurotoxic effect upon the phrenic-nerve diaphragm preparation in vitro, and in the present experiments a vigorous response of the rabbit diaphragm to stimulation of the phrenic nerve immediately following death confirms this finding. In addition, the increased ventilation which so often occurred after venom injection and which declined only slowly until shortly before "death" is evidence against a developing peripheral neurotoxicity. However, the apparent difficulty of respiration (as demonstrated by the pronounced hyperpnoea) could be readily interpreted as an effort to maintain an otherwise failing respiratory system, and it is probably this aspect of the envenomation response which has led to suggestions of neurotoxic action. Rejection of this concept leaves a need for some explanation of the respiratory effects, and three factors are relevant. Hypotension itself is a respiratory stimulant and is probably involved in the initial stages. However in many experiments the increased ventilation continued after the hypotension reversed, suggesting a more generalized central stimulation. Thirdly, and contributary for perhaps the whole of the post-venom phase, the progressive hypoxia
Effects of Gaboon viper venom
39
Table 2. The effect of venom upon isolated perfused hearts Preparation No. 1 2a b 3a b 4 5 6 7 8 9 10
Species Monkey Monkey Monkey Monkey Monkey Rabbit Rabbit Rabbit Rabbit Rabbit Cat Cat
Volume* (ml) 110 50 50 50 50 50 30 28 66 36 52 98
Venom cone. (~tg/ml) 30 250 547 220 440 200 200 277 146 146 292 345
Time of venom contact (min) 5.5 4"5 4.5 2'0 4"5 2-3 3"5 ca 4 6"0 4.0 4"5 7.5
Response 80% fail in 25 min Ineffective Fail 18 min Ineffective Fail 15 min Fail 2-3 min Fail 7 min Fail 4 min Fail 8 min Fail 18 min Fail 4-5 min Fail 6-7 min
* Volume represents that containingvenom which passed through the heart. In experiments 1 and 3 it was re-cycled twice; in all others no re-cycling occurred. Failure time calculated from the start of venom perfusion. would provide a powerful stimulus to respiration from peripheral receptors. Only when blood oxygen levels were very low would this reflex response to chemoreceptor drive fail; this would be expected to occur very suddenly after such a period of hypoxia, and indeed does so. The fall in oxygen consumption and venous appearance of the animal suggests effects of venom at a pulmonary site rather than an overall diminution of cell oxygen requirements. Qualitatively, blood oxygenation was depressed during this phase, whereas if tissue oxygen usage was declining, blood would retain its normal oxygenated appearance. Apart from the possibility of phospholipase, which, if present, could seriously damage pulmonary epithelium, other enzymes are probably involved, as well as left heart failure which is itself conducive to pulmonary oedema. Cardiac failure was demonstrated by the BP fall, change in ECG record, beating right but quiescent and contracted left ventricle at death as well as by the Langendorff preparations mentioned. These latter results suggest that a powerful cardiotoxin is present in this venom, as in that from many other snake venoms (Jiminez Porras, 1970). The widespread formation of haemorrhagic petechiae following the administration of venom would also be likely to cause structural damage to lung alveoli and pulmonary capillaries. Whilst specific enzymes of a protease nature have not yet been clearly identified in venom of B. gabonica, they are widespread in snake venoms generally, including those of the Viperidae (see Jiminez Porras, 1970) and our own unpublished .experiments have demonstrated proteolytic activity in several fractions isolated using Sephadex and CM columns: so too has Mebs (1969). Blood sugar changes have been described before
as a result of Bitis spp. envenomation (Grasset & Goldstein, 1947) but no analysis of the occurrence has been attempted. If a specific hyperglycaemic factor was liberated, an early peak and slower decline might be expected. Alternatively perhaps, catecholamine release would occur and contribute to the hyperglycaemic response; however the apparent vasodilation and unchanged heart rate is not entirely consonant with this and the phenomenon requires further study. Blood coagulation changes appear to depend upon venom concentration and upon whether in rive or in vitro conditions are used. In vitro, low doses stimulate and larger amounts prevent clotting. Grasset & Zoutendyke (1938) observed a coagulant effect and Christensen (1955) obtained accelerated clotting with large doses and a reduced clotting with small doses. Forbes et aL (1969) reported only an anti-coagulant effect in vitro. More recent experiments (Marsh & Whaler, 1972, 1974) have shown that both pro- and anti-coagulant factors can be demonstrated in vitro; under in vivo conditions whole venom seems to produce only defibrination. The mechanisms of these responses are being investigated. A substantial clinical report (Staley, 1929) comments upon some of the matters raised above. This report discussed the clinical findings in terms of physiological effects, and many of the responses described are found in the present results, even though species were different. The nature and cause of the haemorrhagic areas needs further investigation. I n appearance this presents itself as mild to gross reddening of many tissues, particularly the diaphragm and gut, an effect which increases roughly with the time during which the venom has acted prior to death. Large doses, killing rapidly, show no effect, whereas those animals
B. C. WHALER in which death takes 20--200 min to occur show extensive haemorrhage. At an early stage in development, these appear as small petechiae in body wall, diaphragm and alimentary canal, but later the induration can be so extensive---and this is seen very clearly in mice used for assay purposes after i.p. venom--as to mask virtually all appearance o f normal structure as seen by the naked eye. The extensive release of myoglobin (as described by Reid (1961) after sea snake bites) could account for this, but the invasion o f tissues by erythrocytes from damaged capillaries is an equally satisfactory explanation, and one which is supported indirectly by two further pieces o f evidence. One is that little or no haemoglobin is present in plasma after venom, and at a time when haemorrhage is extensive; secondly, the haematuria which so frequently occurred was never accompanied by free haemoglobin or myoglobin. These considerations make it unlikely that myoglobin release on an extensive scale was occurring; moreover the haematuria which probably originated from glomerular damage indicates that gross capillary damage elsewhere could occur. However further investigations on these points are continuing. If the findings here can be extrapolated to man, it would seem that the major problems associated with envenomation, and which should require primary clinical attention are: (a) cardiac damage and failure, and (b) changes in the lungs, whereby oxygenation of blood is prevented. If these can be alleviated then other factors are not so crucial. In principle, defibrination per se does not cause haemorrhage, as shown by Reid & Chart (1968) for the Malayan Pit vipers. The evidence of survival of mice used for assay purposes, in spite of extensive tissue haemorrhage, suggests that this factor too may not be crucial in causing death. As far as man is concerned Doucet (1963) published a list of ten cases of G a b o o n viper bite in W. Africa; there were no fatalities. In view of the warnings expressed by both Ionides (quoted by Lane, 1963) and by Pitman (1938 and personal communication) about the deadly nature of this species, Doucet's findings are surprising, and one suspects two sub-species, the East African one having a more lethal venom than that from West Africa. The sensitivity of the monkey (200-300 ~tg/kg is lethal) and even more so the baboon (50-100 ~tg/kg, Whaler, unpublished) would suggest that man might be equally affected by the venom, yet no deaths have been corroborated, and indeed no eases even documented from East Africa. Route of administration of the venom is clearly important. A snake would inject intra-venously only by chance; however they could inject subcutaneously or intra-muscularly some tens or hundreds of milligrams at a strike---a dose which would be expected to cause extremely rapid intoxication; the absence of human fatalities thus remains puzzling
and may, as Pitman suggests, reflect the extraordinary placidity of the animal.
SUMMARY
G a b o o n viper venom produces hypotension which may or may not reverse. Irrespective of this the animal eventually dies in a markedly hypotensive state associated with cardiac failure. The cardiac effect occurs also in vitro. Oxygen consumption declines, often to levels only 10-20yo of the prevenom value; in rabbits (but not monkeys) this change is accompanied by hyperventilation terminating abruptly with. the appearance of pulmonary oedema fluid. Plasma fibrinogen levels fall and blood is eventually incoagulable. Extensive haemorrhage occurs, initially in visceral tissues and later spreading to skeletal muscle, especially the diaphragm. The significance of the results is discussed particularly with respect to treatment of humans bitten by this snake. Surprisingly there are no formal reports o f deaths, in spite of the widespread fear of this snake and its reputedly deadly nature expressed by East Africans. Acknowledgements--A research grant from Makerere University, Uganda, made this work possible; in addition, the author is indebted to Professor P. G. Wright and his staff for their hospitality, and to Mr. B. S. Ssebbuggwaawo for excellent technical assistance.
REFERENCES CHRISTENSENP. A. (1955) South African Snake Venoms and Antivenins. South African Institute for Medical Research, Johannesburg, S.A. DOUCET J. (1963) Les serpents de la Republique de Cote d'Ivoire. Acta Tropica XX, 201-259. EDITORIAL(1954) 30,000--40,000 snake bite deaths every year. S. Aft. Med. J. 28, 654--655. FORBES C. D., TURPIE A. G. G., FERGUSON J. C., McNxCOL G. P. & DOUOLAS A. S. (1969) Effect of Gaboon viper, Bitis gabonica, venom on blood coagulation, platelets and the fibrinolytic enzyme system. J. clin. Path. 22, 312-316. GRASS~T E. (1946) La vipere du Gabon. Acta Tropica. 11I, 97-115. GRASSET E. & GOLDSTEIN L. (1947) Hyperglycaemic action of snake venoms in relation to their toxic and antigenic properties. Trans. R. Soc. Trop. Med. Hyg. 40, 771-788. GRASSET E. & ZOUTENDYK A, (1938) Studies on the Gaboon viper (Bitis gabonica) and the preparation of a specific therapeutic antivenene. Trans. R. Soc. Trop. Med. Hyg. 31, 445--450. J~MINEZ PORRAS J. M. (1970) Biochemistry of snake venoms. Clin. Toxicol. 3, 389--431. LANE M. (1963) In Life with lonides, Chap. III. Hamish Hamilton, London. MANGILIG. (1959) I velami ofidici e la coagulazione del sangue. Arch. Zool. Ital. XLIV, 165-214.
Effects of Gaboon viper venom MARSH N. A. & WHALERB. C. (1972) Separation from Gaboon viper venom of two fractions affecting haemostasis. XIV Internat. Congress of Haematology; Abstracts. S~to Paulo, Brazil. MARSH N. A. & WHALER B. C. (1974) Separation and partial characterisation of a coagulant enzyme from Bitis gabonica venom. Br. J. Haemat. 26, 295-306. MEns D. (1969) Ober Schlangengift-Kallikreine: heinigung und Eigenschaften eines Kinin-freisetzenden Enzyms ans dem Gift der Viper Bitis gabonica. Hoppe-Seyler's Z. PhysioL Chem. 350, 1563-1569. PITMAN C. R. S. (1938) A Guide to the Snakes of Uganda. The Uganda Soc., Kampala, Uganda. REID H. A. (1961) Myoglobinuria and sea snake-bite poisoning. Br. Med. 3". I, 1284.
41
REID H. A. & C'nAR K. E. (1968) The paradox in therapeutic defibrination. Lancet i, 485. STALEY F. H. (1929) A case report of Gaboon viper poisoning with recovery. Bull. Antivenin Inst. Am. HI, 31-39. WrtALER B. C. (1971) Venom yields from captive Gaboon vipers. The Uganda Journal, Kampala 35, 195-206. WHALERB. C. (1972) Gaboon viper venom and its effects. J. PhysioL, Lond. 222, 61P.
Key Word Index Venom; snake venom; vipers; cardiovascular; Bitis gabonica.