Effects of Russell's viper venom fractions on systemic and renal hemodynamics.

Changes in systemic and renal hemodynamics induced by Russell's viper venom are well established. The component of the venom responsible for hemodynamic alteration has not been identified. By Sephadex column chromatography five fractions of Russell's viper (Daboia russellii siamensis) venom were isolated. Each venom fraction consisted of phospholipase A2, proteolytic enzyme, phosphomonoesterase, phosphodiesterase, arginine ester hydrolase and hyaluronidase of varying activities. Hemodynamic effects of each venom fraction including mean arterial pressure, cardiac output, systemic and renal vascular resistance, renal blood flow and glomerular filtration rate were studied in five groups of dogs; each group had four dogs. Minimal hemodynamic changes were observed in dogs receiving venom fraction I. Increased renal vascular resistance with diminution of renal blood flow and glomerular filtration rate was observed in dogs receiving venom fractions II, III, IV and V. A markedly increased renal vascular resistance with maximal decrease in renal blood flow and glomerular filtration rate was caused by fraction III of the venom with highest PLA2 and proteolytic enzyme activities. However, renal hemodynamic changes appeared to correlate better with proteolytic enzyme activity than PLA2 activity. The findings suggested the proteolytic enzyme as an important determinant of hemodynamic alteration. Fractional excretion of Na was increased in dogs injected with venom fraction IV, and is presumed to be due to the inhibition of tubular reabsorption of Na by a natriuretic factor in this venom fraction.     

Effect of Russell's viper (Vipera russelli siamensis) venom on renal hemodynamics in dogs

The effect on renal hemodynamics of Russell's viper (Vipera russelli siamensis) venom was studied in 8 mongrel dogs. The venom (0.10 mg/kg) was injected i.v. Measurements of general circulation and renal function were carried out over 48 hr. During the initial post-injection period, mean arterial blood pressure, pulse pressure and heart rate decreased. Total peripheral vascular resistance and renal vascular resistance showed a tendency to increase. There was no change in cardiac output. Thereafter the blood pressure and heart rate returned to the control level 2 hr after injection and remained stable throughout the experiment. The cardiac output remained unchanged, but the pulse pressure later increased. Renal blood flow, glomerular filtration rate, renal fraction and the rate of urine flow were decreased 24 hr after venom injection and rose to the control levels at 48 hr. Renal vascular resistance remained relatively increased, while peripheral resistance decreased, at 24 hr. Blood volume was unchanged throughout the 48 hr. Disseminated intravascular coagulation was not observed, although the clotting time was prolonged. Renal histological studies showed no remarkable changes; tubular necrosis was not seen. The renal hemodynamic changes may initially be due to catecholamine release and later to renin-angiotensin activation with renal vasoconstriction.     

Pathophysiological effects of Russell's viper venom on renal function

Pathophysiological effects of Russell's viper venom (RVV) on renal function are reviewed. The evidence in experimental animals on the mechanisms of venom action in relation to changes in either extrarenal or intrarenal factors is considered. The cardiovascular system and renal hemodynamics are affected by the venom. Mean arterial blood pressure, heart rate, cardiac output, and renal hemodynamics decrease, while total peripheral vascular resistance and renal vascular resistance increase in the initial post-injection period of envenoming. After the transitory decrease, all parameters for general circulation gradually increase, returning to near normal level within 30 min, while an increase in renal vascular resistance and decreases in renal blood flow and glomerular filtration rate are still apparent 48 h after envenomation. The effects of venom on renal vasoconstriction are discussed. Possible factors, especially humoral factors, inducing these changes are considered. Russell's viper venom is also able to affect renal tubular cells directly. This can be explained based on available data from in vitro studies. Different studies have been performed to investigate venom action in the isolated perfused kidney, changes in the characteristic polarization of the cell membrane, changes in mitochondria activity and changes in Na, K-ATPase activity of the tissue of both the renal cortex and medulla during envenomation.     

Acute effect of Russell's viper (Vipera russelli siamensis) venom on renal hemodynamics and autoregulation of blood flow in dogs

Renal hemodynamics and autoregulation of blood flow were investigated following intravenous injection of Russell's viper venom (0.1 mg/kg) in dogs anesthetized with sodium pentobarbital. After venom injection, the glomerular filtration rate fell significantly throughout the experimental period of three hr. Urine flow rate and renal blood flow also decreased and the filtered load of electrolytes declined significantly. The fractional excretion of sodium, potassium and phosphorus increased following venom administration. These data suggest that the venom may depress both glomerular and tubular functions. The renal autoregulation of blood flow was maintained during the experimental reduction of renal arterial pressure. We conclude that the ability of renal vasculature to autoregulate renal blood flow is not inhibited by Russell's viper venom, even though renal function is depressed.     

Histological and ultrastructural changes of the kidney in renal failure after viper envenomation

Forty experimental animals injected with lethal doses of Russell's viper venom and seven human victims of viper snake bite were studied in order to elucidate the pathogenesis of renal tubular necrosis due to viper envenomation. Autopsy, light and electron microscopic examinations were performed on animals and on 4 patients that died; renal biopsy studies were also carried out on the 3 patients that recovered. General features of disseminated intravascular coagulation, consumption coagulopathy and paradoxic haemorrhage were seen as a result of the strong coagulant effects of the venom. The salient features in the kidney are intraglomerular deposition of fibrin, fibrin degradation products and coagulation. The obstruction of glomerular capillaries by coagulated material is the most likely cause of reduction of blood supply to the renal tubules. This tubular ischemia results in tubular necrosis and subsequent renal failure.     

Relationship of administered dose to blood venom levels in mice following experimental envenomation by Russell's viper (Vipera russelli) venom

I125 labelled Russell's viper venom was administered i.m. into mice at various doses. Radioactivity in the blood at different intervals was determined and related to the amount injected. A mathematical equation was formulated to represent the relationship of administered dose to blood venom levels. This study suggests that the derivation of such an equation is also possible in humans in order to predict the amount envenomated.     

Some biochemical properties of Russell's viper (Daboia russelli) venom from Eastern India: correlation with clinico-pathological manifestation in Russell's viper bite.

In the present study, some biochemical properties and pathological effects of Daboia russelli venom from Burdwan district of West Bengal, eastern India are presented. The clinical features of Russell's viper envenomation observed in patients admitted to Burdwan Medical College & Hospital are also reported. In vitro, whole venom exerts strong trypsin inhibitory, phospholipase A2 and procoagulant activities in addition to moderate adenosine monophosphatase and adenosine triphosphatase activities. Lethality (LD50) of this venom sample is 0.7 mg kg (i.v.) of mice. Significant local tissue damaging effects including edema, hemorrhage and necrosis are observed in experimental animal models. An increase in the level of serum enzymes, such as aspartate transaminase, alkaline phosphatase, creatine phosphokinase, lactate dehydrogenase after D. russelli venom injection in albino rats is indicative of cell or tissue damage. High incidence of intravascular hemolysis in addition to hemostasis, haemoptysis and haematuria are observed as the most prominent features of RVV envenomation from this part of India. The present study reinforces the hypothesis that variation in the venom composition of RVV from eastern India with respect to venom samples of Russell's vipers from other parts of India is responsible for the differences in the clinical manifestation in patients from eastern India.     

Multiple thrombotic occlusions of vessels after Russell's viper envenoming

Systemic bleeding due to consumption coagulopathy and thrombocytopenia due to activation of procoagulants is the leading manifestation and cause of death in Russell's viper systemic envenoming. Thrombotic occlusion of the blood vessels is rare in cases of snakebite. In this report, two adult patients with Russell's viper systemic envenoming presented multiple cerebral infarctions, digital gangrenes and ischaemic organs in addition to typical clinical manifestations of bleeding diathesis and renal involvement. Our findings in these two special cases suggest that the venom-induced coagulopathy and endothelium damage, predisposed by toxin-induced vasoconstriction, might be the possible mechanism of multiple thrombotic vascular occlusions in systemic envenoming of Formosan Russell's viper.     

Haemorrhagic protein of Russell's viper venom with fibrinolytic and esterolytic activities.

A haemorrhagic toxin specifically active on skin and muscle at the site of introduction in mice has been purified from Vipera russelli russelli (Indian subspecies of Russell's viper) venom by CM-Sephadex C-50 ion exchange chromatography and size exclusion (SE)-HPLC. This toxic protein also has strong fibrinolytic and arginine esterolytic activities. The purified preparation was a single polypeptide chain of molecular weight 73,000, as revealed by SDS-PAGE and SE-HPLC under native and denatured conditions. It has been named as VRR-73. Atomic absorption spectrometry indicated the existence of Mg(2+) in a mol per mol ratio. Antiserum was effective in neutralizing haemorrhage when administered immediately following VRR-73 but was ineffective in inhibiting fibrinolytic and esterolytic activities. On the other hand phenylmethyl sulphonyl fluoride and EDTA inhibited fibrinolysis and esterolysis but did not affect haemorrhage. Thermal denaturation of VRR-73, after exposure at 100 degrees C for 10 min, led to inactivation of all of its activities. Fibrinolytic and esterolytic activities, but not the haemorrhagic activity, were slowly regained after cooling at 25 degrees C. Thus the two pathological activities of VRR-73 appear to be associated with two different regions of the molecule.     

Amount of venom injected by Russell's viper (Vipera russelli)

The total yield of venom (desiccated) in 25 adult V. russelli (mean length 111 +/- 1.8 cm (S.E.) ranges from 21-268 mg (127 +/- 13 mg, mean +/- S.E.) and that of 13 juvenile snakes (mean length 79 +/- 2.8 cm) is 8-79 mg (45 +/- 7 mg). The venom yield per milking correlated well with the length of the snake. The average amount of venom injected in the first bite of 31 adults (mean length 107 +/- 1.4 cm) is 63 +/- 7 mg (range 6-147 mg) and by 17 juvenile snakes (mean length 83 +/- 1.1 cm) is 41 +/- 8 mg (range 3-138 mg). Adults inject 45% of the glands' content in the first bite. More than 75 mg of venom (desiccated) was injected by 11 out of 31 adults and 2 out of 17 juvenile snakes. This indicates that in a substantial proportion of cases of envenomation, 40 ml of Burma Pharmaceutical Industry monovalent antivenom, which is commonly used, may not be adequate.     

Active immunization of rabbit with gamma irradiated Russell's viper venom toxoid

Russell's viper venom detoxified by gamma (gamma)-radiation (100 kR or 200 kR) was used as a toxoid for active immunization of rabbits following a short or long schedule of immunization without any adjuvant. Effective neutralization of venom toxin by immune sera of rabbits was observed with both schedules. Sera of rabbits immunized with 100 kR irradiated venom toxoid (100 kR toxoid antisera) were more potent than 200 kR toxoid antisera. The presence of antibody in the immune sera was detected by immunoelectrophoresis. The effect of gamma-radiation on some enzymes and venom protein profiles was studied. Phosphodiesterase, protease and hyaluronidase were inhibited by radiation though phospholipase A activity remained unaffected. Radiation did not produce any gross change in the protein profile of crude viper venom. Phosphodiesterase and protease activities of viper venom were neutralized more effectively by 100 kR toxoid antisera in the short schedule than in the long schedule of immunization.     

Studies on pharmacological effects of Russell's viper and Saw-scaled viper venom and its neutralization by chicken egg yolk antibodies

Antivenom antibodies were raised in 24-week-old white leghorn chickens against hemotoxic venoms of Russell's viper and Saw-scaled viper snakes. Booster injections of increasing concentrations of venom were given at 14days of time interval to raise the antivenom level in egg yolk. Antibodies were extracted from immunized chicken egg yolk by Polson et al. (Polson A., Von Wechmar M.B., Van Regenmortel M.H.V. Isolation of viral IgY antibodies from yolks of immunized hens. Immunological Communications 1980; 9:475-493.) and further purified by DEAE cellulose ion exchange column chromatography, which gave pure (180-200kDa) specific antibodies against venom. High titre of more than 1:10,000 antibodies were detected by ELISA at the 135th day of observation. The lethal toxicity and various pharmacological activities like hemorrhagic activity, phospholipase activity, edema and procoagulant activities of venom were carried out by both in vivo and in vitro methods. The effectiveness of antivenom in neutralizing these effects was carried out involving pre-incubation type experiments. The median effective dose (ED50) for Russell's viper venom was 0.96mg/2LD50/18g mice and for Saw-scaled viper venom it was 1.28mg/2LD50/18g mice. One millilitre of specific antivenom was effective in neutralizing 0.110mg of Russell's viper and 0.137mg of Saw-scaled viper venoms respectively (PD50). Antivenom was effective in neutralization assays in a dose dependent manner. The results indicate that antibodies raised in chicken could effectively neutralize the pharmacological effects induced by venoms and chickens therefore present an alternative and cheaper source of specific antibody generation.     

Russell's viper snakebite in Taiwan: differences from other Asian countries.

Formosan Russell's viper (Daboia russelli siamensis) is the sixth most frequent cause of snakebite in Taiwan. Its venom has been thought to have both neurotoxic and hematoxic properties. This viper's snakebite is rare and thus scarcely subjected to systemic studies. In this paper, we retrospectively analyzed and described 18 cases of viper snakebite from 1987 to 1999. Like that of the Russell's viper snakebite in other South East Asian areas, varied degrees of acute renal failure, incoagulable blood with bleeding diathesis and hemolysis were the major symptoms found in the systemic envenoming patients. Systemic thrombosis seems to be the distinguishing feature in Formosan Russell's viper snakebite. Neither symptoms nor signs of neuromuscular junction blocking effects were observed, which is another difference from symptoms observed after bites of some other Russell's viper subspecies, suggesting a significant geographic variation. These findings confirmed the clinical importance of Russell's viper snakebite in Taiwan.     

Effect of purified Russell’s viper venom-factor X activator (RVV-X) on renal hemodynamics, renal functions, and coagulopathy in rats

Acute renal failure (ARF) is the most frequent and a serious complication in victims of Russell’s viper snakebites. Russell’s viper venom-factor X activator (RVV-X) has been identified as a main procoagulant enzyme involving coagulopathy, which might be responsible for changes in renal hemodynamics and renal functions. Here, we purified RVV-X from crude Russell’s viper venom to study renal hemodynamics, renal functions, intravascular clot, and histopathological changes in Sprague-Dawley rats. Changes in renal hemodynamics and renal functions were evaluated by measuring the mean arterial pressure, glomerular filtration rate (GFR), effective renal plasma flow (ERPF), effective renal blood flow (ERBF), renal vascular resistance (RVR), and fractional excretion of electrolytes. After 10 min, rats receiving both crude venom and purified RVV-X decreased GFR, ERPF, and ERBF and increased RVR. These changes correlated to renal lesions. Along with the determination of intravascular clot, rats injected with purified RVV-X increased the average D-dimer level and reached a peak at 10 min, declined temporarily, and then reached another peak at 30 min. The temporal association between clots and renal dysfunction was observed in rats within 10 min after the injection of purified RVV-X. These findings suggested RVV-X as a major cause of renal failure through intravascular clotting in the renal microcirculation.     

Synergistic interaction of a protease and protease inhibitors from Russell's viper (Vipera russelli) venom

An acidic proteolytic enzyme, RVVX, was purified from Vipera russelli venom by successive chromatography on CM-Sephadex C-25, DEAE-cellulose and Sephadex G-100 columns. RVVX is a glycoprotein with a mol. wt of 79,000. It exhibited caseinolytic and factor X activating properties. Two trypsin inhibitors, TI-I and TI-II, were purified from V. russelli venom in a single step by CM-Sephadex C-25 column chromatography. The trypsin inhibitors interacted with the proteolytic enzyme RVVX. TI-I inhibited only the factor X activating property of RVVX while TI-II inhibited both, the caseinolytic and also factor X activating properties of RVVX. The edema inducing activity of RVVX increased markedly in the presence of non-edema inducing doses of TI-I and TI-II. RVVX, TI-I and TI-II were non-lethal in mice. The combination of RVVX and TI-II demonstrated enhanced toxicity.     

Risk indicators after envenomation in humans by Echis coloratus (mid-east saw scaled viper).

To determine the frequency, severity and predictors of bleeding and azotemia after envenomation in humans by Echis coloratus, a retrospective survey of 68 cases in Israel between 1970 and 1989 was carried out. We used univariate and multivariate analyses of clinical variables on admission for the outcome variables of bleeding, hemoglobin and platelet levels, and blood urea. Within hours or days after envenomation, a major bleeding episode occurred in 18% of the victims, a drop in hemoglobin to 10 g/dliter or less in 14%, and an increase in blood urea to 9 mmole/liter or more in 15%. These complications correlated with time interval between envenomation and hospital admission, and the following admission variables: degree of bleeding, hemoglobin level, platelet and white blood cell counts, blood urea and proteinuria. Complications were unlikely in patients who were presented with all of the following: a hemoglobin level of 13 g/dliter or more, a platelet count of 100,000/mm3 or more, a blood urea level of 7 mmole/liter or less, no proteinuria and no bleeding. Treatment on admission with a specific monovalent antiserum was associated with a shorter duration of hemostatic failure and a reduced incidence of anemia and thrombopenia. Infusion of fresh frozen plasma on admission did not appear to be effective in preventing complications.     

Geographical variation in India in the composition and lethal potency of Russell's viper (Vipera russelli) venom

Venom samples of Russell's viper (Vipera russelli) from three localities in India were analysed for their composition and toxicity. Column chromatographic fractionation on CM-Sephadex C-25 showed the absence of three fractions in the venom samples of southern India compared with the samples from northern and western India. The SDS-PAGE pattern of southern Indian venom samples also showed lack of three protein bands corresponding to molecular weights of 66,000, 39,000 and 9000. Venom samples from northern and western India possessed high acidic phospholipase activity while acidic phospholipase activity was absent in the samples from southern India, which in contrast showed large basic fractions with phospholipase activity. Proteolytic activity was present in all the venom samples; however, this activity, as well as trypsin inhibitor activity, was very low in the southern Indian samples. The ratio of proteolytic activity to inhibitor activity remained constant in most of the venom samples studied. LD50 values for most of the venom samples from northern and western India were twice as high as that of the samples from southern India. High phospholipase activity correlated with high lethal potency in the venom samples studied.     

Neurohormonal activation in severe scorpion envenomation: correlation with hemodynamics and circulating toxin

We studied the effects of scorpion (Androctonus australis hector) venom on hemodynamics and on the release of catecholamines, neuropeptide Y (NPY), endothelin-1 (ET-1) and atrial natriuretic peptide (ANP) in dog model of severe scorpion envenomation. Nine mongrel anesthetized dogs were submitted to mechanical ventilation through intubation and were administered intravenously purified dried scorpion venom (Androctonus autstralis) 0.05 mg/kg. Measurements including pulmonary artery catheter derived parameters, serum toxin levels and humoral variables were performed at baseline (before venom injection) and 5, 15, 30 and 60 min after venom injection. Humoral variables included: serum lactate, epinephrine (EP), norepinephrine (NE), NPY, ET-1 and ANP plasma concentrations. Scorpion venom caused rapid and transient increase of mean arterial pressure (MAP) and PAOP associated with a marked and sustained decline in cardiac output (-55% at 60 min; P < 0.001). Hemodynamic changes were associated with a rapid and significant increase of all measured hormones. The highest increase was for NE (28-fold) and EP (25-fold). MAP was closely correlated with NE and less significantly correlated with toxin levels. Similarly, significant correlation was observed between PAPO and ANP plasma levels. These findings support the implication of excessive catecholamines release in hemodynamic disturbances of severe SE and suggest that NPY and ET-1 could be involved in this process. Serum toxin does not appear to consistently contribute to these effects. Through its correlation with PAOP, ANP could be a reliable and useful marker of cardiac dysfunction in SE.     

Use of "Near Middle East Antivenom" to treat African bush viper envenomation

Venom from an African bush viper is primarily hemotoxic and potentially life threatening. Existing, commercialy available antivenoms may not neutralize venom of this genus. A 25-y-old male was brought to the emergency room diaphoretic and hypotensive (70/40 mmHg) after a bite from a pet African bush viper. A puncture wound on the left thumb was leaking slightly, but there was no evidence of blood loss, edema or bruising. Approximately 100 min after exposure, the patient experienced a small amount of proximal swelling. Six h after envenomation, he was admitted to the intensive care unit for monitoring. At 10 h after the bite prothrombin time (PT > 100 sec) and international ratio (INR = 9.2) were elevated. The patient was unable to coagulate. He received fresh frozen plasma, cryoprecipitate, and Near Middle East Antivenom. Improvement in clinical status and laboratory parameters were observed after each of 3 doses of antivenom (d-dimer > 1000 and fibrinogen = 137 mg/dL). The patient was monitored overnight, did not require additional antivenom and was discharged as laboratory parameters, vital signs and spread of the necrotic lesion stabilized. Near Middle East Antivenom appears effective in treatment of the hematologic sequelae secondary to African bush viper envenomation.