Abstract

THE USE OF ANTIVENOM REVERSES HEMATOLOGICAL AND OSMOTIC FRAGILITY CHANGES OF ERYTHROCYTES CAUSED BY INDIAN RED SCORPION Mesobuthus tamulus concanesis POCOCK IN EXPERIMENTAL ENVENOMING

K. RADHA KRISHNA MURTHY¹ CORRESPONDENCE TO: K. RADHA KRISHNA MURTHY - Professor of Physiology, Department of Physiology, Seth G.S.Medical College & K.E.M.Hospital, Parel, Mumbai 400 012, India. , M. ABBAS ZARE²

1 Department of Physiology, Seth G.S.Medical College & K.E.M.Hospital, Parel, Mumbai 400 012, India, 2 Institut D'Betat Des Serum ET Vaccine Razi, Hessark, Boita Postal: 656, Teheran, Iran.

ABSTRACT: Acute myocarditis was induced in experimental dogs by subcutaneous (SC) injection of 3mg/kg of scorpion venom from Mesobuthus tamulus concanesis POCOCK (earlier called Buthus tamulus). An increase in hemoglobin (Hb), mean corpuscular hemoglobin concentration (MCHC), packed cell volume (PCV), plasma hemoglobin (Plasma Hb) levels, and increased osmotic fragility of erythrocytes in vivo was observed after envenoming. An increase in osmotic fragility of red blood cells (RBC) was also observed when the blood in vitro was incubated with different concentrations of scorpion venom. Species- specific scorpion antivenom (SAV) was administered to different groups of animals at different time intervals following scorpion envenoming. This resulted in a decrease in Hb, MCHC, PCV, and plasma Hb levels in the envenomed animals and reversal of osmotic fragility changes of erythrocytes. It has been suggested that scorpion venom causes an autonomic storm releasing massive amounts of counter-regulatory hormones, such as catecholamines, angiotensin-II, glucagon, cortisol, and changes in insulin secretion resulting in hematological and osmotic fragility changes of erythrocytes. Administration of SAV effectively neutralized, prevented, and reversed scorpion venom toxicity and related osmotic fragility changes of erythrocytes.

KEY WORDS: Antivenom, scorpion venom, Mesobuthus tamulus concanesis POCOCK, osmotic fragility, hematology, PCV, MCHC, Hb, Plasma Hb, ECG changes.

INTRODUCTION

About 800 scorpions species from 6 families have been described all over the world. The largest family of scorpions of medical importance is the Buthidae, with the most toxic species, such as the Androctonus, Centruroides, Leiurus quinquestriatus and Tityus (1,2,4,8-18). India harbors 99 scorpion species from all the 6 families, of which 45 species belong to the Buthidae family. The scorpion Mesobuthus tamulus concanesis POCOCK (earlier called Buthus tamulus) stings are responsible for a number of deaths in infants, children, and adults in rural India (32,34,38). Scorpion envenomation results in acute myocarditis (21-23), disseminated intravascular coagulation (DIC) (29), transient hypertension followed by hypotension (24,25), pulmonary edema (32), acute pancreatitis (30), encephalopathy, and many other clinical manifestations leading to death. An increase in catecholamines (1,2), angiotensin II (26), glucagon, cortisol (35), thyroxine, triiodothyronine (36), changes in insulin secretion (28-31), and increased osmotic fragility of red blood cells (RBC) (24,25,27) in experimental scorpion envenoming have been reported. There are also reports that scorpion stings result in haematuria in human victims (35), but the mechanisms responsible for it are not well understood. Alterations in osmotic fragility of RBC could be due to changes in the erythrocyte Na⁺ - K⁺ pump activity dependent on hormonal environment (40).

So far, there is no effective specific treatment for scorpion envenoming in India. We demonstrate here the increased osmotic fragility of RBC in vitro and reversal of increased erythrocyte osmotic fragility and related hematological changes by the administration of species-specific scorpion antivenom (SAV) in vivo.

MATERIALS AND METHODS

Lyophilized crude venom from the scorpion Mesobuthus tamulus concanesis POCOCK was obtained from the Haffkine Institute, Mumbai, India. SAV was produced at the Haffkine Biopharmaceutical Corporation Ltd., Mumbai, India. The content of one vial of SAV was capable of neutralizing 12-18 mg of crude venom and was used in all the experimental animals. Venom (3mg/kg body weight) was injected SC, whereas SAV was administered IV.

Thirty-three mongrel dogs of either sex weighing 8±3 kg were anesthetized after overnight fast. These animals were randomly divided into four groups. Group 1 (n=9) received neither venom nor SAV. Blood from these animals was utilized for in vitro study of osmotic fragility changes of RBC (20). This was performed using 0.014 mg, 0.024 mg, 0.036 mg, and 0.04 mg of venom per cubic centimeter of blood and incubated in vitro for osmotic fragility (20).

Blood samples from Groups 2 (n=8), 3 (n=8), and 4 (n=8) were collected before envenomation for use as control. Blood was collected from Group 3 after 30 min and from Group 4 after 30 and 60 min following venom injection to study the effect of venom on osmotic fragility of RBC, hemoglobin (Hb), mean corpuscular hemoglobin concentration (MCHC), packed cell volume (PCV), and plasma hemoglobin (Plasma Hb) levels.

Group 2 received venom and antivenom simultaneously. SAV was administered to Groups 3 and 4 30 and 60 min following venom injection, respectively. Thereafter, the blood was collected every 30 minutes for a further 120 minutes. These blood samples were used to study the effect of SAV on venom-induced changes on osmotic fragility of erythrocytes, Hb, MCHC, PCV, and plasma Hb.

Hb (Drabkin cyanmethohemoglobin method), MCHC, PCV, and plasma Hb (Peroxidase method) were measured, as described by Dacie and Lewis (6). Limb Lead II electrocardiogram (ECG) (Encardiorite, Lucknow, India) was recorded continuously in Group 4 before and following envenoming and up to 120 min after SAV administration. Osmotic fragility of RBC was performed 0 hours and after incubation at 370ºC for 24 hours (20). The results were analyzed statistically by Student's paired 't' test.

RESULTS

Many abnormal ECG changes following scorpion envenoming were observed in Group 4. These changes were reverted following SAV administration. The changes in Hb, MCHC, PCV, and plasma Hb levels following envenoming and SAV administration are shown in Table 1. An increase was observed in Hb, MCHC, PCV, and plasma Hb in Group 3 after 30 min and in Group 4 after 30 and 60 min following envenoming.

No changes in Hb, MCHC, PCV, and plasma Hb levels 60, 90, and 120 min following envenoming and SAV administration were observed in Group 2. An increase in Hb and MCHC was observed only in Group 2 at 30 min.

A significant increase in Hb, MCHC, PCV, and plasma Hb levels was observed in Groups 3 and 4 30 min following envenoming. A sustained increase in all these parameters was observed in Group 4 60 min following envenoming.

SAV administration resulted in a decrease of Hb, MCHC, and PCV 120 min both in Groups 3 and 4. Plasma Hb levels in Group 4 120 min following antivenom returned to the values observed before venom injection. However, plasma Hb levels were increased in Groups 2 and 3 following SAV.

OSMOTIC FRAFILITY CHANGES OF ERYTHROCYTES (IN VIVO) (30-MINUTE INCUBATION)

No significant change in osmotic fragility of RBC was observed in Group 2 (Figure 1). An increase in osmotic fragility of RBC was observed in Group 3 30 min following venom injection (Figure 2). Group 4 also showed a similar increase in osmotic fragility of RBC 30 and 60 min following venom injection (Figure 3).

SAV administration showed a tendency in reduction of osmotic fragility changes of erythrocytes in Groups 3 and 4 after 60 min. These values returned to normal within 120 min following SAV (Figure 2 and Figure 3).

OSMOTIC FRAGILITY CHANGES OF ERYTHROCYTES (IN VIVO) (24-HOUR INCUBATION)

A significant increase in erythrocyte osmotic fragility was observed following venom injection in Groups 3 and 4 (Figure 4 and Figure 5). A significant reduction of osmotic fragility of RBC was observed in Groups 3 and 4 following SAV administration (Figure 4 and Figure 5).

OSMOTIC FRAGILITY CHANGES OF ERYTHROCYTES (IN VITRO)

All the concentrations of scorpion venom from 0.012 mg/ml to 0.04 mg/ml of blood led to increased osmotic fragility of RBC. However, the rise in osmotic fragility was not statistically significant at 0.012 mg/ml of venom, while at 0.024 mg/ml was significant. Venom (0.036 mg/ml) resulted in highly significant increased osmotic fragility changes of erythrocytes (Figure 6). Similar changes were observed in the blood samples incubated at 37ºC for 24 hours (Figure 7).

DISCUSSION

The envenomed animals manifested lacrimation, increased nasal secretions, thick mucous salivary secretions, frequent urination, fasciculation, clonus and tetany-like contractions of skeletal muscle, acute myocarditis, initial transient hypertension followed by hypotension, and many other clinical disorders. These observations are in agreement with earlier results (21,23,28,29,31).

OSMOTIC FRAGILITY CHANGES OF ERYTHROCYTES (IN VIVO) (30-MIN INCUBATION)

The results of this study indicated an increase in osmotic fragility of RBC in Groups 3 and 4 following venom injection. This is in agreement with our previous reports (24,25,27).

The osmotic fragility of freshly taken RBC reflects their ability to absorb water without lysis. The behavior of a RBC in hypotonic saline depends on the initial ratio of surface area to volume and not on the cell absolute size. The ability of the normal RBC to withstand hypotonicity results from its biconcave shape to allow a cell to increase its volume by about 70% before the surface membrane is stretched further (6).

The increase in erythrocyte osmotic fragility is thus due to its shape and is independent of the shape of RBC (6). On the other hand, the shape of the cell does not influence the resistance to hemolysis by lysolecithin, as the hemolytic process is chemical in nature (3). Additionally, pH, temperature, hypertonicity, and blood viscosity in a complex manner that affect the osmotic fragility (20) are shown to be altered in scorpion envenoming (15-17,22,24,25,27,29-31). The presence of phospholipase A2 in scorpion venom (5,39) has been reported. This enzyme is a powerful hemolytic agent and contributes to the increased osmotic fragility of RBC. In addition, the increase in PCV, Hb, plasma Hb, and MCHC in the envenomed animals could also account for the observed increase in osmotic fragility of erythrocytes.

OSMOTIC FRAGILITY CHANGES OF ERYTHROCYTES (IN VIVO) (24-HOUR INCUBATION)

The increased osmotic fragility of RBC after incubation at 37°C for 24 hours (Figure 3 and Figure 5) is in agreement with earlier reports (24,25,27). This increase probably reflects a change in volume to surface area ratio of RBC, but the factors which altered this ratio are more complicated than in fresh blood samples processed for osmotic fragility (6). During incubation for 24 hours, RBC metabolism is under stress and the pumping mechanisms may fail (27). The inhibition of cardiac sarcolemmal (21) and erythrocyte Na⁺-K⁺ATPase, Mg⁺⁺ ATPase and Ca⁺⁺ ATPase activities are demonstrated in the envenomed animals (27). A reduction in the erythrocyte Na⁺-K⁺ ATPase activity has been reported in a scorpion sting victim (27). Pande and Mead (19) have observed the inhibition of Na⁺-K⁺ ATPase activity by elevated free fatty acids (FFA). Through their detergent properties, FFA inhibit this enzyme activity. An increase in FFA following scorpion envenoming has been observed (27-29,31,33).

The increase in PCV, Hb, Plasma Hb, and MCHC might also have contributed to the observed increase in the osmotic fragility of RBC, as seen in Groups 3 and 4 after envenoming. An increase in hematocrit as indicated by PCV measurements reflected an increase in packed cell volume. Therefore, when a fixed unit volume of blood is utilized for measurement of osmotic fragility, we are taking a sample of increased volume of packed red cells circulating in the envenomed animals. It also explained a rise in plasma Hb concentration in the envenomed animals. All these factors might have contributed to the increased color of the supernatant fluid, when the blood samples were incubated with different dilutions of saline and estimated colorimetrically for osmotic fragility measurements.

The rise in PCV, Hb, and MCHC after envenoming could be due to hemoconcentration caused by a massive release of catecholamines (8-13) and angiotensin II (26). Angiotensin II produces a significant decrease in the blood volume and an increase in the extravascular fluid, leading to peripheral circulatory failure and pulmonary edema (7). Angiotensin II also stimulates the release of catecholamines. Catecholamines and angiotensin II may synergize or amplify each other's action and these may act, at least in part, at similar sites (7), resulting in hematological changes.

IN VTRO STUDIES OF OSMOTIC FRAGILITY

The enzyme phospholipase present in the scorpion venom (5,39) could be the agent responsible for the increased hemolysis in vitro. This is in contrast to the large number of factors contributing to the increased hemolysis, as discussed above. Certain venoms, such as the cobra snake venom release the enzyme phosphatidase A, which converts lecithin to lysolecithin, a powerful hemolytic substance (3). Increased osmotic fragility of RBC due to direct action of scorpion venom in vitro is a new finding. The results indicate that scorpion venom at the concentrations ranging from 0.012 to 0.04 mg/ml of blood increased osmotic fragility of erythrocytes. This could be the cause of intravascular hemolysis responsible for hematuria (35). Toxicity due to scorpion envenoming is dose dependent. However, many factors, such as species, venom dose, age, weight, sex of the victim, envenomation route, scorpion age and health, and season contribute to lethality (42,45).

ANTIVENOM ADMINISTRATION

In India, no effective and specific antivenom is available to treat metabolic, cardiovascular, and many other clinical manifestations due to scorpion envenoming. There are conflicting reports about the effectiveness of scorpion antivenom either in experimental animals or scorpion sting victims. Freire-Maia and his co-investigators (8,9), Amaral et al. (1,2), and Ismail (16,17) are of the opinion that serotherapy (antivenom) (scorpion antivenom) (SAV) is effective. Gueron and his co-investigators (10-12) reported that serotherapy is ineffective.

According to the Haffkine Biopharmaceutical Corporation Ltd., Mumbai India, the amount of antivenom present in each vial is sufficient to serve as antidote for scorpion sting victims. Therefore, in this experimental study, this much quantity of antivenom was administered IV. Scorpions usually inject the venom into the interstitial space (producing an immediate and sometimes insupportable pain) and not directly into the blood circulation. Therefore, as suggested by Freire-Maia and Campos (8), the antivenom was injected directly into the vein to neutralize the circulating venom, and the venom that is being absorbed from the sting site. Moreover, it is likely that antivenom administered IV could act on the tissues later on.

The effects of antivenom in reversing the osmotic fragility changes of erythrocytes are difficult to explain. However, it may be argued here that the antivenom will neutralize the effect of venom, and therefore can prevent a further rise in osmotic fragility, but it may not be able to reverse the changes in osmotic fragility of RBC, which has already taken place.

The results of PCV, MCHC, and Hb after antivenom administration showed a definite reversal. These changes could be due to a fall in angiotensin II level, leading to hemodilution. At the same time, an increase in physiologically active insulin levels has been observed (results observed but not presented). Insulin administration resulted in reversal of elevated angiotensin II levels in the experimentally envenomed dogs (26). Changes in insulin secretion (hypoinsulin secretion and hyperinsulin secretion at different time intervals following scorpion envenoming), resulting in hyperglycemia have been observed in this study (unpublished results). The rennin-angiotensin-aldosterone system is involved in scorpion sting victims (12,44). Changes in potassium levels have been demonstrated after scorpion envenoming (22). Insulin influences the rennin-angiotensin-aldosterone system through insulin-induced potassium changes (41). There are also reports suggesting that insulin induces stimulation of the Na⁺-K⁺ ATPase activity. The interaction of insulin with its receptor elicits the release of a mediator, which converts the Na⁺-K⁺ ATPase into an activated form, operating with a higher affinity for Na⁺ on its inner surface. This leads to transient increase in the rate of active Na+-K+ transport, establishing a new steady state with a steeper concentration gradient for Na⁺ across the cell membrane (43).

The maintenance of optimum skeletal muscle function-like excitability, contractile performance, and metabolism is essentially through the rate of active Na⁺-K⁺ transport, with the help of the Na⁺-K⁺ ATPase (the Na-K pump) activity (43). The activity of transport system located primarily in the sarcolemma, but also in the transverse tubules, is subject to acute regulation by epinephrine, norepinephrine, and insulin (13,37). Thus, the observations of fasciculation, clonus, and tetany-like contractions in the skeletal muscle of the envenomed animals could be due to changes in the basal Na⁺-K⁺ ATPase activity.

An imbalance between an increase in secretion of catabolic counter-regulatory hormones (catecholamines-epinephrine and norepinephrine, glucagon, cortisol, thyroxine (T4), triiodothyronine (T3), etc.) and a reduction in an anabolic hormone-like insulin secretion (34), which might have contributed to increased osmotic fragility of erythrocytes and consequent hemolysis. The resulting changes in hyperinsulin secretion -insulin sensitivity-euglycemia (unpublished results) are brought about after antivenom administration in envenomed animals. This might have contributed to the reversal of osmotic fragility changes of RBC.

Thus, scorpion envenoming with multi-system-organ-failure (MSOF) characterized by a massive release of catecholamines and inhibition of insulin secretion is a condition of fuel energy deficits and inability to effectively utilize the existing metabolic substrate (34). Antivenom administration, in our hands, under laboratory conditions effectively neutralized, prevented, and reversed the cardiovascular, hemodynamic, metabolic, and electrocardiographic changes in acute myocarditis induced by Indian red scorpion (Buthidae family) venom (33). Antivenom is found to be effective in preventing or abolishing the various clinical manifestations in human envenoming in clinically controlled trials (40).

E-mail: kradhakrishnamurthy@yahoo.com

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