Hyperhidrosis–Hypertension–Hypotension in Scorpion Envenoming Syndrome: Insulin–Glucose Infusion Reverses All the Clinical Manifestations Caused by Massive Release of Counter-Regulatory Hormones due to Killer Scorpion (Buthidae family) Stings

Death due to poisonous scorpion (Buthidae family) stings is common in many of the developing countries all over the world. Severe uncontrollable pain at the site of sting (without local oedema) results in autonomic storm, release massive quantities of catecholamines, angiotensin II, ACTH, glucocorticoids, glucagon, ADH, aldosterone, either suppressed insulin secretion/or hyperinsulinemia – insulin resistance causing hyperglycemia and a sudden increase in Free Fatty Acid levels (FFA). The increase in catecholamine and angiotensin II hormonal levels cause hyperhidrosis, initial transient hypertension, hyper salivation, hypotension, mydriasis, miosis, DIC, acute pancreatitis, and many other clinical manifestations. Suddenly increased FFA levels are toxic, produce inactivation of Na+–K+ATPase activities, arrhythmias, conduction defects, myocardial infarction, cardiogenic pulmonary oedema, Acute Respiratory Distress Syndrome (ARDS), multisystem organ failure and death. Hyperhidrosis is harmful and wasteful loss of fluid and electrolytes resulting in peripheral circulatory failure, hypotension and death. Based on our animal experimental studies and treating the scorpion sting victims with insulin glucose infusion, we consider that insulin has a primary metabolic role in preventing and reversing hyperhidrosis, hypertension, hypotension, cardiovascular changes, cardiogenic and non-cardiogenic pulmonary (ARDS) oedema. Treatment: Continuous infusion of regular crystalline insulin at the rate of 0.3 U/g glucose and glucose at the rate of 0.1 g/kg body weight/hour, for 48–72 hour, supplementation of potassium (if required), and maintenance of acid–base fluid and electrolyte balance.

2-Deoxy-D-glucose reverses the Indian red scorpion venom-induced cardiopulmonary abnormalities in anesthetized rats.

Role of 2-Deoxy-D-glucose (2-DG) in reversing the Indian red scorpion (Mesobuthus tamulus concanesis Pocock, MBT) venom-induced toxicity was examined. Femoral arterial pressure, ECG and respiratory movements were recorded in urethane anesthetized rats. Plasma glucose and serum insulin levels were also estimated. Intravenous injection of 5 mg/kg MBT venom produced immediate decrease in mean arterial pressure, heart rate and respiratory frequency  followed by an increase and subsequent progressive decrease. ECG pattern exhibited ischaemic changes. There was hyperinsulinemia after venom without corresponding decrease in plasma glucose. The animals died within 37±9 min and demonstrated significant increase in pulmonary water content. 2-DG pretreatment (0.5 g/kg, iv) improved the cardiopulmonary abnormalities induced by venom and the animals survived for nearly 120 min. There was no hyperinsulinemia and increased pulmonary water content in these animals. In insulin (2 IU/kg) treated rats, the MBT venom-induced cardiopulmonary abnormalities were attenuated and ECG abnormalities were reversed. The pulmonary water content in these animals exhibited a decreasing trend and the animals survived for 120 min. Repaglinide (10 mg/kg, iv) pretreatment failed to reverse the venom-induced cardiopulmonary changes including the increased pulmonary water content. The survival time was similar to venom only group. The present results reveal that 2-DG reverses the venom-induced cardiopulmonary toxicity probably by restoring insulin sensitivity.

Consequences of Mesobuthus tamulus gangeticus (Pocock, 1900) envenomation in albino mice

The present study aimed to investigate the effects of Mesobuthus tamulus gangeticus Pocock (Buthidae) venom on albino mice (NIH strain). Whole venom was obtained by electrical stimulation and its toxicity was determined in albino mice by subcutaneous envenomation. The venom LD50 was 2.5 mg kg-1 of mouse body weight. Toxic effects on different biochemical and enzymatic parameters in blood serum and other tissues of albino mice were determined after experimental envenomation with sublethal doses of M. tamulus gangeticus venom. Increased levels of glucose, uric acid and cholesterol, as well as decreased serum total proteins, were observed at 2 and 4 hours after the envenomation. In the liver and muscles, glycogen content dropped after venom injection. Moreover, M. tamulus gangeticus venom elevated the enzymatic activity of acid phosphatase (ACP), lactic dehydrogenase (LDH) and alanine aminotransferase (ALT) in the serum of albino mice. In conclusion, M. tamulus gangeticus can be considered a lethal scorpion species.(AU)

Group III PLA2 from the scorpion, Mesobuthus tamulus: cloning and recombinant expression in E. coli

Phospholipases A2 (PLA2) are enzymes that specifically hydrolyze the sn-2 fatty acid acyl bond of phospholipids, producing a free fatty acid and a lyso-phospholipid. We report the cloning and expression of a secretory phospholipase A2 (sPLA2) from Mesobuthus tamulus, Indian red scorpion. The nucleotide sequence codes for a 167 residue enzyme. The open reading frame codes for a 31 amino acid signal peptide followed by a mature portion of the protein. The primary structure shows the calcium binding motif, catalytic residues, 8 highly-conserved cysteines and C-terminal extension which classify it as a group III PLA2. The entire transcript was expressed in Escherichia coli and was purified by metal affinity chromatography under denaturing conditions. The protein was refolded by serial dilutions in the refolding buffer to its active form. Hemolytic assays indicate that the protein adopts a functional conformation. The functional requisites such as optimum pH of 8 and calcium dependency are shown. This report provides a simple but robust methodology for recombinant expression of toxic proteins.

Intraspecific variation in protein pattern of red scorpion (Mesobuthus tamulus, coconsis, pocock) venoms from Western and Southern India

Red scorpions Mesobuthus tamulus (Coconsis, Pocock) were obtained from different regions of West and South India (Ratnagiri, Chiplun and Ahmednagar from Maharashtra and Chennai from Tamil Nadu, respectively). Their venoms composition was analyzed using gel electrophoresis (SDS-PAGE). All venom samples shared six bands of 170, 80, 60, 57, 43, and 38 kDa molecular weights. Bands of 115 kDa and 51.5 kDa were characteristic of venoms obtained from red scorpions of Chiplun region, and the 26kDa band was absent in scorpion venom from Tamil Nadu. The separated protein band patterns suggest that the venoms from Ratnagiri, Ahmednagar and Tamil Nadu had high similarities in their biochemical composition but differed from that of Chiplun region. These data were also supported by the Jaccard (J) index. The J value was 0.33 for venom obtained from Ratnagiri-Ahmednagar, 0.31 for venom from Ratnagiri-Tamil Nadu, and 0.3 for venom from Ratnagiri-Chiplun region. This suggests the existence of genetic variation among the different strains of red scorpion in western and southern India. The antiserum produced by Haffkine Biopharmaceuticals Corporation Ltd. completely neutralized proteins of venoms from all the regions studied.(AU)

Excitatory effects of Buthus C56 toxin on Drosophila larval neuromuscular junction

Buthus C56 toxin from venom of the Indian red scorpion Mesobuthus tamulus was studied for its effects on spontaneous miniature excitatory junctional potentials (MEJP) on Drosophila larval neuromuscular junctions. C56 toxin was isolated on CM-Cellulose with linear gradient of ammonium acetate buffer, pH 6.0. Toxin purity was determined on SDS slab gel electrophoresis. Effective concentration of C56 toxin was based on contraction paralysis units (CPU) in Drosophila 3rd instar larvae by microinjection (0.1 CPU/ml = 2 x 10-6 g/ml). The toxin-induced excitatory junctional potentials were studied for calcium dependency (0.2 mM to 1.2 mM Ca2+) in Drosophila Ringer. Excitatory junctional potential amplitude was increased with increasing calcium concentration; maximum increase in the frequency at 0.4 mM Ca2+/4 mM Mg2+ Drosophila Ringer. It was suggested that while amplitude of excitatory junctional potentials was increased with concentration, maximum frequency increase at 0.4 mMCa2+/4 mM Mg2+ Drosophila Ringer may be due to augmented Ca2+ influx in 0.4 mM Ca2+, when NMDA receptors were maximally activated in C56 toxin-treated Drosophila larval neuromuscular junction.

Investigations on the role of insulin and scorpion antivenom in scorpion envenoming syndrome

Acute myocardiopathy in alloxan treated experimental dogs and rabbits was induced by subcutaneous (SQ) injection of scorpion venom from Mesobuthus tamulus concanesis, Pocock. Envenoming resulted in an initial transient hypertension (180-320 mm Hg.) followed by hypotension. Simultaneous administration of venom and species-specific scorpion antivenom (SAV) prevented hypertension and hypotension. Hypotension did not occur when SAV was given 60 min after envenoming. Blood glucose, triglycerides, cholesterol, amylase, insulin, glucagon, cortisol, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, red blood cell (RBC) count, hemoglobin (Hb), 2,3-diphosphoglycerate (2,3-DPG), and glutathione levels were increased 60 and 90 min after envenoming. Total white blood cell (WBC) count was reduced 60 min and increased 90 min after envenoming. Simultaneous administration of venom and SAV did not alter Hb, MCHC, and packed cell volume (PCV) levels, or ECG, and cardiovascular, biochemical, metabolic, and hormonal changes. Hematological parameters were reversed when SAV was given 30 and 60 min after envenoming. PCV, Hb, and MCHC values returned to normal 120 min after SAV. Alloxan-treated dogs showed increased blood glucose, cholesterol, glucagon, cortisol levels; reduced glycogen content of liver, cardiac and skeletal muscles; and reduced insulin levels and insulin/ glucagon ratio (I/G ratio). Envenoming in the alloxan pre-treated dogs further increased these levels and reduced tissue glycogen content, insulin levels, and I/G ratio. Administration of 4 U of insulin to alloxan pre-treated envenomed rabbits caused a biochemical and clinical improvement and increased glycogen content of all tissues in comparison with the values from those administered with SAV to alloxan pre-treated envenomed animals. SAV administration to envenomed alloxan pre-treated rabbits did not cause clinical or biochemical improvement. Severe scorpion envenoming causes an autonomic storm with a massive release of catecholamines and other counter-regulatory hormones; changes in insulin secretion resulting in fuel energy deficits producing multi-system-organ-failure (MSOF); and death. Administration of either insulin or SAV (through the release of insulin) appears to be the physiological basis for the control of the metabolic support to control the adverse effects triggered by counter-regulatory hormones.