EFFECTS OF THREE FRACTIONS OBTAINED FROM Naja haje VENOM ON HEMOLYSIS AND LIPID METABOLISM IN RABBITS

Original paper

EFFECTS OF THREE FRACTIONS OBTAINED FROM Naja haje VENOM ON HEMOLYSIS AND LIPID METABOLISM IN RABBITS

A. A. EL-AAL

, A. R. EZZAT

1 Zoology Department, Faculty of Science, Ain Shams University, Cairo, Egypt.

ABSTRACT. Three fractions (F1, F2 and F3) were obtained from the venom of the Egyptian cobra Naja haje by gel filtration. F1 and F2 had indirect hemolytic effects on rabbit erythrocytes. The HU50 (the amount of the fraction causing 50% hemolysis ) values of F1 and F2 were 12.04 ± 3.89 and 36.57 ± 2.20 µg, respectively. The effects of the three cobra venom fractions on lipid metabolism were studied after 1, 2, 3 and 4 hours. Serum total lipids and cholesterol were significantly decreased at almost all tested times, but variable changes were observed in the serum triglycerides. Serum inorganic phosphorus levels were significantly increased with F1 and F2 administration more than that with the F3 fraction. These results suggest that F1 and F2 possess phospholipase A2-like activities and their effects on the lipid metabolism is more intense than that of F3.

KEY WORDS: Naja haje venom fractions, hemolysis, total lipids, cholesterol, triglycerides, inorganic phosphorus.

INTRODUCTION

A number of protein fractions isolated from snake venoms having phospholipase A2 activity are highly toxic per se or when complemented by other components in venoms (13,28). Three protein fractions of Naja haje venom were isolated using gel filtration by Abu Sinna et al. (2). It was reported that these fractions induced hyperglycemia concomitant with hyperinsulinemia (15). Al Nagdy et al.(3) studied the effects of these three cobra venom fractions on protein metabolism and on individual amino acid profile in rabbits. These authors concluded that these fractions possessed variable potencies in altering protein synthesis, inducing gluconeogensis from amino acids and enhancing protein catabolism. Various Naja venoms have been found to possess variable levels of phospholipase A2 activity(12,17). Condrea et al.(7,8) found that Naja naja atra phospholipase A2 hydrolyzed phospholipids in intact erythrocytes of guinea-pig. Naja haje venom was reported to alter lipid metabolism in rats(1,29). Phospholipase A2-containing fractions isolated from different cobra venoms were found to have little or no direct hemolytic effects (23,34) in spite of their ability to induce indirect hemolysis(4,31).

Consequently, the present work was initiated to study the hemolytic effects of three fractions isolated from Naja haje venom on rabbit erythrocytes. Furthermore, the effects of these fractions on the levels of serum total lipids, total cholesterol, triglycerides and inorganic phosphorus were studied in rabbits.

MATERIALS AND METHODS

VENOM: Naja haje venom was collected from snakes housed in the Venom Research Laboratory Serpentarium of the Department of Zoology, Faculty of Science, at Ain Shams University, Cairo, Egypt. For this reason, healthy adult snakes were collected from Giza and Fayum areas in Egypt. The venom was obtained by a technique in which the snake was allowed to bite through a nylon diaphragm on the edge of a chilled container into which the venom was ejected. The venom was then lyophilized and stored at 0°C in a desiccator.

VENOM FRACTIONATION: Crude venom was fractionated by gel filtration on a G-75 Sephadex (Sigma, St. Louis, USA.) column according to the method used by Abu Sinna et al.(2), and the effluents of each fraction were collected at 4°C. Protein concentration of each fraction was then measured by the method of Lowry et al.(27) using bovine serum albumin (Sigma, St. Louis, USA) as standard.

HEMOLYTIC ACTIVITY: Direct and indirect hemolytic activities of the venom fractions were assayed as described by Vishwanath et al.(38) using freshly-washed rabbit red blood cells (RBC). The HU50 values (the amount of each fraction causing 50% hemolysis) were calculated according to the method of Vaughan et al.(37). Three determinations were carried out in duplicate in amounts of venom fractions ranging from 10 µg to 120 µg.

EFFECTS OF COBRA VENOM FRACTIONS ON LIPID METABOLISM: Four experimental groups made up of eight male rabbits each (1-1.5 kg) were used. Each animal of the first group (control) received 0.2 ml of saline vehicle. The other three animal groups were i.v. injected with a single sub-LD50 dose of either one of the cobra venom fractions. The doses were 96, 65 and 35µg/kg of body weight for F1, F2 and F3, respectively. Blood samples were collected by heart puncture after 1, 2, 3 and 4 hours and were allowed to coagulate. Sera were separated by centrifugation and stored deep frozen pending analysis. Total lipids in the serum were estimated using the method of Zollner and Kirsch(39). Total cholesterol was determined according to the procedure of Kattermann(24). Estimation of serum inorganic phosphorus was carried out according to the method described by Wootton(36). Determination of serum triglycerides was performed by the method of Wahlefeld(35).

STATISTICAL ANALYSIS: Statistical significance of the results was assessed by the Student's t-test. Values of p < 0.05 were considered to be statistically significant(33).

RESULTS

Figure 1 shows the fractionation profile and the protein content of 100 mg of lyophilized Naja haje venom. Lyophilized Naja haje venom (100 mg) was fractionated on a G-75 Sephadex column and three fractions (F1, F2 and F3 ) were collected. No direct hemolytic activity was detected for any of the three cobra venom fractions (Figure 2). Figure 3 shows the indirect hemolysis of rabbit erythrocytes induced by different concentrations of cobra venom fractions. The indirect hemolytic activity was in the following sequence: F1 > F2 >F3. The maximum indirect hemolysis corresponding to 100% for F1 and 94% for F2 was found to occur using 120 µg of each one of these fractions. On the other hand, the maximum hemolysis of F3 was only 13% and was detected using 40µg . In the presence of the egg yolk phospholipids, the estimated HU50 values for F1 and F2 (mean ± S.D.) were 12.04 ± 3.89 and 36.57 ± 2.20µg, respectively, whereas F3 was ineffective.

FIGURE 1.
G-75 Sephadex gel filtration of Naja haje venom. 100 mg of venom was fractionated on a 1.8 x 60 cm column using 0.3 M phosphate buffer at a flow rate of 20 ml h

FIGURE 2.
Effect of three fractions from Naja haje venom on direct hemolysis of rabbit erythrocytes.

FIGURE 3.
Effect of three fractions from Naja haje venom on indirect hemolysis of rabbit erythrocytes.

All fractions decreased serum total lipids significantly at all tested times (Table 1). Serum total cholesterol was significantly decreased by F1 after 1, 2, 3 and 4 hours (Table 2). This decrease was only evident with F2 after 1 and 2 hours. F3 was not effective in altering this parameter after 1, 2 and 3 hours, whereas a significant increase in total cholesterol was found with this fraction after 4 hours of treatment. Table 3 shows the effects of cobra venom fractions on serum triglycerides. F1 significantly decreased the levels of serum triglycerides after 1 and 2 hours, while F2 significantly increased the levels of serum triglycerides after 3 and 4 hours. F3 induced an initial significant increase after 1 hour followed by a significant decrease after 2, 3 and 4 hours. Table 4 shows the effect of cobra venom fractions on serum inorganic phosphorus. Serum inorganic phosphorus was significantly increased by F1 after 2, 3 and 4 hours, but this increase was evident with F2 at all tested times. F3 was found to increase serum total phosphorus after 1 and 2 h. A significant decrease of serum total inorganic phosphorus was unexpectedly found with F3 after 4 hours of treatment.

TABLE 1.
The effects of three Naja haje venom fractions on serum total lipids (mg/100ml) in rabbits.

TABLE 2.
The effects of three Naja haje venom fractions on serum total cholesterol (mg/100ml) in rabbits.

TABLE 3.
The effects of three Naja haje venom fractions on serum triglycerides (mg/100 ml) in rabbits.

TABLE 4.
The effects of three Naja haje venom fractions on serum total phosphorus (mg/100ml) in rabbits.

DISCUSSION

In the present study, the three venom fractions obtained from the Egyptian cobra Naja haje are similar to those previously reported by Kochva et al.(26) and Abu Sinna et al.(2). Two fractions of cobra venom, F1 and F2, were found to possess hemolytic effects. The indirect hemolytic activity of these fractions is likely to be the result of their contents of phospholipase A2 or the enzyme-like activity. Phospholipase A2 was found in cobra venom by Kamel(22) and Kochva et al.(26). In addition, Fletcher et al.(16,17) reported the presence of phospholipase A2-like activity in snake venoms. The third fraction was devoid of any hemolytic activity which coincides with the findings of Kamel(22) and Kochva et al.(26), who found that this fraction was mainly neurotoxic and cardiotoxic in nature.

On the contrary, all of the three fractions of Naja haje venom were devoid of a direct hemolytic effect. This is in agreement with the work of Bhat and Gowda(4) where no direct hemolytic effect was found for Naja naja naja venom fractions. In general, snake venoms can hemolyze red blood cells only when a source of phosphatidylcholine is added to the preparation of RBCs(6,11). This may account for the indirect hemolytic activity observed in the present work. Moreover, the lack of the hemolytic activity of snake venom phospholipases in the absence of exogenous phospholipids was also reported by Bhat and Gowda(4), Kasturi and Gowda(23) and Mohamed and Kamel(31). The intense decrease in serum total lipids observed herein with the three venom fractions at all tested times probably results from venom-induced lipolysis. This lipolytic action could have occurred either as a direct effect of the venom on the hepatic and peripheral tissue lipids or by the activation of the adrenal secretions. Adrenal medullary catecholamines as well as the adrenocorticosteroids are known as potent lipolytic hormones(10). This conclusion is supported by the results of Mohamed et al.(32) who reported a stimulatory effect of Naja haje venom on rat adrenals. Circulating levels of cortisol were reported to increase by Naja haje venom in rabbits(14). Furthermore, a direct venom lipolytic effect was also reported by Mohamed et al.(29) where Naja haje venom caused the release of free fatty acids from tissues. Mohamed et al.(30) believed that Naja nigricollis venom-induced release of fatty acids from tissues was due to the enzymatic activity of phospholipase A2 in the venom.

Our results indicated that F1 and F2 tended to lower serum total cholesterol, which agrees with a previous report(14) about the same snake venom. Ezzat and Abd El-Aal(14) found that a sublethal dose of Naja haje venom decreased serum total cholesterol in rabbits. These authors explained their findings by the activation of the pituitary-adrenal axis resulting in increased circulatory levels of ACTH and cortisol. ACTH induces specific membrane receptors in the cells of the adrenal cortex to increase serum cholesterol uptake(19,20). The increased level of T4 from the thyroid gland following administration of Naja haje venom fractions(15) may also explain the reason for lowering serum cholesterol level. An inverse correlation between serum thyroid hormones and cholesterol levels has been reported by several authors(10,18).

In the present study, F1 and F3 resulted in a decrease in serum triglyceride levels, whereas F2 had an opposite effect. These discrepant effects might be related to the relative action of each fraction on the hepatic synthesis of triglycerides or to the activity of lipoprotein lipase which regulates tissue uptake of triglycerides(10)

Serum total phosphorus was variably increased by the three venom fractions with F2, being by far the most effective. This effect is likely to be a result of the venom phospholipase A2 activity. The highest activity of the venom phospholipase A2 was found in the present work associated with F2, which may explain the drastic increase in total phosphorus in response to treatment with that fraction. Phospholipids hydrolysis by venoms phospholipase A2 was previously reported(5,7,8,9,21,34,38). Moreover, Klibansky et al.(25) found that injection of phospholipase A2 of Vipera palestinae venom into rabbits caused an increase in plasma phosphorus level.

REFERENCES

01 ABD EL-AAL A.Effect of Egyptian cobra Naja haje venom on some physiological aspects. 1985. in Shams University, Cairo, Egypt. [Thesis. Ph.D]

02 ABU SINNA G., AL-ZAHABY A., ABD EL-AAL A., ABD EL-BASET A., SABER T. Short term effects of animal venoms on the mitotic index of the duodenal mucosa of albino rats. Nat. Toxins., 1992, 1, 111-8.

03 AL NAGDY SA., EZZAT AR., SALEH F., ABD EL-AAL A. Serum proteins and amino acid profile in rabbits treated with three fractions of Naja haje venom. J. Egypt. Soc. Toxicol., 1993, 11, 21-30.

04 BHAT MK., GOWDA TV. Purification and characterization of a myotoxic phospholipase A2 from Indian cobra (Naja naja naja) venom. Toxicon, 1989, 27, 861-73.

05 CHEN SZJ., GOPALAKRISHNAKONE P., GWEE MCE. Ultrastructural changes in skeletal muscle, heart muscle and kidney induced by phospholipase A2 isolated from venom of the King brown snake (Pseudechis australis). Toxicon, 1995, 33, 301 [Abstract].

06 CONDREA E. Hemolytic effects of snake venoms. In: Lee CY. Ed. Snake venoms. Berlim : Springer, 1979: 448-79.

07 CONDREA E., FLETCHER JE., RAPUANO BE., YANG CC., ROSENBERG P. Effect of modification of one histidine residue on the enzymatic and pharmacological properties of a toxic phospholipase A2 from Naja nigricollis snake venom and less toxic phospholipase A2 from Hemachatus haemachatus and Naja naja atra snake venoms. Toxicon, 1981, 19, 61-71.

08 CONDREA E., FLETCHER JE., RAPUANO BE., YANG CC., ROSENBERG P. Dissociation of enzymatic activity from lethality and pharmacological properties by cartbamylation of lysines in Naja nigricollis and Naja naja atra snake venom phospholipase A2. Toxicon, 1981, 19, 705.

09 CONDREA E., YANG CC., ROSENBERG P. A comparison of a relatively toxic phospholipase A2 from Naja nigricollis snake venom with that of a relatively toxic phospholipase A2 from H. Haemachatus snake venom. I. Enzymatic activity on free and membrane bound substrates. Biochem. Pharmacol., 1980, 29, 1555-63.

10 DEVLIN TM.Biochemisitry with clinical correlations . New York: John Wiley, 1993.

11 DE VRIES A., KIRSCHMANN C., KLIBANSKY C., CONDREA E., GITTER S. Hemolytic action of indirect lytic snake venom in vivo. Toxicon, 1962, 1, 19-23.

12 DURKIN JP., PICKWELL GV., TROTTER JT., SHIER WT. Phospholipase A2 electrophoretic variants in reptile venoms. Toxicon, 1981, 19, 535-46.

13 EAKER D. Studies on presynaptically neurotoxic and myotoxic phospholipase A2. In: LT CH Ed. Versatility of proteins. New York : Academic Press, 1978: 413-31

14 EZZAT AR., ABD EL-AAL A. Effect of cobra Naja haje venom on the adrenal activity in rabbits. Qatar Univ. Bull., 1989, 9, 169-76.

15 EZZAT AR. Changes in rabbit serum glncose, insulin, T4 and T3 in response to three ionopheretic fractions of Naja haje venom. J. Egypt. Soc. Toxicol., 1993, 11, 31-7.

16 FLETCHER JE., JIANG MS., SETLISTRE DE ARAUJO HS., OWNBY CL. Effects of three myotoxins on membrane phospholipid hydrolysis in cell culture systems. Toxicon, 1995, 33, 301. [Abstract].

17 FLETCHER JE., RAPUANO BE., CONDREA E., YANG CC., ROSENBERG P. Relationship between catalysis and toxicological properties of three phospholipases from elapid snake venoms. Toxic. Appl. Pharmacol., 1981, 59, 375-85.

18 GANONG WF.Review of medical physiology. 15ed. Norwalk, Com: Appleton & Lange, 1991: 296-311.

19 GWYNNE JT., HESS B. Binding and degradation of human HDL by rat adrenocortical cells.Metabolism., 1978, 27, 1593-600.

20 GWYNNE JT., HUGHES T., ROUNDTREE R., HESS B. ACTH regulation of the HDL receptors of the rat adrenal cortex . In: INGBAR SH. Ed. Contemporary endocrinology. New York: Plenum Medical Book, 1979: 196-203.

21 IBRAHIM SA., THOMPSON RHS. Action of phospholipase A2 on human red cell ghosts and intact erythrocytes. Biochim. Biophys. Acta, 1965, 99, 331-41.

22 KAMEL A. Fractionation of Egyptian cobra venom. Toxicon, 1974, 12, 495-99.

23 KASTURI S., GOWDA TV. Purification and characterization of a major phospholipase A2 from Russell's viper (Vipera russelli ) venom. Toxicon, 1989, 27, 229-37.

24 KATTERMANN R. Boehringer Mannheim instruction Manual. Boehringer Mannheim Gmbh Diagnostica , Germany. J. Clin. Chem. Clin. Biochem., 1984, 22, 245.

25 KLIBANSKY C., CONDREA E., DE VRIES A. Changes in plasma phospholipids after intravenous phospholipase A2 injection in the rabbit. Am. J. Physiol., 1962, 203, 114-8.

26 KOCHVA E.,TONSING L., LOUW AL., LIEBENBERG NVDW., VISSER L. Biosynthesis, secretion and in vivo isotopic labelling of venom of the Egyptian Cobra, Naja haje annulifera. Toxicon, 1982, 20, 615-36.

27 LOWRY OH., ROSENBROUGH NJ., FORR AL., RANDALL RJ. Protein measurement with Folin phenol reagent. J. Biol. Chem., 1951, 93, 265-75.

28 MEBS D. Myotoxic activity of phospholipase A2 isolated from cobra venoms: Neutralization by polyvalent antivenoms. Toxicon, 1986, 24, 1001-8.

29 MOHAMED AH., HANI-AYOBE M., BESKAROUN MA., EL-DAMARAWY NA. Effects of cobra venom (Naja haje) on rat liver metabolism in vitro. Ain Shams Med. J., 1975, 26, 503-6.

30 MOHAMED AH., HANI-AYOBE M., MOHAMED FA. Diabetogenic actions of nigricollis venom. II. Effect of Naja nigricollis venom on lipolysis. Ain Shams Med. J., 1974, 25, 201-3.

31 MOHAMED AH., KAMEL A. Direct hemolysis induced by some Egyptian snake venoms and other allied venoms. Indian J. Med. Res., 1972, 60, 1759-63.

32 MOHAMED AH., SALEH AM., AHMED S., BESHIR SR. Histopathological effects of Naja haje snake venom and a venom gland extract of the scorpion Buthus quinquestriatus on the liver, suprarenal gland and pancreas of mice. Toxicon, 1978, 16, 253-61.

33 PARKER RE. Introductory statistics for biology. London: Esward Arnoid, 1973.

34 ROSENBERG P. Phospholipases. In: SHIER WT., MEBS D. Eds. Handbook of toxinology, New York: Marcel Dekker, 1990: 68-277.

35 WAHLEFELD AW.Triglycerides determination after enzymatic hydrolysis. New York: Academic Press, 1974: 18-31.

36 WOOTTON IDP.Microanalysis in medical biochemistry. 5ed. London: Churchill Livingstone, 1974: 70.

37 VAUGHAN GT., SCULLEY TB.,TIRRELL R. Isolation of a hemolytic, toxic phospholipase from the venom of the Australian red-bellied black snake (Pseudechis porphyriacus). Toxicon, 1981, 19, 95-101.

38 VISHWANATH BS., KINI RM., GOWDA TV. Characterization of three edema-inducing phospholipase A2 enzymes from habu (Trimeresurus flavoviridis) venom and their interaction with the alkaloid aristolochic acid. Toxicon, 1987, 25, 501-15.

39 ZOLLNER N., KIRSCH K. Bochringer Mannheim instruction manual. Bochringer annheim Gmtt Diagnostica Germany. Z. Gesamte. Exp. Med., 1962, 135, 545-60.

Received 22 November 1996

Accepted 01 April 1997

• 02 ABU SINNA G., AL-ZAHABY A., ABD EL-AAL A., ABD EL-BASET A., SABER T. Short term effects of animal venoms on the mitotic index of the duodenal mucosa of albino rats. Nat. Toxins., 1992, 1, 111-8.
• 03 AL NAGDY SA., EZZAT AR., SALEH F., ABD EL-AAL A. Serum proteins and amino acid profile in rabbits treated with three fractions of Naja haje venom. J. Egypt. Soc. Toxicol., 1993, 11, 21-30.
• 04 BHAT MK., GOWDA TV. Purification and characterization of a myotoxic phospholipase A2 from Indian cobra (Naja naja naja) venom. Toxicon, 1989, 27, 861-73.
• 06 CONDREA E. Hemolytic effects of snake venoms. In: Lee CY. Ed. Snake venoms. Berlim : Springer, 1979: 448-79.
• 07 CONDREA E., FLETCHER JE., RAPUANO BE., YANG CC., ROSENBERG P. Effect of modification of one histidine residue on the enzymatic and pharmacological properties of a toxic phospholipase A2 from Naja nigricollis snake venom and less toxic phospholipase A2 from Hemachatus haemachatus and Naja naja atra snake venoms. Toxicon, 1981, 19, 61-71.
• 08 CONDREA E., FLETCHER JE., RAPUANO BE., YANG CC., ROSENBERG P. Dissociation of enzymatic activity from lethality and pharmacological properties by cartbamylation of lysines in Naja nigricollis and Naja naja atra snake venom phospholipase A2. Toxicon, 1981, 19, 705.
• 09 CONDREA E., YANG CC., ROSENBERG P. A comparison of a relatively toxic phospholipase A2 from Naja nigricollis snake venom with that of a relatively toxic phospholipase A2 from H. Haemachatus snake venom. I. Enzymatic activity on free and membrane bound substrates. Biochem. Pharmacol., 1980, 29, 1555-63.
• ¹⁰
  10 DEVLIN TM.Biochemisitry with clinical correlations . New York: John Wiley, 1993.
• 11 DE VRIES A., KIRSCHMANN C., KLIBANSKY C., CONDREA E., GITTER S. Hemolytic action of indirect lytic snake venom in vivo Toxicon, 1962, 1, 19-23.
• 12 DURKIN JP., PICKWELL GV., TROTTER JT., SHIER WT. Phospholipase A2 electrophoretic variants in reptile venoms. Toxicon, 1981, 19, 535-46.
• 13 EAKER D. Studies on presynaptically neurotoxic and myotoxic phospholipase A2. In: LT CH Ed. Versatility of proteins. New York : Academic Press, 1978: 413-31
• 14 EZZAT AR., ABD EL-AAL A. Effect of cobra Naja haje venom on the adrenal activity in rabbits. Qatar Univ. Bull., 1989, 9, 169-76.
• 15 EZZAT AR. Changes in rabbit serum glncose, insulin, T4 and T3 in response to three ionopheretic fractions of Naja haje venom. J. Egypt. Soc. Toxicol., 1993, 11, 31-7.
• 17 FLETCHER JE., RAPUANO BE., CONDREA E., YANG CC., ROSENBERG P. Relationship between catalysis and toxicological properties of three phospholipases from elapid snake venoms. Toxic. Appl. Pharmacol., 1981, 59, 375-85.
• 19 GWYNNE JT., HESS B. Binding and degradation of human HDL by rat adrenocortical cells.Metabolism., 1978, 27, 1593-600.
• 20 GWYNNE JT., HUGHES T., ROUNDTREE R., HESS B. ACTH regulation of the HDL receptors of the rat adrenal cortex . In: INGBAR SH. Ed. Contemporary endocrinology. New York: Plenum Medical Book, 1979: 196-203.
• 21 IBRAHIM SA., THOMPSON RHS. Action of phospholipase A2 on human red cell ghosts and intact erythrocytes. Biochim. Biophys. Acta, 1965, 99, 331-41.
• 22 KAMEL A. Fractionation of Egyptian cobra venom. Toxicon, 1974, 12, 495-99.
• 23 KASTURI S., GOWDA TV. Purification and characterization of a major phospholipase A2 from Russell's viper (Vipera russelli ) venom. Toxicon, 1989, 27, 229-37.
• 24 KATTERMANN R. Boehringer Mannheim instruction Manual. Boehringer Mannheim Gmbh Diagnostica , Germany. J. Clin. Chem. Clin. Biochem., 1984, 22, 245.
• 25 KLIBANSKY C., CONDREA E., DE VRIES A. Changes in plasma phospholipids after intravenous phospholipase A2 injection in the rabbit. Am. J. Physiol., 1962, 203, 114-8.
• 26 KOCHVA E.,TONSING L., LOUW AL., LIEBENBERG NVDW., VISSER L. Biosynthesis, secretion and in vivo isotopic labelling of venom of the Egyptian Cobra, Naja haje annulifera. Toxicon, 1982, 20, 615-36.
• 27 LOWRY OH., ROSENBROUGH NJ., FORR AL., RANDALL RJ. Protein measurement with Folin phenol reagent. J. Biol. Chem., 1951, 93, 265-75.
• 28 MEBS D. Myotoxic activity of phospholipase A2 isolated from cobra venoms: Neutralization by polyvalent antivenoms. Toxicon, 1986, 24, 1001-8.
• 29 MOHAMED AH., HANI-AYOBE M., BESKAROUN MA., EL-DAMARAWY NA. Effects of cobra venom (Naja haje) on rat liver metabolism in vitro. Ain Shams Med. J., 1975, 26, 503-6.
• 30 MOHAMED AH., HANI-AYOBE M., MOHAMED FA. Diabetogenic actions of nigricollis venom. II. Effect of Naja nigricollis venom on lipolysis. Ain Shams Med. J., 1974, 25, 201-3.
• 31 MOHAMED AH., KAMEL A. Direct hemolysis induced by some Egyptian snake venoms and other allied venoms. Indian J. Med. Res., 1972, 60, 1759-63.
• 32 MOHAMED AH., SALEH AM., AHMED S., BESHIR SR. Histopathological effects of Naja haje snake venom and a venom gland extract of the scorpion Buthus quinquestriatus on the liver, suprarenal gland and pancreas of mice. Toxicon, 1978, 16, 253-61.
• ³³
  33 PARKER RE. Introductory statistics for biology London: Esward Arnoid, 1973.
• ³⁴
  ROSENBERG P. Phospholipases. In: SHIER WT., MEBS D. Eds. Handbook of toxinology, New York: Marcel Dekker, 1990: 68-277.
• 35 WAHLEFELD AW.Triglycerides determination after enzymatic hydrolysis. New York: Academic Press, 1974: 18-31.
• 36 WOOTTON IDP.Microanalysis in medical biochemistry. 5ed. London: Churchill Livingstone, 1974: 70.
• 37 VAUGHAN GT., SCULLEY TB.,TIRRELL R. Isolation of a hemolytic, toxic phospholipase from the venom of the Australian red-bellied black snake (Pseudechis porphyriacus). Toxicon, 1981, 19, 95-101.
• 38 VISHWANATH BS., KINI RM., GOWDA TV. Characterization of three edema-inducing phospholipase A2 enzymes from habu (Trimeresurus flavoviridis) venom and their interaction with the alkaloid aristolochic acid. Toxicon, 1987, 25, 501-15.
• 39 ZOLLNER N., KIRSCH K. Bochringer Mannheim instruction manual. Bochringer annheim Gmtt Diagnostica Germany. Z. Gesamte. Exp. Med., 1962, 135, 545-60.

CORRESPONDENCE TO:

A. ABD EL-AAL - Zoology Department, Faculty of Science, Ain Shams University, Abbassia, Cairo, Egypt.

Publication Dates

• Publication in this collection
  08 Jan 1999
• Date of issue
  1997

History

• Received
  22 Nov 1996
• Accepted
  01 Apr 1997