129
Atherosclerosis, 84 (1990) 129-134
Elsevier Scientific Publishers Ireland, Ltd. ATHERO 04528
Effect of snake venom of Agkistrodon ha@ on atherosclerosis and blood characteristics in Japanese quail * Li-Guang Sun I, Wen-Xue Hao
1
and Jason C.H. Shih 2
’Department of Biochemistry China Medical Unioersity, Shenyang. Liaoning (People’s Republic of China), and ’ University Biotechnology Program and Department of Poultry Science, North Carolina State University, Raleigh, NC 276957608 (U.S.A.)
(Received 18 June, 1989) (Revised, received 2 May, 1990) (Accepted 12 May, 1990)
Extracts of snake venom have been widely used for the treatment of vascular thrombotic diseases, yet the therapeutic mechanism is not clear. The effect of snake venom fractions on atherosclerosis in Japanese quail was studied. The venom of Agkistrodon halys was fractionated by DEAE-cellulose chromatography and the pooled protein fractions that resulted were injected intravenously into the quail with aortic atherosclerosis induced by dietary cholesterol. After 7 weeks of injections on every other day, the quail were killed, blood clotting times and serum cholesterol levels were determined, and aortic atherosclerosis and fatty liver were scored. The results showed that while no regression of atherosclerosis was observed, the lowering of serum cholesterol, prolonged blood clotting time and reduced fatty liver were significantly affected by the injection of one of the pooled protein fractions. This venom fraction contained two major protein components, one of which had arginine esterase activity. From this study we conclude that snake venom has little effect on the regression of atherosclerosis, but it prolongs blood clotting and lowers serum cholesterol.
Key words: Snake venom; Atherosclerosis;
Blood characteristics;
Japanese quail; Agkistrodon halys
Introduction * Paper No. 12245 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695 7643, U.S.A. Correspondence to: Dr. Jason H.C. Shih, Department of Poultry Science, North Carolina State University, Raleigh, NC 276957608, U.S.A. Phone: (919) 737-2623. 0021-9150/90/$03.50
In China, more than 3000 cases have been reported in which extracts of snake venom are effective in the treatment of patients with vascular diseases [l]. The curative rate was about 90% and no serious toxic effect was found. Similar treat-
0 1990 Elsevier Scientific Publishers Ireland, Ltd.
130 ments against thrombotic disease by the anticoagulant fraction purified from the Malayan pit viper (Agkistrodon rhodostoma) have been reported in England [2,3]. The principle of the therapy is believed to be related to the fibrinolytic activity in snake venoms [2-51. On the other hand, the arginine esterase active fraction of Agkistrodon ha&s was found to be effective in the treatment of cerebral thrombosis and thromboangitis [6,7]. In a study with dogs, snake venom preparations decreased the viscosity, fibrinogen and lipid levels in the blood [8,9]. The lipid lowering effect is interesting and has also been observed in human patients treated with venom extracts [lo-121. The mechanism of anti-thrombotic activity of snake venom may be more complex than a simple process of proteolysis. The effect of snake venom extracts on atherosclerosis has never been investigated, although atherosclerosis is known to narrow the lumen of blood vessels which usually sets the stage for the development of thrombosis. The Japanese quail (Coturnix coturnix japonica) is a laboratory animal suitable for atherosclerosis research [1316]. Strains of quail which are genetically susceptible (SUS) and resistant (RES) to the disease have been developed and the pathology of quail atherosclerosis is similar to the human disease [15]. Recent studies have indicated that the genetic susceptibility may be linked to a latent infection of Marek’s disease herpes virus (MDV), an avian herpes virus [16,17]. Because of its small size, short life cycle and susceptibility to the disease, the genetically selected SUS quail is particularly useful for the study of anti-atherosclerotic treatment. In this communication we report the test results of the effect of snake venom fractions on aortic atherosclerosis in the Japanese quail. Materials and methods
Animals and atherosclerosis Atherosclerosis susceptible (SUS) strain Japanese quail (Coturnix coturnix japonica) were bred and raised in the Department of Poultry Science, NCSU. Aortic lesions were induced in adult male quail by feeding them an atherogenic diet containing 0.5% cholesterol as previously described [15]. After 9 weeks the quail developed severe aortic
lesions and were then switched to a diet containing 0.25% cholesterol. This level of dietary cholesterol allowed the birds to maintain a moderately high level of serum cholesterol (700-800 mg/dl) without further progression or regression of atherosclerosis. At this stage the quail were given injections of the snake venom preparation to test for the regression of aortic lesions. At the end of the test period (7 weeks) the quail were killed and blood clotting times measured, serum cholesterol determined, and aortic atherosclerosis and fatty liver were scored as described previously [15]. Aortic atherosclerosis was scored according to the percent of lesioned area: 0, normal; 1, a few lesions; 2, lesioned area < 50%; 3, lesioned area > 50%; and 4, plaque formation. Fatty liver was scored according to its fatty appearance from 0, normal to 4, whitish and enlarged. Fractionation of snake venom DEAE-cellulose column chromatography was employed to fractionate the venom [18]. Venom which was freeze-dried into a powder (5.5 g) was dissolved in 10 ml 5 mM sodium phosphate buffer (pH 7.5) containing 10 mM EDTA. After standing at room temperature for 30 min, the sample was dialysed at 5 “C for 12 h against 50 mM sodium phosphate buffer (pH 7.5) containing 0.1 mM EDTA. The dialysed sample was loaded on a column (2.5 X 60 cm) of 180 g DEAE-cellulose 52 (Whatman International, Maidstone, KY) preequilibrated with the same buffer solution. The column was eluted with discontinuous molar gradient solutions of NaCl (0, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.8, 1.0 M) in the same buffer. The flow rate was 2 ml/mm and 560 X lo-ml fractions were collected. Each fraction was assayed for protein (A,,,) and arginine esterase activity [18]. A total of 12 protein peaks were detected and these were pooled into 5 fractions corresponding to the following NaCl concentrations: A, O-0.04 M; B, 0.06-0.08 M; C, 0.1-0.2 M; D, 0.4-0.6 M; and E, 0.8-1.0 M. These fractions were dialyzed in deionized water, concentrated in the dialysis bag against polyethylene glycol flasks (Fisher Scientific, Fair Lawn, NJ) and then lyophilized to dry powder. In a separate experiment, only fraction D was collected. The same column loaded with 5.5 g
131 snake venom was pre-eluted with 0.2 M NaCl and then eluted with 0.4-0.6 M NaCl for the collection of fraction D. Injections The lyophilized fractions were individually dis-
solved in sterile phosphate-buffered saline (50 mM sodium phosphate, 0.75 M NaCl, pH 7.0) to produce a concentration of 62 mg/ml, except for fraction A which was 16 mg/ml. In the first injection experiments, 5 groups of 10 atherosclerotic quail (9 weeks on atherogenic diet) were injected with the venom fractions. A 6th group was injected with phosphate buffered saline and used as a control. Each bird was injected with 0.03 ml of the solution intravenously (15.5 mg/kg) every other day for a total of 7 weeks. For the second injection experiment, a separate set of birds was injected with fraction D only or the control (phosphate-buffered saline). Each group contained 24 birds. The birds were maintained on the same diet for the same period of time as the birds in the first experiment.
Sephactyl gel chromatography
Fraction D from DEAE-cellulose column was further fractionated by gel permeation. 150 ml Sephacryl HR200 (Pharmacia Piscataway, NJ) was diluted in 2 x vols 10 mM Tris-HCl buffer, pH 7.0, packed into a column (1.5 x 90 cm), and equilibrated with 2 column vols of the same buffer solution. 100 mg fraction D redissolved in a minimum volume of the buffer was loaded in the column. It was eluted with 10 mM Tris-HCl buffer, pH 7.5, containing 1.0 NaCl at a flow rate of 0.3 ml/mm and 100 x 1.7 ml fractions were collected and measured for AzgO. Gel electrophoresis
Snake venom and fraction D were analyzed by electrophoresis in the SDS-polacrylamide gradient gel (5-178) [21]. Electrophoresis was performed wit,h a Bio-Rad Protean II electrophoresis system at 30 mA for 4-5 h in 25 mM Tris, 19.2 mM glycine and 0.1% SDS. The gel was stained by 0.1% Coomassie blue overnight and then destained in 10% glacial acetic acid and 5% methanol. Results and discussion
Arginine esterase activity Arginine esterase activity was measured in each
fraction of snake venom by the method of Guan and Qie [ 181 using TAME ( p-tosyl-arginine methyl ester from Sigma Chemical Co., St. Louis, MO) as the substrate. TAME (0.05 ml of 10 mM) was added to 0.91 ml 46 mM Tris-HCl buffer, pH 8.1, in a l-ml quartz cuvette at room temperature in a Carl Zeiss P6M spectrophotometer. The increase of absorbance at 247 nm after the addition of 0.04 ml of the test sample solution was followed to determine the hydrolysis of TAME.
Fractionation of snake venom The chromatographic fractionation
of the snake venom by a DEAE cellulose column is shown in Fig. 1. A total of 560 tubes were collected and analyzed for protein (A,,,) and arginine esterase activity. There were 8 major and 4 minor peaks of protein and at least 5 peaks of esterase activity.
Blood parameters
Serum cholesterol was assayed according to the method of Zlatkis et al. [19]. Blood clotting time was determined by the method of Lee and White [20]. When the quail were sacrificed by decapitation, 1.0 ml blood was collected in a test tube (8.0 mm diameter) pre-rinsed with physiological saline (8.5% NaCl). The tube was tilted every 30 sec. The end point was reached when the tube could be inverted without displacing the clot.
TUBE NfJU8ER Fig. 1. DFAE-Cellulose 52 column chromatography of the venom of Agkistrodon hnlys.
132
number (n = 10) of quail tested. However, fraction D produced the lowest numerical score of aortic atherosclerosis (2.4 f 0.3) and serum cholesterol (691 + 89). Therefore, the experiment was repeated in more quail (n = 24) injected with fraction D (Table 2). Again, no regression of aortic atherosclerosis was observed (score 1.8 + 0.3 vs. 2.2 + 0.3 of the control). However, the clotting time was significantly increased (108 f 11 vs. 80 rt 3 set for the control) and interestingly the fatty liver score (1.2 & 0.3 vs. 2.1 f 0.3 of the control) and serum cholesterol (658 f 47 vs. 861 + 61 in the control) were significantly reduced by the treatment. The second injection experiment was conducted with more animals and the results are more uniform as indicated by smaller SEM values and less variability. The anti-coagulating effect of snake venom has been well established by many studies [2-51. This was also found to be true in quail. The anticoagulating mechanism is possibly due to the fibrinolytic activity as detected in fraction D. The cholesterol lowering effect in human patients has also been reported in many cases in China [lo-121 and we observed a similar response in quail. The cause of lipid reduction is not clear. It may involve lipid metabolism in the liver which indirectly reduces serum cholesterol and fatty liver caused by the high intake of cholesterol.
The protein yields for each pool were: A, 1.7 g; B, 1.2 g; C, 1.0 g; D, 0.76 g; and E, 0.56 g. The fractions indicated by letters A-E were pooled into 5 major fractions as previously mentioned. Further analyses of fraction D were done by using a Sephacryl HR200 column and SDS-PAGE. Both methods confirmed the presence of 2 major proteins having molecular weights of 63000 and 51000. Arginine esterase activity was detected in the lower molecular weight protein. Fig. 2 illustrates the results of the SDS gradient gel. Fraction D was found to be positive when tested for fibrinolytic activity by a quick test of Astrup and Millertz [22]. It produced a lysed zone on the surface of the clot formed by the reaction between fibrinogen and thrombin. Such an antithrombin or fibrinolytic activity may explain a possible mechanism of the effective treatment of thrombosis by snake venom [2-81. A prolonged clotting time of the quail blood was also observed in the fraction D-injected birds. Effects on atherosclerosis
The results of the tests for the regression of atherosclerosis by the intravenous injection of venom fractions are summarized in Tables 1 and 2. In the first injection experiment (Table 1) 5 venom fractions had no statistical effect on the treatment of atherosclerotic quail possibly due to the lower
TABLE 1 EFFECTS
OF VENOM FRACTIONS
ON QUAIL ATHEROSCLEROSIS
Protein fractions collected by DEAEceIlulose of quail. Fractions Atheroscl./total Atherosclerosis * Clotting time (set) Serum cholesterol (mg/dB Body wt. (9) before injection after injection Liver wt. (g) Fatty liver
A
B
7/10 3.3f 84* 786
cohunn chromatography. All values are presented as mean f SEM, except the number
0.6 6
*138
119 f 130 f 1.9* 3.1*
9/10 3.1* 0.4 92 +12 872
4 5 0.1 0.3
C
f126
117 f 126 f 2.1* 2.6*
lO/lO 3.4* 88 f 756
4 4 0.1 0.4
E
D 0.3 8
*156
123 f 128 f 2.0f 2.7&
9/10 2.4* 77 f 691
4 3 0.1 0.4
’ Score from 0 (normal) to 4 (severe). No statistical differences (P < 0.05) were found among different treatments.
0.3 8
f89
117 f 124 f 1.7* 2.0*
8/9 3.6* 76 f 718
3 3 0.1 0.3
Control 0.3 4
*75
126 f 127 f 1.9* 2.3&
8/8 2.3* 68 f 726
2 3 0.1 0.3
0.6 8
+19
128 f 132 f 1.9* 2.7k
3 2 0.1 0.6
133 Quail model In this study it becomes clear that the Japanese quail, especially the genetically susceptible strain [15], is a valuable animal model for this kind of study. It is possible to test a small quantity of chemicals such as snake venom and other drugs [23] and to test nutritional prevention with chemically defined diets [14]. Other studies have also shown that viral infections are associated with atherosclerosis in chickens [24] and quail [16,17]. The quail model warrants more attention in its utility for atherosclerosis research. In summary, the extracts of Agkistrodon halys venom did not regress aortic atherosclerosis in-
TABLE
2
EFFECT OF VENOM ATHEROSCLEROSIS
FROM
FRACTION
D ON QUAIL
Ah values are presented as mean + SEM, except the number of quail. Fraction D Atheroscl./totaI Atherosclerosis a Clotting time (set) Serum cholesterol
24/24 1.8* 0.3 108 +ll
(mg/dI) Body wt. (g) before injection after injection Liver wt. (g) Fatty liver a
658
+41
128 f 130 + 2.1 f 1.2*
1 2 0.1 0.3
P
Control 23/24 2.2* 0.3 80 + 3 861
+61
127 f 126 + 2.2* 2.1+
2 2 0.3 0.3
> 0.05 > 0.05 i 0.05 i 0.05 > > >
Atherosclerosis, 84 (1990) 129-134
Elsevier Scientific Publishers Ireland, Ltd. ATHERO 04528
Effect of snake venom of Agkistrodon ha@ on atherosclerosis and blood characteristics in Japanese quail * Li-Guang Sun I, Wen-Xue Hao
1
and Jason C.H. Shih 2
’Department of Biochemistry China Medical Unioersity, Shenyang. Liaoning (People’s Republic of China), and ’ University Biotechnology Program and Department of Poultry Science, North Carolina State University, Raleigh, NC 276957608 (U.S.A.)
(Received 18 June, 1989) (Revised, received 2 May, 1990) (Accepted 12 May, 1990)
Extracts of snake venom have been widely used for the treatment of vascular thrombotic diseases, yet the therapeutic mechanism is not clear. The effect of snake venom fractions on atherosclerosis in Japanese quail was studied. The venom of Agkistrodon halys was fractionated by DEAE-cellulose chromatography and the pooled protein fractions that resulted were injected intravenously into the quail with aortic atherosclerosis induced by dietary cholesterol. After 7 weeks of injections on every other day, the quail were killed, blood clotting times and serum cholesterol levels were determined, and aortic atherosclerosis and fatty liver were scored. The results showed that while no regression of atherosclerosis was observed, the lowering of serum cholesterol, prolonged blood clotting time and reduced fatty liver were significantly affected by the injection of one of the pooled protein fractions. This venom fraction contained two major protein components, one of which had arginine esterase activity. From this study we conclude that snake venom has little effect on the regression of atherosclerosis, but it prolongs blood clotting and lowers serum cholesterol.
Key words: Snake venom; Atherosclerosis;
Blood characteristics;
Japanese quail; Agkistrodon halys
Introduction * Paper No. 12245 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695 7643, U.S.A. Correspondence to: Dr. Jason H.C. Shih, Department of Poultry Science, North Carolina State University, Raleigh, NC 276957608, U.S.A. Phone: (919) 737-2623. 0021-9150/90/$03.50
In China, more than 3000 cases have been reported in which extracts of snake venom are effective in the treatment of patients with vascular diseases [l]. The curative rate was about 90% and no serious toxic effect was found. Similar treat-
0 1990 Elsevier Scientific Publishers Ireland, Ltd.
130 ments against thrombotic disease by the anticoagulant fraction purified from the Malayan pit viper (Agkistrodon rhodostoma) have been reported in England [2,3]. The principle of the therapy is believed to be related to the fibrinolytic activity in snake venoms [2-51. On the other hand, the arginine esterase active fraction of Agkistrodon ha&s was found to be effective in the treatment of cerebral thrombosis and thromboangitis [6,7]. In a study with dogs, snake venom preparations decreased the viscosity, fibrinogen and lipid levels in the blood [8,9]. The lipid lowering effect is interesting and has also been observed in human patients treated with venom extracts [lo-121. The mechanism of anti-thrombotic activity of snake venom may be more complex than a simple process of proteolysis. The effect of snake venom extracts on atherosclerosis has never been investigated, although atherosclerosis is known to narrow the lumen of blood vessels which usually sets the stage for the development of thrombosis. The Japanese quail (Coturnix coturnix japonica) is a laboratory animal suitable for atherosclerosis research [1316]. Strains of quail which are genetically susceptible (SUS) and resistant (RES) to the disease have been developed and the pathology of quail atherosclerosis is similar to the human disease [15]. Recent studies have indicated that the genetic susceptibility may be linked to a latent infection of Marek’s disease herpes virus (MDV), an avian herpes virus [16,17]. Because of its small size, short life cycle and susceptibility to the disease, the genetically selected SUS quail is particularly useful for the study of anti-atherosclerotic treatment. In this communication we report the test results of the effect of snake venom fractions on aortic atherosclerosis in the Japanese quail. Materials and methods
Animals and atherosclerosis Atherosclerosis susceptible (SUS) strain Japanese quail (Coturnix coturnix japonica) were bred and raised in the Department of Poultry Science, NCSU. Aortic lesions were induced in adult male quail by feeding them an atherogenic diet containing 0.5% cholesterol as previously described [15]. After 9 weeks the quail developed severe aortic
lesions and were then switched to a diet containing 0.25% cholesterol. This level of dietary cholesterol allowed the birds to maintain a moderately high level of serum cholesterol (700-800 mg/dl) without further progression or regression of atherosclerosis. At this stage the quail were given injections of the snake venom preparation to test for the regression of aortic lesions. At the end of the test period (7 weeks) the quail were killed and blood clotting times measured, serum cholesterol determined, and aortic atherosclerosis and fatty liver were scored as described previously [15]. Aortic atherosclerosis was scored according to the percent of lesioned area: 0, normal; 1, a few lesions; 2, lesioned area < 50%; 3, lesioned area > 50%; and 4, plaque formation. Fatty liver was scored according to its fatty appearance from 0, normal to 4, whitish and enlarged. Fractionation of snake venom DEAE-cellulose column chromatography was employed to fractionate the venom [18]. Venom which was freeze-dried into a powder (5.5 g) was dissolved in 10 ml 5 mM sodium phosphate buffer (pH 7.5) containing 10 mM EDTA. After standing at room temperature for 30 min, the sample was dialysed at 5 “C for 12 h against 50 mM sodium phosphate buffer (pH 7.5) containing 0.1 mM EDTA. The dialysed sample was loaded on a column (2.5 X 60 cm) of 180 g DEAE-cellulose 52 (Whatman International, Maidstone, KY) preequilibrated with the same buffer solution. The column was eluted with discontinuous molar gradient solutions of NaCl (0, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.8, 1.0 M) in the same buffer. The flow rate was 2 ml/mm and 560 X lo-ml fractions were collected. Each fraction was assayed for protein (A,,,) and arginine esterase activity [18]. A total of 12 protein peaks were detected and these were pooled into 5 fractions corresponding to the following NaCl concentrations: A, O-0.04 M; B, 0.06-0.08 M; C, 0.1-0.2 M; D, 0.4-0.6 M; and E, 0.8-1.0 M. These fractions were dialyzed in deionized water, concentrated in the dialysis bag against polyethylene glycol flasks (Fisher Scientific, Fair Lawn, NJ) and then lyophilized to dry powder. In a separate experiment, only fraction D was collected. The same column loaded with 5.5 g
131 snake venom was pre-eluted with 0.2 M NaCl and then eluted with 0.4-0.6 M NaCl for the collection of fraction D. Injections The lyophilized fractions were individually dis-
solved in sterile phosphate-buffered saline (50 mM sodium phosphate, 0.75 M NaCl, pH 7.0) to produce a concentration of 62 mg/ml, except for fraction A which was 16 mg/ml. In the first injection experiments, 5 groups of 10 atherosclerotic quail (9 weeks on atherogenic diet) were injected with the venom fractions. A 6th group was injected with phosphate buffered saline and used as a control. Each bird was injected with 0.03 ml of the solution intravenously (15.5 mg/kg) every other day for a total of 7 weeks. For the second injection experiment, a separate set of birds was injected with fraction D only or the control (phosphate-buffered saline). Each group contained 24 birds. The birds were maintained on the same diet for the same period of time as the birds in the first experiment.
Sephactyl gel chromatography
Fraction D from DEAE-cellulose column was further fractionated by gel permeation. 150 ml Sephacryl HR200 (Pharmacia Piscataway, NJ) was diluted in 2 x vols 10 mM Tris-HCl buffer, pH 7.0, packed into a column (1.5 x 90 cm), and equilibrated with 2 column vols of the same buffer solution. 100 mg fraction D redissolved in a minimum volume of the buffer was loaded in the column. It was eluted with 10 mM Tris-HCl buffer, pH 7.5, containing 1.0 NaCl at a flow rate of 0.3 ml/mm and 100 x 1.7 ml fractions were collected and measured for AzgO. Gel electrophoresis
Snake venom and fraction D were analyzed by electrophoresis in the SDS-polacrylamide gradient gel (5-178) [21]. Electrophoresis was performed wit,h a Bio-Rad Protean II electrophoresis system at 30 mA for 4-5 h in 25 mM Tris, 19.2 mM glycine and 0.1% SDS. The gel was stained by 0.1% Coomassie blue overnight and then destained in 10% glacial acetic acid and 5% methanol. Results and discussion
Arginine esterase activity Arginine esterase activity was measured in each
fraction of snake venom by the method of Guan and Qie [ 181 using TAME ( p-tosyl-arginine methyl ester from Sigma Chemical Co., St. Louis, MO) as the substrate. TAME (0.05 ml of 10 mM) was added to 0.91 ml 46 mM Tris-HCl buffer, pH 8.1, in a l-ml quartz cuvette at room temperature in a Carl Zeiss P6M spectrophotometer. The increase of absorbance at 247 nm after the addition of 0.04 ml of the test sample solution was followed to determine the hydrolysis of TAME.
Fractionation of snake venom The chromatographic fractionation
of the snake venom by a DEAE cellulose column is shown in Fig. 1. A total of 560 tubes were collected and analyzed for protein (A,,,) and arginine esterase activity. There were 8 major and 4 minor peaks of protein and at least 5 peaks of esterase activity.
Blood parameters
Serum cholesterol was assayed according to the method of Zlatkis et al. [19]. Blood clotting time was determined by the method of Lee and White [20]. When the quail were sacrificed by decapitation, 1.0 ml blood was collected in a test tube (8.0 mm diameter) pre-rinsed with physiological saline (8.5% NaCl). The tube was tilted every 30 sec. The end point was reached when the tube could be inverted without displacing the clot.
TUBE NfJU8ER Fig. 1. DFAE-Cellulose 52 column chromatography of the venom of Agkistrodon hnlys.
132
number (n = 10) of quail tested. However, fraction D produced the lowest numerical score of aortic atherosclerosis (2.4 f 0.3) and serum cholesterol (691 + 89). Therefore, the experiment was repeated in more quail (n = 24) injected with fraction D (Table 2). Again, no regression of aortic atherosclerosis was observed (score 1.8 + 0.3 vs. 2.2 + 0.3 of the control). However, the clotting time was significantly increased (108 f 11 vs. 80 rt 3 set for the control) and interestingly the fatty liver score (1.2 & 0.3 vs. 2.1 f 0.3 of the control) and serum cholesterol (658 f 47 vs. 861 + 61 in the control) were significantly reduced by the treatment. The second injection experiment was conducted with more animals and the results are more uniform as indicated by smaller SEM values and less variability. The anti-coagulating effect of snake venom has been well established by many studies [2-51. This was also found to be true in quail. The anticoagulating mechanism is possibly due to the fibrinolytic activity as detected in fraction D. The cholesterol lowering effect in human patients has also been reported in many cases in China [lo-121 and we observed a similar response in quail. The cause of lipid reduction is not clear. It may involve lipid metabolism in the liver which indirectly reduces serum cholesterol and fatty liver caused by the high intake of cholesterol.
The protein yields for each pool were: A, 1.7 g; B, 1.2 g; C, 1.0 g; D, 0.76 g; and E, 0.56 g. The fractions indicated by letters A-E were pooled into 5 major fractions as previously mentioned. Further analyses of fraction D were done by using a Sephacryl HR200 column and SDS-PAGE. Both methods confirmed the presence of 2 major proteins having molecular weights of 63000 and 51000. Arginine esterase activity was detected in the lower molecular weight protein. Fig. 2 illustrates the results of the SDS gradient gel. Fraction D was found to be positive when tested for fibrinolytic activity by a quick test of Astrup and Millertz [22]. It produced a lysed zone on the surface of the clot formed by the reaction between fibrinogen and thrombin. Such an antithrombin or fibrinolytic activity may explain a possible mechanism of the effective treatment of thrombosis by snake venom [2-81. A prolonged clotting time of the quail blood was also observed in the fraction D-injected birds. Effects on atherosclerosis
The results of the tests for the regression of atherosclerosis by the intravenous injection of venom fractions are summarized in Tables 1 and 2. In the first injection experiment (Table 1) 5 venom fractions had no statistical effect on the treatment of atherosclerotic quail possibly due to the lower
TABLE 1 EFFECTS
OF VENOM FRACTIONS
ON QUAIL ATHEROSCLEROSIS
Protein fractions collected by DEAEceIlulose of quail. Fractions Atheroscl./total Atherosclerosis * Clotting time (set) Serum cholesterol (mg/dB Body wt. (9) before injection after injection Liver wt. (g) Fatty liver
A
B
7/10 3.3f 84* 786
cohunn chromatography. All values are presented as mean f SEM, except the number
0.6 6
*138
119 f 130 f 1.9* 3.1*
9/10 3.1* 0.4 92 +12 872
4 5 0.1 0.3
C
f126
117 f 126 f 2.1* 2.6*
lO/lO 3.4* 88 f 756
4 4 0.1 0.4
E
D 0.3 8
*156
123 f 128 f 2.0f 2.7&
9/10 2.4* 77 f 691
4 3 0.1 0.4
’ Score from 0 (normal) to 4 (severe). No statistical differences (P < 0.05) were found among different treatments.
0.3 8
f89
117 f 124 f 1.7* 2.0*
8/9 3.6* 76 f 718
3 3 0.1 0.3
Control 0.3 4
*75
126 f 127 f 1.9* 2.3&
8/8 2.3* 68 f 726
2 3 0.1 0.3
0.6 8
+19
128 f 132 f 1.9* 2.7k
3 2 0.1 0.6
133 Quail model In this study it becomes clear that the Japanese quail, especially the genetically susceptible strain [15], is a valuable animal model for this kind of study. It is possible to test a small quantity of chemicals such as snake venom and other drugs [23] and to test nutritional prevention with chemically defined diets [14]. Other studies have also shown that viral infections are associated with atherosclerosis in chickens [24] and quail [16,17]. The quail model warrants more attention in its utility for atherosclerosis research. In summary, the extracts of Agkistrodon halys venom did not regress aortic atherosclerosis in-
TABLE
2
EFFECT OF VENOM ATHEROSCLEROSIS
FROM
FRACTION
D ON QUAIL
Ah values are presented as mean + SEM, except the number of quail. Fraction D Atheroscl./totaI Atherosclerosis a Clotting time (set) Serum cholesterol
24/24 1.8* 0.3 108 +ll
(mg/dI) Body wt. (g) before injection after injection Liver wt. (g) Fatty liver a
658
+41
128 f 130 + 2.1 f 1.2*
1 2 0.1 0.3
P
Control 23/24 2.2* 0.3 80 + 3 861
+61
127 f 126 + 2.2* 2.1+
2 2 0.3 0.3
> 0.05 > 0.05 i 0.05 i 0.05 > > >
