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Antithrombotic activity of an unsaturated fatty acid preparation
THROMBOSIS RESEARCH 58; 505510,199O 0049-3848190 $3.00 + .OOPrinted in the USA. Copyright (c) 1990 Pergamon Press pk. All rights reserved.
ANTITHROMBOTIC ACTIVITY OF AN UNSATURATED FATTY ACID PREPARATION J. Hladovei
and F. Kornalii
4th Department of Internal Medicin; and Institute of Pathophysiolog;,
Charles University
U nemocnice 2, Prague, 12808
CZECHOSLOVAKIA
(Received 53.1990; accepted in revised form 16.3.1990 by Editor J. Dyr)
ABSTRACT An unsaturated fatty acid preparation from fish oil, ” Epavit”, completely prevented arterial thrombosis induced in the rat aorta by a combined stenosis, extensive endothelial perturbation and i.v. serotonin, at an optimum dose of 0.1 ml / 200 g orally. A partial effect lasted more than 18 hours. After repeated administrations the acute completely inhibitory effect was decreased but the partial and chronic one remained unchanged. Aspirin (ASA) showed no synergic activity. Venous thrombosis ( one-sided ligature of the caval vein combined with extensive endothelial perturbation ) was inhibited to a similar degree as arterial thrombosis. Epavit also prevented the destabilizing effect of i.v. citrate on the endothelial lining.
INTRODUCTION The therapeutic and preventive effects of unsaturated fatty acids from fish oil have been in the centre of atiention especially for the last ten years. As the antiatheroscierotic effect was not equivocally confirmed increasing attention has been given to the supposed antithrombotic activity. Many preparations based on fish oil have been markeied in spite of insufficient documentation of their antkhrombotic effect. If many ex vivo studies of platelet functions, blood clotting and fibrinolysis are dismissed as problematic from the point of view of methodology and interpretation, it remains to mention similarly equivocal results of several small clinical studies and some attempts to use animal modeis (1). The authors have used a new and relatively advanced set of models to study the antithrombotic activity of Epavit, a combined fish oil and plant unsaturated fatty acid preparation (2). MATERIAL AND METHODS The fish oil preparation “Epavit” (Tukovy prfimysl, k.p., Severoceske tukove zavody, Ustf nad Labem, Czechoslovakia) contalned In 100 g 12 - 15 g of linoleic acid, 6 - 11 g of alpha - linolenic acid, ________________--_____--___-----------Key - words: Fish oil fatty acids, antithrombotlc
activity 505
506
ANTITHROMBOTIC ACTIVITY
Vol. 58, No. 5
3 - 7 g eicosapentaenoic acid, 2 - 2.5 g docosahexaenoic acid with 10 mg vitamin E added as an antioxidant. The oral administration of 0.025 - 1.O ml / 209 g was used in the study. Female Wistar rats, weighing 180 - 220 g were used, eight animals in a group. All animals were under deep urethane anesthesia (1900 mg / kg s.c.). Two thrombosis models in rats and that of endothelial lesion were used, all of them based on a unified pathogenetlc concept (3 , 4). a/ Endothelemia model (5). Estimation of the count of circulating carcasses of endothelial cells was used as an indicator of endothelial lesion. Blood was collected from right hearts using siliconized cdlecting material. In principle, the elements were isolated together with platelets and the latter were removed by the addition of adenosined diphosphate. After centriiugation the sediment was resuspended in a small vdume of procaine solution and the elements were counted in a Biirker’ s chamber under phase contrast. The number of elements was obtained as a mean of four counts and expressed as the count in 9 j.rI plasma. increased endothelemia (endotheiial perturbation) was induced by intravenous injection of citrate (1.52 %, 2 ml / 200 g). b/ Arterial thrombosis model (6, 7). Rectal temperature was measured at a room temperature of 22.0 - 22.5 degrees Celsius. After opening the abdominal cavity a modified minibulldog clip with a minimal distance of branches of 0.7 mm was placed on the aorta. Hypotonic saline ( 0.225 % NaCI, 2 ml / 200 g ) together with 20 ug / kg serotonin creatinine sulfate was injected intravenously five minutes after the clip insertion. Sixty minutes after provisional closure of abdomen rectal temperature was measured again. c/ Venous thrombosis model (8). One minute after intravenous administration of hypotonic saline (0.225 NaCl, 2 ml / 200 g) into the femoral vein, the abdominal cavity was opened and a tight ligature was placed on the vena cava. After provisional closure the cavity was reopened 10 min later. A vein segment was ligated also 2 cm below the first ligature and removed from the body. It was opened in a Petri dish, the thrombus was removed and weighed after 2 h-incubation in a wet chamber. Contrd values were obtained in a series of experiments with the clip alone in the arterial model and with sham operation and intravenous injection of physiological saline in the venous model. The oil was administered by gavage one hour before the start of experiment. Results were expressed as means with standard deviations and statistical siginficance was evaluated by the two-tailed t-test.
RESULTS In the arterial thrombosis model in rats the unsaturated fatty acid preparation from fish oil, roughly to the clinical dose level. A dose of 0.1 ml / 200 g administered orally 1 h in advance of thrombus induction had a maximum ( 100 % ) effect while a lower dose of 0.025 ml and a higher dose of 1.0 ml had a tendency to a decreasing effect ( Fig. 1).
Epavit, induced complete inhibition of thrombus formation at doses corresponding
The effect of the optimum dose lasted at least 18 hours at a somewhat lower and relatively stable level( Fig. 2). If the preparation was administered daily for 4 days and thrombus induction was carried out either on the fiih day without oil administration or one hour after the fifth administration , it was evident that the acute effect was decreased while some activity of the previous 4day administration was still observed (Fig. 3).
Vol. 58, No. 5
ANTITHROMBOTIC
ACTIVITY
507
27 OC
20
29 Contr. Cthrombosis)
Clip only
0.025 0.1 Epavit -ml/2OOg p.0.
1.0
FIG. 1 The effect of EpaVfi On arterial thrombosis in rats. Means with standard deviations. n = 8.
OC 27 .A * ,I 20 -
29
I
I
0
1
Epavit
I
3 0.1ml /2OOg
‘tJ=
J
18tl
p.0.
FIG. 2 Duration of the antithrombotic effect of Epavit on arterial thrombosis in rats. Asterisks: Statistically significant differences to the control.
508
Vol. 58, No. 5
28
29
Contr. Clip Single Doily Daily with only dose jdays _fdays, throlllbus @t@ddGthesth Epavit 0,025m1!3,009 FIG. 3 The effect of oral Epavit administration for 4 days with arterial thrombosis induced on the ffh day and with or without additional administration one hour in advance. Another question was the possibility of the interaction of oil and aspirin administration ( Fig. 4 ). If a still significantly effective dose of ASA was chosen and combined with the lower dose of Epavit ( 0.025 ml ) showing already some antithrombotic effect, then the combination was devoid of synergic activiiy and, on the contrary, some tendency to antagonism could be observed. In the venous thrombosls model the dose of 0.1 ml p.o. completely formation just as in the arteriil model ( Fig. 5 ).
inhibited thrombus
28
Epavit Epavit ASA Cad. C\i with my 0.025 + lmglkg thrornb. rnlbZCDg ASA
P
FIG. 4 The effect on arterial thrombosis of Epavit or asoirin alone or in combination.
Vol. 58, No. 5
ANTITHROMBOTIC
ACTIVITY
509
2, mg
Conk Contr. 0.1ml C0.2250/0Iphysiol.Epavit NaCIi.X) dinC) p.0. i.v.
FIG. 5. The effect of oral Epavit on venous thrombosis in rats. Asterisks: Statistically significant differences to the control. In order to contribute to the understanding of the Epavit effect the activii in the endotheiial model was investigated after induction of increased counts by intravenous citrate injection ( Fig. 6). While Epavit had no effect altogether on control values with physiological saline, it inhibited the increased endothdemia after citrate by about 75 % of the difference between controls I and II.
y.p*&()05
. . . . . .i : : : :
c
FIG. 6 The effect of oral Epavit on endothelemia increased by i.v. citrate ( 1.52 %, L ml / 200 g).
510
ANTITHROMBOTIC
Vol. 58, No. 5
ACTIVITY
DISCUSSION It may be concluded that Epavit is a highly effective antithrombotic agent in both thrombosis models at doses corresponding approximately to the clinical level. Two kinds of effect can ba distinguished. First, an acute one, evidently having a pharmacological character and resulting in a complete inhibition of thrombus formation. Repeated administration decreased this acute effect (tachyphylaxis?) while producing a second kind of effect of a lower degree but prolonged duration. This could possibly correspond to the delayed incorporation of exogenous fatty acids into platelet and endothelial membranes. The fact that the preparation was effective in both thrombosis models seems to indicate that neither platelets , whose role prevails in the arterial model, nor blood clotting and ffbdndysis playing a predominant part in the venous model, are the only factor in the antithrombotic activity of the preparation. While endothellal function is important in both thrombosis models, it was of particular Interest to study the effect of Epavlt on endothelial stability. The results confirmed the supposed presence of this activii. The question of the intimate mechanism of this stabilizing effect on the endothelium remains unanswered and experiments designed to detect the effect on the destabilizing activii of oxygen radicals are being developed.
REFERENCES 1. HORNSTRA, G. Influence of dietary fish oil on arterial thrombosis and atherosclerosis models and in man. J. intern. Me&. 225, Suppl.731, 53 - 59, 1989.
in animal
2: HRABAK, P., fiK. A., ZEMAN, M. and MARES, P. Dietery supplementation with natural polyunsaturated fatty acids: Hypolipidemic effects of a new czechoslovak dietetic “Epavit”. CVD Epidemioloov Newsletter. Council on EDidemiOlWV. Amer.Heart Assoc.,No 44,83 - 84,1988. 3. HlADOVEC, J. Antfthrombotic Druas in Thrombosis Models, CRC Press, Boca Raton, 1989. 4. HLADOVEC, J. Models of thrombosis. Minireview. Gen. Pharmacol., 19, 171 - 175, 1988. 5. HlADOVEC, J. and ROSSMANN, P. Circulating endothelial cells isolated together with platelets and the experimental modification of their counts in rats. Thromb. Res., 3,665 - 670, 1973. 6. HLADOVEC, J. A new model of arterial thrombosis. Thromb. Res., 41,659 - 664, 1986. 7. HLADOVEC, J. The effect of antithrombotics 41,665 -670, 1986.
in a new model of arterial thrombosis. Thromb. Res.,
8. HLADOVEC, J, A sensitive model of venous thrombosis in rats. Thromb. Res., 43,539 - 544,1986.
ANTITHROMBOTIC ACTIVITY OF AN UNSATURATED FATTY ACID PREPARATION J. Hladovei
and F. Kornalii
4th Department of Internal Medicin; and Institute of Pathophysiolog;,
Charles University
U nemocnice 2, Prague, 12808
CZECHOSLOVAKIA
(Received 53.1990; accepted in revised form 16.3.1990 by Editor J. Dyr)
ABSTRACT An unsaturated fatty acid preparation from fish oil, ” Epavit”, completely prevented arterial thrombosis induced in the rat aorta by a combined stenosis, extensive endothelial perturbation and i.v. serotonin, at an optimum dose of 0.1 ml / 200 g orally. A partial effect lasted more than 18 hours. After repeated administrations the acute completely inhibitory effect was decreased but the partial and chronic one remained unchanged. Aspirin (ASA) showed no synergic activity. Venous thrombosis ( one-sided ligature of the caval vein combined with extensive endothelial perturbation ) was inhibited to a similar degree as arterial thrombosis. Epavit also prevented the destabilizing effect of i.v. citrate on the endothelial lining.
INTRODUCTION The therapeutic and preventive effects of unsaturated fatty acids from fish oil have been in the centre of atiention especially for the last ten years. As the antiatheroscierotic effect was not equivocally confirmed increasing attention has been given to the supposed antithrombotic activity. Many preparations based on fish oil have been markeied in spite of insufficient documentation of their antkhrombotic effect. If many ex vivo studies of platelet functions, blood clotting and fibrinolysis are dismissed as problematic from the point of view of methodology and interpretation, it remains to mention similarly equivocal results of several small clinical studies and some attempts to use animal modeis (1). The authors have used a new and relatively advanced set of models to study the antithrombotic activity of Epavit, a combined fish oil and plant unsaturated fatty acid preparation (2). MATERIAL AND METHODS The fish oil preparation “Epavit” (Tukovy prfimysl, k.p., Severoceske tukove zavody, Ustf nad Labem, Czechoslovakia) contalned In 100 g 12 - 15 g of linoleic acid, 6 - 11 g of alpha - linolenic acid, ________________--_____--___-----------Key - words: Fish oil fatty acids, antithrombotlc
activity 505
506
ANTITHROMBOTIC ACTIVITY
Vol. 58, No. 5
3 - 7 g eicosapentaenoic acid, 2 - 2.5 g docosahexaenoic acid with 10 mg vitamin E added as an antioxidant. The oral administration of 0.025 - 1.O ml / 209 g was used in the study. Female Wistar rats, weighing 180 - 220 g were used, eight animals in a group. All animals were under deep urethane anesthesia (1900 mg / kg s.c.). Two thrombosis models in rats and that of endothelial lesion were used, all of them based on a unified pathogenetlc concept (3 , 4). a/ Endothelemia model (5). Estimation of the count of circulating carcasses of endothelial cells was used as an indicator of endothelial lesion. Blood was collected from right hearts using siliconized cdlecting material. In principle, the elements were isolated together with platelets and the latter were removed by the addition of adenosined diphosphate. After centriiugation the sediment was resuspended in a small vdume of procaine solution and the elements were counted in a Biirker’ s chamber under phase contrast. The number of elements was obtained as a mean of four counts and expressed as the count in 9 j.rI plasma. increased endothelemia (endotheiial perturbation) was induced by intravenous injection of citrate (1.52 %, 2 ml / 200 g). b/ Arterial thrombosis model (6, 7). Rectal temperature was measured at a room temperature of 22.0 - 22.5 degrees Celsius. After opening the abdominal cavity a modified minibulldog clip with a minimal distance of branches of 0.7 mm was placed on the aorta. Hypotonic saline ( 0.225 % NaCI, 2 ml / 200 g ) together with 20 ug / kg serotonin creatinine sulfate was injected intravenously five minutes after the clip insertion. Sixty minutes after provisional closure of abdomen rectal temperature was measured again. c/ Venous thrombosis model (8). One minute after intravenous administration of hypotonic saline (0.225 NaCl, 2 ml / 200 g) into the femoral vein, the abdominal cavity was opened and a tight ligature was placed on the vena cava. After provisional closure the cavity was reopened 10 min later. A vein segment was ligated also 2 cm below the first ligature and removed from the body. It was opened in a Petri dish, the thrombus was removed and weighed after 2 h-incubation in a wet chamber. Contrd values were obtained in a series of experiments with the clip alone in the arterial model and with sham operation and intravenous injection of physiological saline in the venous model. The oil was administered by gavage one hour before the start of experiment. Results were expressed as means with standard deviations and statistical siginficance was evaluated by the two-tailed t-test.
RESULTS In the arterial thrombosis model in rats the unsaturated fatty acid preparation from fish oil, roughly to the clinical dose level. A dose of 0.1 ml / 200 g administered orally 1 h in advance of thrombus induction had a maximum ( 100 % ) effect while a lower dose of 0.025 ml and a higher dose of 1.0 ml had a tendency to a decreasing effect ( Fig. 1).
Epavit, induced complete inhibition of thrombus formation at doses corresponding
The effect of the optimum dose lasted at least 18 hours at a somewhat lower and relatively stable level( Fig. 2). If the preparation was administered daily for 4 days and thrombus induction was carried out either on the fiih day without oil administration or one hour after the fifth administration , it was evident that the acute effect was decreased while some activity of the previous 4day administration was still observed (Fig. 3).
Vol. 58, No. 5
ANTITHROMBOTIC
ACTIVITY
507
27 OC
20
29 Contr. Cthrombosis)
Clip only
0.025 0.1 Epavit -ml/2OOg p.0.
1.0
FIG. 1 The effect of EpaVfi On arterial thrombosis in rats. Means with standard deviations. n = 8.
OC 27 .A * ,I 20 -
29
I
I
0
1
Epavit
I
3 0.1ml /2OOg
‘tJ=
J
18tl
p.0.
FIG. 2 Duration of the antithrombotic effect of Epavit on arterial thrombosis in rats. Asterisks: Statistically significant differences to the control.
508
Vol. 58, No. 5
28
29
Contr. Clip Single Doily Daily with only dose jdays _fdays, throlllbus @t@ddGthesth Epavit 0,025m1!3,009 FIG. 3 The effect of oral Epavit administration for 4 days with arterial thrombosis induced on the ffh day and with or without additional administration one hour in advance. Another question was the possibility of the interaction of oil and aspirin administration ( Fig. 4 ). If a still significantly effective dose of ASA was chosen and combined with the lower dose of Epavit ( 0.025 ml ) showing already some antithrombotic effect, then the combination was devoid of synergic activiiy and, on the contrary, some tendency to antagonism could be observed. In the venous thrombosls model the dose of 0.1 ml p.o. completely formation just as in the arteriil model ( Fig. 5 ).
inhibited thrombus
28
Epavit Epavit ASA Cad. C\i with my 0.025 + lmglkg thrornb. rnlbZCDg ASA
P
FIG. 4 The effect on arterial thrombosis of Epavit or asoirin alone or in combination.
Vol. 58, No. 5
ANTITHROMBOTIC
ACTIVITY
509
2, mg
Conk Contr. 0.1ml C0.2250/0Iphysiol.Epavit NaCIi.X) dinC) p.0. i.v.
FIG. 5. The effect of oral Epavit on venous thrombosis in rats. Asterisks: Statistically significant differences to the control. In order to contribute to the understanding of the Epavit effect the activii in the endotheiial model was investigated after induction of increased counts by intravenous citrate injection ( Fig. 6). While Epavit had no effect altogether on control values with physiological saline, it inhibited the increased endothdemia after citrate by about 75 % of the difference between controls I and II.
y.p*&()05
. . . . . .i : : : :
c
FIG. 6 The effect of oral Epavit on endothelemia increased by i.v. citrate ( 1.52 %, L ml / 200 g).
510
ANTITHROMBOTIC
Vol. 58, No. 5
ACTIVITY
DISCUSSION It may be concluded that Epavit is a highly effective antithrombotic agent in both thrombosis models at doses corresponding approximately to the clinical level. Two kinds of effect can ba distinguished. First, an acute one, evidently having a pharmacological character and resulting in a complete inhibition of thrombus formation. Repeated administration decreased this acute effect (tachyphylaxis?) while producing a second kind of effect of a lower degree but prolonged duration. This could possibly correspond to the delayed incorporation of exogenous fatty acids into platelet and endothelial membranes. The fact that the preparation was effective in both thrombosis models seems to indicate that neither platelets , whose role prevails in the arterial model, nor blood clotting and ffbdndysis playing a predominant part in the venous model, are the only factor in the antithrombotic activity of the preparation. While endothellal function is important in both thrombosis models, it was of particular Interest to study the effect of Epavlt on endothelial stability. The results confirmed the supposed presence of this activii. The question of the intimate mechanism of this stabilizing effect on the endothelium remains unanswered and experiments designed to detect the effect on the destabilizing activii of oxygen radicals are being developed.
REFERENCES 1. HORNSTRA, G. Influence of dietary fish oil on arterial thrombosis and atherosclerosis models and in man. J. intern. Me&. 225, Suppl.731, 53 - 59, 1989.
in animal
2: HRABAK, P., fiK. A., ZEMAN, M. and MARES, P. Dietery supplementation with natural polyunsaturated fatty acids: Hypolipidemic effects of a new czechoslovak dietetic “Epavit”. CVD Epidemioloov Newsletter. Council on EDidemiOlWV. Amer.Heart Assoc.,No 44,83 - 84,1988. 3. HlADOVEC, J. Antfthrombotic Druas in Thrombosis Models, CRC Press, Boca Raton, 1989. 4. HLADOVEC, J. Models of thrombosis. Minireview. Gen. Pharmacol., 19, 171 - 175, 1988. 5. HlADOVEC, J. and ROSSMANN, P. Circulating endothelial cells isolated together with platelets and the experimental modification of their counts in rats. Thromb. Res., 3,665 - 670, 1973. 6. HLADOVEC, J. A new model of arterial thrombosis. Thromb. Res., 41,659 - 664, 1986. 7. HLADOVEC, J. The effect of antithrombotics 41,665 -670, 1986.
in a new model of arterial thrombosis. Thromb. Res.,
8. HLADOVEC, J, A sensitive model of venous thrombosis in rats. Thromb. Res., 43,539 - 544,1986.