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Dietary fats and arterial thrombosis
THROMBOSIS Ptd. in USA
RESEARCH
Supplement Number 1 LIPIDS AND THROMBOSIS, noms+
Vol. 4, 1974 Pergamon Preaa
DIETARY FATS AND ARTERIAL THROMBOSIS
S. Renaud Unit6 de Recherche de Physio-Pathologie Vasculaire (INSERM, Unit6 63), 22, Avenue du Doyen-Lgpine, 69500 - Lyon-Bron, France.
ABSTRACT. Epidemiologic studies have shown that coronary heart disease (CHD), particularly coronary deaths where presumably arterial thrombosis plays a prominent role, was closely associated with the saturated fat intake. Conclusions from experiments, with totally unrelated techniques, appear to be unanimous in that fats, with long chain saturated fatty acids, particularly stearic acid, affect platelet behavior and clotting in vitro and in vivo, and predispose animals to thrombosis by long term feeding. Linoleic and linolenic acids might have anti-thrombotic effects. One mechanism by which the long-chain saturated fatty acids predispose to thrombosis could be by changing the fatty acid pattern of platelet phospholipids, a result demonstrated both in man and animals. Since the formation of platelet aggregates seems to be the earliest event in thrombosis, the platelet phospholipids, because of their structural role in platelet membranes, might play a prominent role in the observed increased susceptibility of platelets to aggregation. In addition, it has been shown that modifications in the phospholipids fatty acid composition of platelets have an effect on their clotting activity, i.e. the lipid moiety of platelet factor 3 (PF3). An increase in the activity of PF3 which has been shown to cause in vitro hypercoagulability, was found in CHD as well as in animals fed the long chain fatty acids. Finally, prevention studies have indicated that by changing the type of fat in the diet, the death-rate from CHD can be consistently decreased. The results mentioned above are concordant with the hypo+.hasis that some dietary saturated fats, in practice chiefly butter and dairy products, predispose to arterial thrombosis, essentially through an effect on blood platelets.
Supplement Number 1, Vol. 4.1974
26
Introduction Numerous retrospectiveepidemiologicalstudies have shown that coronary heart
disease
(CID) is related to the level of serum cholesteroland to the
consumptionof dietary saturated fats ai reviewed in recent papers (1, 2). In addition, a five year large-scaleprospective internationalstudy was carried out, in seven countries (3))
on the relationshipbetween diet and the
incidence of CHD. The results indicate that CHD deaths(coronarythrombosis is presumed to play a prominent role) ma all
CHD.cases,were closely related
to the saturated fat intake. In the mechanisms by which dietary fats might influence CID, considerable emphasishas been placed on atherosclerosis(1). Nevertheless,coronary thrombosis appears to be one of the most dramatic event in CHD, although quite often a thrombus cannot be founkiin acute myocardial ischemia (4, 5).
However,
platelet aggregates in epicardial (6) ma intramyocardialarteries (7) are frequentlyfound in sudden coronary death. In addition, it is well documented that mural
thrombi can contributeto the formation of atheroscleroticplaques
(8, 9). Therefore arterial thrombosis appears to have an important clinical significance in populations susceptibleto CHO. The question is whether dietary fats predisposeto CHD through an effect on thrombosis apart from the vessel wall changes they induce.
Jvihence from in vitro studies In this field, the attention has been focused on the effect of certain fat constituentson platelets,the initial component of a thrombus, and on clot: ting. It has been shown that only the long chain saturated fatty acids (from 14 carbons atoms) were effectiveto induce platelet aggregation (10, il, 12, 13)
and to accelerate coagulation (14) by activatingHageman (15) and other
clotting factors (16). Among the commonest fatty acids of dietary fats, stearic acid (C 18) appears to be the most effective in inducing platelet aggregation ma promoting coagulation. These sttiies underline the possibilitythat free fatty acids (FFA) in plasma could initiate thrombosis and that dietary fats could ikluence this triggering effect by changing the concentrationor the type of these
27
Supplement Number 1, Vol. 4,1974
fatty acids. -Furthermore, it is expected that changes in the FFA compositionmight modify the behavior of platelets by changingtheir lipid fatty acid composition. Rapid changes in the platelet fatty acids have been observed in vitro by incubatingplatelets with various fatty acids (17).
-Svidencefrom studies in animals Jn vivo deleteriouseffects of long chain saturated fatty acids have been observed in dogs after the infusion of these fatty acids which resulted in massive thrombosis of the heart and large vessels (14). Perhaps more important in the relationshipbetween dietary fats and thrombosis in man might be the thrombotic tendency resulting from the long-term feeding of high-fat diets in animals. Mustard and Coil. have examined the effect of lard and egg yolk fed to pigs with an extracorporealcirculation (18).
Under these conditions., the
tendency of blood to form thrombi was increasedby the high fat diet. A similar result was obtained by Mathues et al. (19) in rabbits fed coconut oil. In rats fed a purified diet rich in a coconut fat mixture, an increased incidence of arteriolaroccluding thrombi, initiatedby the intravenousinjection of adenosine diphosphate,was observed by Nord6y et al. (20). Hartz-oft(20) induced cardiac and coronary thrombosisby feeding rats a high fat diet supplemented with thiouracil and cholic acid. In this experimentalmodel (21), cacao butter was the most thrombogenicdietary fat, followed by butter while the animals fed corn oil and cottonseedoil were completelyprotected.Unfortunately, these last studies were not further elaboratedto determinewhat and how constituents of the dietary saturated fats were thrombogenic. Recently, Hornstra and Vendelmans-Starrenburg(23)
reported an ingenious
technique to induce occlusive thrombi in the rat by means of a cannula inserted in the abdominal aorta. With this model, the effect of dietary fats on thrombosis has been studied in a systematicway. From the results obtained Iiornstra (24) concludedthat in the fats only three fatty acids, myristic, palmitic and stearic acids were thrombogenic.By contrast linoleic and probably linolenic acids, appeared to have an antithromboticeffect. Under these conditions,the thrombogeniceffect of fats was related to the susceptibilityof platelets to aggregation,at least as determinedby the filter-looptechnique (24).
Supplement Number 1, Vol.
4.1974
-Under our own experimentalconditions,we have observed that the feeding of fats rich in long chain saturated fatty acids predispose rats to large 'thromboseswhich, depending on the triggering agent, occur in veins (endotoxins) (25), in cardiac cavities (epinephrine)(26) or even in coronary arteries (ellagicacid) as shown by experimentsstill in progress. In these studies,
myristic, palmitic but mostly stearic acids were shown to be thrombo-
genie (25, 27) while linoleic acid could be the main antithrombogenicfatty acid. In subsequent studies we have observed that one of the mechanisms by which dietary fatty acids were predisposingrats to thrombosiswas probably through a change in the fatty acid compositionof platelet phospholipids (28, 29). This resulted in an increase of platelet susceptibilityto aggregation, as evaluated by the turbidimetricmethod (26), and an increase in the activity of platelet factor 3 (PF3) (30), the only clotting factor of lipid nature. The increased activity of PF3 appears to be responsible for the in vitro hypercoagulabilityresulting from the feeding of high fat diets (30). Similar result were obtained in rabbits fed various fats (30). In addition in rabbits (31) as in rats (29), the clotting activity of platelet-richplasma or of the platelet phoapholipids (PS + PI) the most active on clotting (321,
was correlatedwith the ratio stearic/linoleicacid in the diet. This
confirms the key role of these two fatty acids in clotting changes, as well as in thrombosis in the rat., Another interestingresult from the studies carried out in rabbits was that butter has been found not only extremely effective in inducing a hypercoagulability,but also the most atherogenicand hypercholesterolemicfat. In previous studies in rat (27) we had observed that stearic acid was the most thrombogenicand palmitic was the most hypercholesterolemicand atherogenic fatty acid. In man it has also been reported that palmitic acid was highly hypercholesterolemic(33).
Consequently,the multiple effects of butter pro-
bably result mostly from its high content in palmitic and stearic acid and low content in linoleic acid. In the rat (29) and rabbit (31),
the effects
of butter on thrombosis and clotting could be related to its ratio of stearicllinoleicacid. Also of interest is the observationthat the long-term feeding of fats rich in palmitic and stearic acids does not solely or necessarilyresult in an increase of the same fatty acids in the platelets. Under these conditions,in rat
Supplement
(28,
29)
29
Number 1, Vol. 4, 1974
and rabbit (31), an increase of the corresponding desaturated fat-
ty acids (palmitoieic and oleic) is usually observed. By contrast, feeding oleic acid does not appear to increase the oleic acid content of platelets. Finally, it seems that it is only linoleic acid (and perhaps linolenic acid) in the diet which is accompanied by a marked increase of the same fatty acid (and also arachidonic acid) in platelets, at least in rats (28). Stearic acid in the diet could be the most thrombogenic fatty acid by increasing mainly the level of oleic acid in platelets. The phospholipids rich in oleic acid and not those rich in saturated fatty acids, have been found to be the most active in clotting (32).
Evidence from studies in human subjects Nordby and RGdset (34) have shown that in human subjects, a dietary change with more unsaturated fatty acids, will be subsequently reflected in the platelets. The change was not characterized by an increase in the platelet cholesterol or phospholipids level but, by an increase in the linoleic acid content of various platelet phospholipids at the expense of palmitic and oleic acids. They also observed that the PF3 activity was consistently decreased in the subjects fed the.unsaturated fat, which agrees with our results from experiments carried out in animals (30, 31, 32). Iacono and Coil. have also noted (35) that in subjects from Cincinnati, highly susceptible to CHD and eating typical American food, the level of stearic and oleic acids in the platelet PS were higher than in platelets subjects from Milan or Sicily. We have shown (28)
that the fatty acid pattern of total
platelets lipids from patients with a recent myocardial infarction, was characterized, as compared to normal subjects having no risk factors for CID, chiefly by an increase in palmitic, stearic and oleic acids at the expense of linoleic and arachidonic acids. Therefore, it seems that in man given saturated fats or presenting CHD, the fatty acid pattern of platelets, mostly platelet phospholipids, is comparable to that of animals fed thrombogenic diets. Nordijyand RCidsethave shown that the activity of PF3 was increased in patients with ischaemic heart disease (36) and in patients with hyperbetalipoproteinemia (37). In a recent study in patients submitted to coronarography (38), we have observed that irrespective of the severity of the lesions, the activity
of PF3 determined in washed platelets (39), was markedly increased and was di-
Supplement Number 1, Vol. 4, 1974
rectly related to the clotting activity of the total platelet phospholipids. This increase in PF3 activity was responsiblefor an in vitro hypercoagulability evaluatedby the recalcificationplasma clotting time. When the platelets were removed (cephalintime) by slow speed centrifugation,a prolongation of the clotting test was observed when performed in siliconizedglassware. This peculiar prolongationof the cephslin time (partialthromboplastin time) has also been reported by other investigatorsin patients with peripheral occlusive atherosclerosis(40). This indicatesthat the plasmatic clotting factors are probably not involved in the state of hypercoagulabilityobserved in CRD. This hypercoagulabilitynoted in coronary patients and evaluated by the (platelet-rich)plasma clotting time in siliconizedmaterial, has also been observed in survivors of myocardial infarction (41). These investigators (41) concluded that neither the fibrinogenlevel nor the Factors II, VII, VIII, or X could be responsible for the shortened clotting time. However, they did not evaluate the activity of PF3. The importanceof PF3 in the hypercoagulabilityobserved in coronary patients is illustratedin figure 1.
Fig.1 Recalcificationplasma clotting time (PCT) and platelet factor 3 clotting activity (F3 CT) in 15 patients with coronary lesions visualized by angiography.The clotting t_estwere performed accordingto techniques already reported (30, 38).
Supplement Number 1, Vol. 4,1974
31
In this figure it is shown that the clotting time of the PRP representing the whole blood clotting activity, is closely correlatedwith the PF3 clotting time which has been determinedon washed platelets from 15 patients submitted to coronarography.These results are concordantwith those we obtained in hyperlipemicrats (29, 30) and in normal human subjects (30). Concerningplate&et aggregation,an increased susceptibilityof platelets to aggregationby ADP (42,
43>for citing solely a few reports) and a defective
platelet disaggregation(44) tive been reported in occlusive arterial disease. In coronary patients, we have found (38) that it was mostly to thrombin as in hyperlipemicanimals (26),
that the platelets were more susceptible,and it
was highly significantin patients with severe stenotic resions (38). An additional evidence of the role of dietary fat in coronsry thrombosis
can be obtain from the prevention studies by dietary modifications.Several reports have shown that by changingthe type of fat and reducing the amount of calories supplied by fats in the diet, the incidence of CHD can be significantly reduced in a population initially free from CID clinical manifestations (45)
or in survivors of qocardial infarction (46)
(for citing only a few stu-
dies). The 12 year Finland prevention trial (47) in two mental Hospitals appears to be of special interest. The well controlleddietary modificationsconsisted mostly in replacing butter and common margarinesby an unsaturatedmargarine, and milk, by an emulsion of soybean oil in skim milk. The most striking difference was a marked reduction in the death-ratefrom CID. This finding suggests that coronary thrombosis was markedly reduced by this relatively simple dietary modification.
32
Supplement Number 1, Vol. 4, 1974
References
4.
STAMLER, J. Epidemiology of coronary heart disease. Med. Clin.: 57., 5.1973.
2.
STORMORKEN, H.Epidemiological studies. Relation between dietary fat and arterial and venous thrombosis. In "Dietary fats and thrombosis". S. Renaud and A. NordZjy (Ed). Basle, S. Karger, 1974.
3.
KEYS, A. Coronary heart disease in seven countries. Circulation.: 41 (suppl.1). 1970.
4.
SPAIN, D.M., and BRADES, V.A. Sudden death from coronary heart disease. Chest.: 58., 107.1970.
5.
BAROLDI, G. Acute coronary occlusion as a cause of myocardial infarct and sudden coronary heart death. Amer. J. Cardiol.: 16., 859.1965.
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HAREM,
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HAEREM, J.W. Platelet aggregates in intrsmyocardial vessels of patients dying suddenly and unexpectedly of coronary artery disease. Atherosclerosis.: 15., 199.1972.
8.
DUGUID, J.B. Thrombosis as a factor in the pathogenesis of aortic atherosclerosis. J. Path. Bact.: 60., 57.1948.
9.
HAUST, M.D., MORE, R.H., and MOVAT, H.Z. The mechanism of fibrosis in arteriosclerosis. Amer.,J. Path.: 35., 265.1959.
10.
WARNER, E.D., HOAK, J.C., and CONNOR, W.E. The role of fatty acids in platelet aggregation and thrombosis. Thromb. Diath. Haemorrh.: 26.,
J.W. Sudden coronary death: The occurence of platelet aggregates in the epicardial arteries of man. Atherosclerosis.: lb., 417.1971.
249.1967.
11.
MAHADEVAN, V., SINGH, H., and LUNDBERG, W.O. Effect of saturated and unsaturated fatty acids on blood platelet aggregation in vitro. Proc. Sot. Exp. Biol. Med.: 121., 82.1966.
12.
KERR, J.W., PIRRIE, R., MacAULAY, I., and BRONTE-STEWART, B. Plateletaggregation by phospholipids and free fatty acids. Lancet.: l., 1296.1965.
13.
PROST-DVOJAKOVIC, R.J., and SAMAMA, M. Clot-promoting and platelet-aggregating effects of fatty acids. In "Dietary fats and thrombosis". S. Renaud and A. Norddy (Ed). Basle, S. Karger, 1974.
14.
CONNOR, W.E., HOAK, J.C. and WARNER, E.D. 8. The effects of fatty acids on blood coagulation and thrombosis. Thromb. Diath. Haemorrh.: 13.,'.09. 1965.
Supplement Number 1, Vol. 4, 1974
15.
BIDISHEIM, P., and MIBASHAN, R.S. Activation of Hageman factor (Factor XII) by long-chain saturated fatty acids. Thromb. Diath. Haemorrh.: f /., 346.1963.
16. KAHN, M.J.P., and HEMKER, H.C. Studies on blood coagulationfactor V. I. The interactionof salts of fatty acids and coagulationfactors. Thromb. Diath. Haemorrh.: 22., 417.1969. 17.
ANDREOLI, V.M., MAFFEI, F., and THONON, G.C. Platelet lipid modification by fatty acids. Experimentalstudies and correlationswith human neuropathology.In "Dietary fats and thrombosis".S. Renaud and A. Nord6y (Ed). Basle, S. Karger, 1974.
18. MUSTARD, J.F., ROWSELL, H.C., MURPHY, E.A. and DOWNIE, H.G. Diet and thrombus formation:Quantitativestudies using an extracorporealcirculation in pigs. J. Clin. Invest.: 42., 1783.1963. 19.
MATHUES, J.K., WOLFF, C.E., CEVALLOS,W.H., and HOLMES, W.L. Platelet adhesivenessand thrombosis in rabbits on an atherogenicdiet. Med. Exp.: 18., 121.1968.
H. The influence 20. NORDO.Y,,A.,, HAMLIN, J.T., CHANDLER,A.B., a.& INIAND, of dietary fats on plasma and platelet lipids and ADP induced platelet thrombosis in the rat. Scand. J. Haemat.: 5., 458.1968. 21. HARTROFT, W.S. Abnormal fat transport.Diabetes.: 7., 221.1958. 22. HARTROFT, W.S., and O'NEAL, R.N. Experimentalproduction of coronary atherosclerosis.Amer. J. Cardiol.: 9., 335.1962. 23.
HORNSTRA, G., and VENDELMANS-STARRENBURG, A. Induction of experimental occlusive thrombi in rats. Atherosclerosis.:17., 369.1973.
24. HORNSTRA, G. Experimentalstudies. Dietary fats and arterial thrombosis In "Dietary fats and Thrombosis".S. Renaud and A. Nord6y (Ed). Basle, S. Karger, 1974. 25.
RENAUD; S. Thrombotic,atheroscleroticand lipemic effects of dietary fats in the rat. Angiology.: 20., 657.1969.
&.RENAUD, S., and GODU, J. Induction of large thrombi in hyperlipemicrats by epinephrineand endotoxin.Lab. Invest.: 21., 512.1969. 27.
RENAUD, S. Thrombogenicityand atherogenicityof dietary fatty acids in rat. J. Atheroscler.Res.: 8., 625.1968.
28,RENAUD, S., KUBA, K., GOULET, C., LEMIRE, Y., and ALLARD, C. Relationship between fatty-acidcompositionof platelets and platelet aggregation in rat and man. Relation to thrombosis.Circ..Res.:26., 552.1970.
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Supplement Number 1, Vol. 4,1974
29.
GAUTHXRON, P., and RENAUD, S. Hyperlipemiainduced hypercoagulablestate
in rat..Role of an increased activity of platelet phosphatidylserine in response to certain dietary fatty acids. Thromb. Res.: l., 353.1972. 30.
RENALID, S., and LECOMPTE, F. Hypercoagulabilityinduced by hyperlipemia in rat, rabbit and man. Role of platelet factor 3. Circ. Res.: 27.,
31.
1003.1970.
RENAUD, S., and GAUTHERON, P. Influence of dietary fats on atheroscle-
rosis, coagulationand platelet phospholipidsin rabbits. Atherosclerosis (In press), 1974. 32.
RENAUD, S., and GAUTHERON, P. Dietary fats and experimental (Cardiac
and Venous) thrombosis.In "Dietary fats and thrombosis".S.'Renaud and NordCiy(Ed). Basle, S. Karger, 1974, ~53. 33.
KEYS, A., ANDERSON, J.T., and GRANDE, F. Serum cholesterolresponse to changes in the diet IV particular saturated fatty acids in the diet. Metabolism.: 14., 776.1965.
34.
NORDOY, A., and RODSET, J.M. The influence of dietary fats on platelets in man. Acta. Med. Stand.: 190., 27.1971.
35*
IACONO, A., and ZELLNER, D.C., PAOLETTI, R., ISHIKAWA, T., FRIGENI, V. and FUMAGALLI, R. Comparison of blood platelet and Erythrocytelipids in man in three age groups from three regions : Milan, Cincinnati and Sicily. In "Dietary fats and thrombosis".S. Renaud and Nordtiy,A. (Ed). Basle, S. Karger,.l974. .
36.
NORDOY, A., and RODSET, J.M.'.'Platelet phospholipidsand their function in patients with ischemic heart disease. Acta. Med. Stand.: 188.,133. 1970.
37.
NORDOY, A., and RODSET, J.M. Platelet function and platelet phospholipids in patients with hyperbetalipoproteinemia. Acta. Med. Stand.: 180.) 385.1971.
38.
RENAUD, S., GAUTHERON, P. ARBOGAST, R., and DUMONT, E. Platelet factor
3 activity and platelet aggregation in patients submittedto coronarography. Stand. J. Haemat.: 12., 85.1974. 39. 40.
RENAUD, S., GAUTHERON,P., and ROSENSTEIN,H. Platelet factor 3 activity in washed platelets. Thromb. Diath. Haemorrh.: 30., 566.1973. COTTON, R.C., BLOOR, K., and ARCHIBALD, G. Intsr-relationshipsbetween platelet response to adenosive diphosphate, blood coagulation and rum lipids in patients with peripheral occlusive atherosclerosis. Atherosclerosis.: 16., 337.1972.
41.
se-
KORSAN-BENGTSEN,K., WILHELMSEN,L., ELMFELDT; D:, and TIBBLIN, G. Blood coagulationand fibrinolysisin man after myocardial infarction compared with a representative-population sample. Atherosclerosis.: 16.) 83.1972.
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35
42.
'DREYFUS, F., and ZAHAVI, J. Adenosive diphosphate induced platelet aggregation in myocardial infarction and ischemic heart disease. Atherosclerosis.: 17., 107.1973.
43.
O'BRIEN, J.R., HEYWOOD, J.B., and HEADY, J.A. The quantitation of platelet aggregation induced by four compounds. A study in relation to myocardial infarction. Thromb. Diath. Haemorrh.: 16., 752.1966.
44.
DAVIS, J.M. Defective platelet disaggregation associated with occlusive arterial disease. Angiology.: 24., 391.1973.
45.
CHRISTAKIS, G., RINZLER, S.H., ARCHER, M., WINSLOW, G., JAMPEL, S., STEPHENSON, J., FRIEDMAN, G., FEIN, H., KRANS, A., and JAMES, G. The anti-coroanry club. A dietary approach to'the prevention of coronary heart disease - a seven year report. Am. J. Publ. Health.: 56., 299.1966.
46.
LEREN, P. The effect of plasma cholesterol lowering diet in male survivors of myocardial infarction. Acta. Med. Stand.: 181(supp1.466)., 1.1966.
47.
MIETTINEN, M., TURPEINEN, O., KARVONEN, M.J., ELOSUO, R., and PAAVILAINEN, E. Effect of cholesterol-lowering diet on mortality from coronary heart disease and other causes. Lancet: l., 835.1972.
RESEARCH
Supplement Number 1 LIPIDS AND THROMBOSIS, noms+
Vol. 4, 1974 Pergamon Preaa
DIETARY FATS AND ARTERIAL THROMBOSIS
S. Renaud Unit6 de Recherche de Physio-Pathologie Vasculaire (INSERM, Unit6 63), 22, Avenue du Doyen-Lgpine, 69500 - Lyon-Bron, France.
ABSTRACT. Epidemiologic studies have shown that coronary heart disease (CHD), particularly coronary deaths where presumably arterial thrombosis plays a prominent role, was closely associated with the saturated fat intake. Conclusions from experiments, with totally unrelated techniques, appear to be unanimous in that fats, with long chain saturated fatty acids, particularly stearic acid, affect platelet behavior and clotting in vitro and in vivo, and predispose animals to thrombosis by long term feeding. Linoleic and linolenic acids might have anti-thrombotic effects. One mechanism by which the long-chain saturated fatty acids predispose to thrombosis could be by changing the fatty acid pattern of platelet phospholipids, a result demonstrated both in man and animals. Since the formation of platelet aggregates seems to be the earliest event in thrombosis, the platelet phospholipids, because of their structural role in platelet membranes, might play a prominent role in the observed increased susceptibility of platelets to aggregation. In addition, it has been shown that modifications in the phospholipids fatty acid composition of platelets have an effect on their clotting activity, i.e. the lipid moiety of platelet factor 3 (PF3). An increase in the activity of PF3 which has been shown to cause in vitro hypercoagulability, was found in CHD as well as in animals fed the long chain fatty acids. Finally, prevention studies have indicated that by changing the type of fat in the diet, the death-rate from CHD can be consistently decreased. The results mentioned above are concordant with the hypo+.hasis that some dietary saturated fats, in practice chiefly butter and dairy products, predispose to arterial thrombosis, essentially through an effect on blood platelets.
Supplement Number 1, Vol. 4.1974
26
Introduction Numerous retrospectiveepidemiologicalstudies have shown that coronary heart
disease
(CID) is related to the level of serum cholesteroland to the
consumptionof dietary saturated fats ai reviewed in recent papers (1, 2). In addition, a five year large-scaleprospective internationalstudy was carried out, in seven countries (3))
on the relationshipbetween diet and the
incidence of CHD. The results indicate that CHD deaths(coronarythrombosis is presumed to play a prominent role) ma all
CHD.cases,were closely related
to the saturated fat intake. In the mechanisms by which dietary fats might influence CID, considerable emphasishas been placed on atherosclerosis(1). Nevertheless,coronary thrombosis appears to be one of the most dramatic event in CHD, although quite often a thrombus cannot be founkiin acute myocardial ischemia (4, 5).
However,
platelet aggregates in epicardial (6) ma intramyocardialarteries (7) are frequentlyfound in sudden coronary death. In addition, it is well documented that mural
thrombi can contributeto the formation of atheroscleroticplaques
(8, 9). Therefore arterial thrombosis appears to have an important clinical significance in populations susceptibleto CHO. The question is whether dietary fats predisposeto CHD through an effect on thrombosis apart from the vessel wall changes they induce.
Jvihence from in vitro studies In this field, the attention has been focused on the effect of certain fat constituentson platelets,the initial component of a thrombus, and on clot: ting. It has been shown that only the long chain saturated fatty acids (from 14 carbons atoms) were effectiveto induce platelet aggregation (10, il, 12, 13)
and to accelerate coagulation (14) by activatingHageman (15) and other
clotting factors (16). Among the commonest fatty acids of dietary fats, stearic acid (C 18) appears to be the most effective in inducing platelet aggregation ma promoting coagulation. These sttiies underline the possibilitythat free fatty acids (FFA) in plasma could initiate thrombosis and that dietary fats could ikluence this triggering effect by changing the concentrationor the type of these
27
Supplement Number 1, Vol. 4,1974
fatty acids. -Furthermore, it is expected that changes in the FFA compositionmight modify the behavior of platelets by changingtheir lipid fatty acid composition. Rapid changes in the platelet fatty acids have been observed in vitro by incubatingplatelets with various fatty acids (17).
-Svidencefrom studies in animals Jn vivo deleteriouseffects of long chain saturated fatty acids have been observed in dogs after the infusion of these fatty acids which resulted in massive thrombosis of the heart and large vessels (14). Perhaps more important in the relationshipbetween dietary fats and thrombosis in man might be the thrombotic tendency resulting from the long-term feeding of high-fat diets in animals. Mustard and Coil. have examined the effect of lard and egg yolk fed to pigs with an extracorporealcirculation (18).
Under these conditions., the
tendency of blood to form thrombi was increasedby the high fat diet. A similar result was obtained by Mathues et al. (19) in rabbits fed coconut oil. In rats fed a purified diet rich in a coconut fat mixture, an increased incidence of arteriolaroccluding thrombi, initiatedby the intravenousinjection of adenosine diphosphate,was observed by Nord6y et al. (20). Hartz-oft(20) induced cardiac and coronary thrombosisby feeding rats a high fat diet supplemented with thiouracil and cholic acid. In this experimentalmodel (21), cacao butter was the most thrombogenicdietary fat, followed by butter while the animals fed corn oil and cottonseedoil were completelyprotected.Unfortunately, these last studies were not further elaboratedto determinewhat and how constituents of the dietary saturated fats were thrombogenic. Recently, Hornstra and Vendelmans-Starrenburg(23)
reported an ingenious
technique to induce occlusive thrombi in the rat by means of a cannula inserted in the abdominal aorta. With this model, the effect of dietary fats on thrombosis has been studied in a systematicway. From the results obtained Iiornstra (24) concludedthat in the fats only three fatty acids, myristic, palmitic and stearic acids were thrombogenic.By contrast linoleic and probably linolenic acids, appeared to have an antithromboticeffect. Under these conditions,the thrombogeniceffect of fats was related to the susceptibilityof platelets to aggregation,at least as determinedby the filter-looptechnique (24).
Supplement Number 1, Vol.
4.1974
-Under our own experimentalconditions,we have observed that the feeding of fats rich in long chain saturated fatty acids predispose rats to large 'thromboseswhich, depending on the triggering agent, occur in veins (endotoxins) (25), in cardiac cavities (epinephrine)(26) or even in coronary arteries (ellagicacid) as shown by experimentsstill in progress. In these studies,
myristic, palmitic but mostly stearic acids were shown to be thrombo-
genie (25, 27) while linoleic acid could be the main antithrombogenicfatty acid. In subsequent studies we have observed that one of the mechanisms by which dietary fatty acids were predisposingrats to thrombosiswas probably through a change in the fatty acid compositionof platelet phospholipids (28, 29). This resulted in an increase of platelet susceptibilityto aggregation, as evaluated by the turbidimetricmethod (26), and an increase in the activity of platelet factor 3 (PF3) (30), the only clotting factor of lipid nature. The increased activity of PF3 appears to be responsible for the in vitro hypercoagulabilityresulting from the feeding of high fat diets (30). Similar result were obtained in rabbits fed various fats (30). In addition in rabbits (31) as in rats (29), the clotting activity of platelet-richplasma or of the platelet phoapholipids (PS + PI) the most active on clotting (321,
was correlatedwith the ratio stearic/linoleicacid in the diet. This
confirms the key role of these two fatty acids in clotting changes, as well as in thrombosis in the rat., Another interestingresult from the studies carried out in rabbits was that butter has been found not only extremely effective in inducing a hypercoagulability,but also the most atherogenicand hypercholesterolemicfat. In previous studies in rat (27) we had observed that stearic acid was the most thrombogenicand palmitic was the most hypercholesterolemicand atherogenic fatty acid. In man it has also been reported that palmitic acid was highly hypercholesterolemic(33).
Consequently,the multiple effects of butter pro-
bably result mostly from its high content in palmitic and stearic acid and low content in linoleic acid. In the rat (29) and rabbit (31),
the effects
of butter on thrombosis and clotting could be related to its ratio of stearicllinoleicacid. Also of interest is the observationthat the long-term feeding of fats rich in palmitic and stearic acids does not solely or necessarilyresult in an increase of the same fatty acids in the platelets. Under these conditions,in rat
Supplement
(28,
29)
29
Number 1, Vol. 4, 1974
and rabbit (31), an increase of the corresponding desaturated fat-
ty acids (palmitoieic and oleic) is usually observed. By contrast, feeding oleic acid does not appear to increase the oleic acid content of platelets. Finally, it seems that it is only linoleic acid (and perhaps linolenic acid) in the diet which is accompanied by a marked increase of the same fatty acid (and also arachidonic acid) in platelets, at least in rats (28). Stearic acid in the diet could be the most thrombogenic fatty acid by increasing mainly the level of oleic acid in platelets. The phospholipids rich in oleic acid and not those rich in saturated fatty acids, have been found to be the most active in clotting (32).
Evidence from studies in human subjects Nordby and RGdset (34) have shown that in human subjects, a dietary change with more unsaturated fatty acids, will be subsequently reflected in the platelets. The change was not characterized by an increase in the platelet cholesterol or phospholipids level but, by an increase in the linoleic acid content of various platelet phospholipids at the expense of palmitic and oleic acids. They also observed that the PF3 activity was consistently decreased in the subjects fed the.unsaturated fat, which agrees with our results from experiments carried out in animals (30, 31, 32). Iacono and Coil. have also noted (35) that in subjects from Cincinnati, highly susceptible to CHD and eating typical American food, the level of stearic and oleic acids in the platelet PS were higher than in platelets subjects from Milan or Sicily. We have shown (28)
that the fatty acid pattern of total
platelets lipids from patients with a recent myocardial infarction, was characterized, as compared to normal subjects having no risk factors for CID, chiefly by an increase in palmitic, stearic and oleic acids at the expense of linoleic and arachidonic acids. Therefore, it seems that in man given saturated fats or presenting CHD, the fatty acid pattern of platelets, mostly platelet phospholipids, is comparable to that of animals fed thrombogenic diets. Nordijyand RCidsethave shown that the activity of PF3 was increased in patients with ischaemic heart disease (36) and in patients with hyperbetalipoproteinemia (37). In a recent study in patients submitted to coronarography (38), we have observed that irrespective of the severity of the lesions, the activity
of PF3 determined in washed platelets (39), was markedly increased and was di-
Supplement Number 1, Vol. 4, 1974
rectly related to the clotting activity of the total platelet phospholipids. This increase in PF3 activity was responsiblefor an in vitro hypercoagulability evaluatedby the recalcificationplasma clotting time. When the platelets were removed (cephalintime) by slow speed centrifugation,a prolongation of the clotting test was observed when performed in siliconizedglassware. This peculiar prolongationof the cephslin time (partialthromboplastin time) has also been reported by other investigatorsin patients with peripheral occlusive atherosclerosis(40). This indicatesthat the plasmatic clotting factors are probably not involved in the state of hypercoagulabilityobserved in CRD. This hypercoagulabilitynoted in coronary patients and evaluated by the (platelet-rich)plasma clotting time in siliconizedmaterial, has also been observed in survivors of myocardial infarction (41). These investigators (41) concluded that neither the fibrinogenlevel nor the Factors II, VII, VIII, or X could be responsible for the shortened clotting time. However, they did not evaluate the activity of PF3. The importanceof PF3 in the hypercoagulabilityobserved in coronary patients is illustratedin figure 1.
Fig.1 Recalcificationplasma clotting time (PCT) and platelet factor 3 clotting activity (F3 CT) in 15 patients with coronary lesions visualized by angiography.The clotting t_estwere performed accordingto techniques already reported (30, 38).
Supplement Number 1, Vol. 4,1974
31
In this figure it is shown that the clotting time of the PRP representing the whole blood clotting activity, is closely correlatedwith the PF3 clotting time which has been determinedon washed platelets from 15 patients submitted to coronarography.These results are concordantwith those we obtained in hyperlipemicrats (29, 30) and in normal human subjects (30). Concerningplate&et aggregation,an increased susceptibilityof platelets to aggregationby ADP (42,
43>for citing solely a few reports) and a defective
platelet disaggregation(44) tive been reported in occlusive arterial disease. In coronary patients, we have found (38) that it was mostly to thrombin as in hyperlipemicanimals (26),
that the platelets were more susceptible,and it
was highly significantin patients with severe stenotic resions (38). An additional evidence of the role of dietary fat in coronsry thrombosis
can be obtain from the prevention studies by dietary modifications.Several reports have shown that by changingthe type of fat and reducing the amount of calories supplied by fats in the diet, the incidence of CHD can be significantly reduced in a population initially free from CID clinical manifestations (45)
or in survivors of qocardial infarction (46)
(for citing only a few stu-
dies). The 12 year Finland prevention trial (47) in two mental Hospitals appears to be of special interest. The well controlleddietary modificationsconsisted mostly in replacing butter and common margarinesby an unsaturatedmargarine, and milk, by an emulsion of soybean oil in skim milk. The most striking difference was a marked reduction in the death-ratefrom CID. This finding suggests that coronary thrombosis was markedly reduced by this relatively simple dietary modification.
32
Supplement Number 1, Vol. 4, 1974
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