Cholesterol synthesis in the pathogenesis of lithocholic acid-induced cholestasis.

Lithocholic acid (LCA)-induced intrahepatic cholestasis is associated with increased de novo synthesis of hepatic cholesterol and augmented cholesterol content of the liver cell plasma membrane fraction enriched in bile canalicular complexes (BCM). To determine whether inhibition of cholesterol synthesis could prevent LCA-induced cholestasis, adult male Wistar rats were treated daily with the hypocholesterolemic agents, clofibrate (250 mg/kg) or mevinolin (25 mg/kg), for one, two or four days. After bile duct cannulation and bile collection for one hr, the animals were injected intravenously with 120 mumoles/kg of LCA or its carrier (albumin). Cholesterol synthesis was measured in liver homogenates, and its contribution to the BCM was estimated. LCA reduced bile flow by 51%, 35% and 25% after clofibrate pretreatment for one, two and four days, respectively, and by 51%, 30% and 42% in mevinolin-pretreated animals after one, two and four days. In control animals, cholesterol synthesis and the contribution of newly synthesized cholesterol in the BCM were increased after LCA injection. However, despite that cholesterol synthesis and the contribution of newly synthesized cholesterol in the BCM were reduced in drug-pretreated rats, LCA injection caused a relative increase in these parameters of a magnitude similar to that observed in controls. Thus, the ability of LCA injection to augment de novo cholesterol synthesis and its transport to the BCM may be an important pathogenetic step in the development of cholestasis.

Effect of the amount and type of dietary fat on platelet function, platelet survival and response to continuous aortic injury in rats.

The effect of giving diets containing 1.5 or 16% safflower or corn oil or 16% milk fat for 15 weeks on changes in the fatty acid composition of platelet phospholipids, in vitro platelet function, platelet survival and thrombosis was examined in rats. The mean plasma cholesterol concentration was not different among the groups. Diets containing 1.5% safflower or corn oil or 16% milk fat were associated with a decrease in 18:2n - 6 and an increase in 18:1n - 9 and the 20:4n - 6/18:2n - 6 ratio in the platelet phospholipids compared with the 16% safflower or corn oil diets. The 16% milk fat diet was associated with an increase in 14:0, 20:3n - 9, 22:3n - 9 and a decrease in 22:4n - 6 in platelet phospholipids compared with the other groups. There were no differences among the groups in the sensitivity of washed platelets to ADP-, thrombin- or collagen-induced aggregation, or thrombin- or collagen-induced release of granule contents or loss of arachidonate from platelet phospholipids. Platelet survival and turnover in rats given the diets were not different among the groups. In response to indwelling aortic catheters neither the percentage reduction in platelet survival nor the platelet accumulation on injured aortae and catheters were different among the groups. No macroscopic thrombi were seen in rats given any of the diets. The results of these studies provide no evidence that diet-induced alterations in fatty acid content (increases in 18:1n - 9, 20:3n - 9, 22:3n - 9, 20:3n - 6, and 20:4n - 6/18:2n - 6 ratio and a decrease in 22:4n - 6) of platelet phospholipids modify in vitro platelet function, platelet survival or turnover or influence thrombosis in rats.

Effect of chronic administration of taurolithocholate on bile formation and liver ultrastructure in the rat.

In some cholestatic syndromes, lithocholic acid (LCA) has been identified in serum and bile and might play a role in the pathogenesis of cholestasis. Thus, we examined the effect of chronic LCA administration on hepatobiliary function in rats. The taurine-conjugate of LCA (TLCA) was given to male rats at the dose of 50 mg/kg body weight i.v. twice daily for 18 days. Bile flow, biliary secretion rate of bile acids, phospholipids and cholesterol, measured 15 h after the last injection, were normal with the exception of phospholipid, which was significantly higher when compared to controls. Individual bile acids measured by GLC showed that the contribution of chenodeoxycholic and muricholic acids increased while that of cholic acid decreased. By electron microscopy, bile canalicular structures appeared normal except for a widening of the pericanalicular ectoplasm. Evaluation of hepatobiliary function 1 h after an additional injection of TLCA to rats that received the bile acid for 18 days, resulted in the typical acute cholestatic response. Thus chronic administration of TLCA does not influence the acute cholestatic effect of TLCA.

The effect of dietary saturated fat and cholesterol on platelet function, platelet survival and response to continuous aortic injury in rats.

Induction of hypercholesterolemia in rats by diets containing milk fat, cholesterol and taurocholate caused increased sensitivity of platelets to thrombin-induced aggregation and release, but not to ADP- or collagen-induced aggregation or release. This hypersensitivity to thrombin persisted in the presence of CP/CPK to convert released ADP to ATP, and aspirin to block formation of thromboxane A2. The increased sensitivity of platelets to thrombin in hypercholesterolemic animals was associated with an increase in 18:1 omega 9, 18:2 omega 6 and 20:3 omega 6 and a decrease in 20:4 omega 6 and 22:4 omega 6 in their phospholipids. Hypercholesterolemic animals also had a shortened platelet survival that did not appear to be due to an alteration in the lipid composition of the platelets. The diet-induced changes in platelet function were not associated with enhanced thrombosis in animals with indwelling aortic catheters, but were associated with increased platelet accumulation on the exposed subendothelium.

Nutrients, platelet function and composition in nine groups of French and British farmers.

In 260 male farmers (40-45 years) divided into 9 groups from different areas in France and Britain, coagulation, platelet aggregation, lipemia, fatty acids from plasma lipids and platelet phospholipids were determined in relation to the food intake evaluated by recall, weighing and chemical analysis of the diet. The clotting activity of platelets and their response to thrombin aggregation was significantly correlated on an individual basis with the intake of saturated fatty acids both in subsamples as well as in the whole study. Serum cholesterol was also significantly correlated with saturated fats but only on a group basis or on the totality of the study. Calcium, linolenic acid and alcohol in the diet were inversely related to certain platelet functions. Linoleic acid was inversely related to serum cholesterol and triglycerides. Dietary saturated fats were associated, with an increase in the platelet phospholipids not in saturated fatty acids but in 20:3 (n-9), known to promote platelet aggregation to thrombin, with a decrease in platelet cholesterol, also apparently regulating platelet functions. The present studies indicate that dietary saturated fats, calcium (hard water) and alcohol, influence platelet behaviour in a way strictly parallel to their known effect on coronary heart disease.

Comparative beneficial effects on platelet functions and atherosclerosis of dietary linoleic and gamma-linolenic acids in the rabbit.

Male rabbits (10 weeks old) were fed, for 20 weeks, purified diets rich in fat (45% of calories) containing saturated fats (butter), polyunsaturated fats (evening primrose oil + butter or sunflower oil + butter) for 20 weeks. Linoleic acid represented respectively 3.6, 33 and 34% of the dietary fatty acids, while gamma-linolenic acid was present (4.4%) solely in the second group. A significant increase in di-homo-gamma-linolenic acid in plasma, platelets and aorta was noted only in the animals fed evening primrose oil. Despite this, the results of the platelet aggregation to thrombin and ADP, the recalcification plasma-clotting time (platelet-rich plasma) and the severity of atherosclerosis were not significantly different from those observed in the group fed sunflower oil. In contrast, in comparison to the butter-fed animals, the two groups fed the polyunsaturated fats showed remarkable improvements in the clotting time (P less than 0.01) and in the severity of atherosclerotic lesions (evening primrose oil P less than 0.001; sunflower oil P less than 0.05). However, the response to thrombin-induced aggregation was significantly decreased (P less than 0.05) only in the evening primrose oil-fed animals. In these long-term studies in young rabbits, dietary gamma-linolenic acid did not seem to have marked beneficial effects, additional to those induced by linoleic acid, on platelet functions or on atherosclerosis.

Platelet functions in relation to diet and serum lipids in British farmers.

Coagulation and platelet aggregation induced by thrombin, ADP, adrenaline, and collagen were studied in three contrasted groups, each of 20 to 22 middle-aged male farmers. Serum lipids were similar in the three groups. In the west of Scotland group, however, platelet reactivity was significantly greater than in the east of Scotland. This was associated with a dietary intake, evaluated by three different techniques, higher in saturated fat but also lower in polyunsaturated fat and alcohol. Platelet function in the southern England group also correlated with dietary fats and in addition inversely with calcium intake. On an individual basis in the 63 farmers, all the platelet function tests were significantly correlated with the intake of saturated fat regulated by that of calcium and alcohol. The dietary effects on platelets appear to be mediated by the fatty acid composition of plasma lipids and of platelet phospholipids. In that fraction, the fatty acids 20:3 omega 9, 22:3 omega 9 and 20:4 were the most closely related to the platelet function tests. the trienoic acid 20:3 omega 9, identified with essential fatty acid deficiency, was also correlated with the intake of saturated fat and calcium. In this study, platelet functions were more dependent upon the dietary factors associated with coronary heart disease such as saturated fats, calcium, and alcohol than upon serum lipids.

Platelet functions and fatty acid composition of platelet phospholipids in spontaneously hypertensive rats fed saturated or polyunsaturated fats.

Spontaneously hypertensive rats (Okamoto-Wistar) as compared to their normotensive controls (Kyoto-Wistar) presented a markedly higher platelet activity both in coagulation (as evaluated by the recalcification plasma clotting time of platelet-rich plasma) and aggregation, as triggered by thrombin. By comparing animals fed saturated or polyunsaturated fat, it could be observed that the saturated fat diet induced similar results on platelet functions to these observed in hypertension. In addition, the saturated fat diet further increased the platelet response of the hypertensive animals. By contrast, the polyunsaturated fat, could neither reduce the hypertension nor completely abolish the platelet reactivity associated with the hypertension. The most significant change induced by the saturated fat diet in the fatty acid composition of the platelet phospholipids was an increase in 20:3 omega 9, further enhanced in the hypertensive animals. In the polyunsaturated diet-fed rats, it was mostly 20:4 which was more elevated in the platelet phospholipids of the hypertensive. As a result, it was the sum of 20:3 omega 9 + 20:4 in the platelet phospholipids, which appeared to be the most significantly related, in the 4 groups of animals, to the response of platelets to thrombin induced aggregation.

A comparison of the effects of dietary short and long chain saturated fatty acids on platelet functions, platelet phospholipids, and blood coagulation in rats.

Rats were fed for 20 weeks balanced purified diets solely enriched in fat, to determine whether, as in diets containing cholesterol and cholic acid, it was mostly the common dietary long chain saturated fatty acids (16:0 and 18:0) which could induce platelet function abnormalities. The dietary fats rich in 16:0 and 18:0 induced increased thrombin and adenosine diphosphate (ADP) platelet aggregation and an acceleration of the platelet-rich plasma clotting time. These effects on platelet functions were coupled with increased levels of 20:3 omega 6 and 20:3 omega 9 in total platelet phospholipids and a decrease of 22:4 and 24:4 omega 6 in the platelet phosphatidyl ethanolamine. Fatty acids that most closely correlated with thrombin aggregation and plasma clotting time (r = 0.99) were 20:3 omega 9 and 22:4 omega 6. There was no relationship between the effects of dietary fats on blood lipids and on blood platelets. Consequently, in different experimental models in rats, the dietary long chain saturated fatty acids appear to be responsible for certain platelet function abnormalities (clotting activity, response to thrombin aggregation). These platelet abnormalities might be due to changes in the platelet phospholipid fatty acid composition.

Dietary fats and platelet function in French and Scottish farmers.

Although the intake of saturated facts still appears to be the environmental factor most closely associated with coronary heart disease (CHD), this does not necessarily mean that CHD is caused essentially or solely by blood lipids, as suggested by several investigators. It seems that blood platelets rather than (or at least in addition to) blood lipids might be the intermediate link between certain environmental factors (saturated fats, hard water) and CHD, through an effect on both thrombosis and atherosclerosis. Our recent studies in French and Scottish farmers, have shown that blood platelet function is more drastically affected by saturated fats than blood lipids. In those studies, platelet function was the only blood parameter correlated on an individual basis with the intake of saturated fat and inversely related to calcium intake. Calcium is probably the cation responsible for the protective effect of hard water against CHD in various countries. The results obtained also indicate that platelet function can be improved by increasing the intake of polyunsaturated fats at the expense of saturated fats. Finally, only platelet function was different from one region of France to another and from our region of Scotland to another; this difference could be related to the reported incidence of CHD in these various regions.

Influence of a hypolipemic drug (procetofene) on platelet functions and lipid composition in type II B hyperlipoproteinemia.

Platelet function was evaluated in platelet-rich plasma and in washed platelets in relation to their lipid composition, in 18 type II B hyperlipoproteinemic men treated with procetofene. The results were compared to those obtained in 15 young, healthy normolipemic controls and in 22 untreated male subjects with type II B hyperlipoproteinemia. In addition to completely normalizing platelet functions, procetofene was able to induce a 28-53% decrease in serum cholesterol and triglycerides. Solely, the aggregation to ADP (secondary), arachidonic acid and epinephrine was higher in the untreated hyperlipoproteinemic subjects. This increased susceptibility to aggregation does not appear to be related to the cholesterol or cholesterol-phospholipid content of platelets.

Dietary fats and platelet functions in relation to atherosclerosis and coronary heart disease.

Results in animals and in man indicate that in many circumstances, lipemia is not closely related to the severity of atherosclerosis nor to the incidence of coronary heart disease (CHD) or the intake of saturated fats as observed in paired studies between farmers from Moselle and Var in France and from West and East Scotland. In rabbits, an increased response of platelets to thrombin occurs before any deposition of cholesterol, as a result of a saturated fat feeding. Under these conditions, the addition of alcohol to the drinking water decreases significantly both the platelet response to thrombin and the severity of atherosclerotic lesions without much affecting plasma cholesterol. In farmers from Moselle and Var (as well as from Scotland), platelet functions, namely the aggregation to thrombin and their clotting activity, i.e. PF3, are closely related to the intake of saturated fats, either as a result of the long-term feeding or of a 1 year change in the diet of Moselle farmers. Certain platelet functions appear to be the only blood parameter related to the incidence of CHD and significantly correlated on a group, as well as on an individual basis, with the intake of saturated fat, and inversely related with that of calcium. Saturated fats and calcium are known to be the two main dietary factors related to CHD. These results suggest that the intermediate link between dietary fats and CHD might be blood platelets rather than serum lipids, through an effect on both thrombosis and atherosclerosis.