Antithrombotic effects of (n-3) polyunsaturated fatty acids in rat models of arterial and venous thrombosis.

The antithrombotic effects of dietary lipids were investigated in rat models of arterial and venous thrombosis. In the arterial model, thrombus formation was evaluated by determination of the occlusion time and the deposition of 111In-labeled platelets and 125I-labeled fibrinogen in a collagen-coated glass capillary inserted into an arterio-arterial shunt. Venous thrombosis was evaluated by measurement of the thrombus weight after administration of thromboplastin as a source of tissue factor and establishment of stasis in the vena cava. Diets were supplemented with saturated (SAT group) or (n-3) fatty acids, the latter being added either as MaxEPA oil (MaxEPA group), or as docosahexaenoic (DHA group) or eicosapentaenoic (EPA group) ethyl ester. Only the MaxEPA group displayed a prolonged occlusion time as compared with all other groups. Platelet accumulation, similar in the MaxEPA, EPA and DHA groups (13.3, 16.7 and 17.7 x 10(6) platelets/shunt, respectively), was significantly higher in the SAT group (25.3 x 10(6) platelets/shunt), while accumulation of fibrinogen-fibrin was similar whatever the group. There was a trend towards a lower venous thrombus weight in MaxEPA fed rats relative to those fed other diets. Our data indicate that the MaxEPA diet had antithrombotic effects in arterial and to a lesser extent venous thrombosis models, best attributed to its multiple targeting of platelets and coagulation.

Simultaneous determination of homologues of vitamin E and coenzyme Q and products of alpha-tocopherol oxidation.

A sensitive procedure is described for the simultaneous determination of vitamin E and coenzyme Q homologues and alpha-tocopherol oxidation products using two-isocratic step high pressure liquid chromatography (HPLC) and electrochemical detection in the oxidative mode. Zinc-catalyzed reduction in a post-column reactor allows the detection of alpha-tocopherolquinone, epoxy-tocopherolquinone, and ubiquinones. This technique was used to quantify lipophilic antioxidants in the liver tissue of rats treated or not with alpha-tocopherolquinone and in a plant oil. Alpha-tocopherolquinone and its epoxide derivatives, formed from alpha-tocopherol during iron-catalyzed phospholipid peroxidation, were also determined in a liposome suspension. The high selectivity and sensitivity of the coulometric detection system enabled use of low oxidation potentials giving little baseline noise, while a fast isolation procedure and quantitative recoveries of all oxidized and reduced forms made it possible to measure a high ubiquinol/ubiquinone ratio in liver tissue. Administration of alpha-tocopherolquinone to rats did not alter the antioxidant status of the liver, despite strong accumulation of both this quinone and its reduced form, alpha-tocopherolhydroquinone. These results indicate the presence of an efficient reductase and suggest that it could contribute to the protection of cellular membranes from oxidative stress.

Diets enriched in (n-3) fatty acids affect rat coagulation factors dependent on vitamin K.

The effects of dietary lipids on haemostasis were investigated in rats fed high fat diets enriched in saturated fatty acids (SAT), oleic acid (OLEIC), MaxEPA oil (MaxEPA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) and results were compared to those for rats fed standard chow (ST). Coagulant activities of factor IIc and factor VII-Xc were reduced by about 70% in the MaxEPA group and 50% in the EPA and DHA groups relative to the OLEIC, SAT and ST groups. Liver vitamin K levels were five times lower in the experimental groups than in the ST group, which would indicate an effect of high fat diets on vitamin K metabolism. However, only (n-3) fatty acids prolonged the prothrombin time. These components could act at the post-translational modification level of vitamin K-dependent plasma clotting factors. The changes in haemostatic factors found in the MaxEPA group were counteracted by vitamin K supplementation.

Quantitative analysis of vitamin E, cholesterol and phospholipid fatty acids in a single aliquot of human platelets and cultured endothelial cells.

A reliable procedure is described for the joint analysis of vitamin E (tocopherols), cholesterol and phospholipids in the same minute sample of human platelets and on human cultured endothelial cells. The whole procedure is based on the extraction of total lipids, thin-layer chromatography of all compounds of interest and microcolumn purification of tocopherols and cholesterol. The combined use of butyl hydroxytoluene and ascorbic acid in the purification steps allowed a complete recovery of the tocopherols analyzed, as well as of cholesterol by high-performance liquid chromatography. The detection of these lipids was performed with fluorometric, spectrophotometric and evaporative light-scattering detectors whose respective sensitivities were compared. The fatty acid composition of phospholipid classes from the same sample, separated on the same silica gel plate, was determined by gas-liquid chromatography. The whole procedure is rapid since it requires about 4 h to analyse tocopherols and cholesterol and to prepare methylated fatty acids, 28 samples being easily completed within one working day. The evaluation of the whole membrane antioxidant status requires as little as one 25 cm2 confluent culture flask (about 0.75 x 10(6) cells) for endothelial cells or two ml of blood (3 x 10(8) platelets).

Food deprivation modifies fatty acid partitioning and beta-oxidation capacity in rat liver.

The involvement of lipids under starvation conditions in the shift from the phase of protein sparing (phase II) to the phase of increased protein breakdown (phase III) has been investigated. Plasma and liver were sampled from fed and unfed rats at two distinct stages which were characterized according to the changes in specific loss in daily body mass and nitrogen excretion. In the two groups of food-deprived rats corresponding to phases II and III, the liver concentration of triglycerides (micromol/g) was significantly lower, that of cholesterol significantly higher and that of the other lipid classes was moderately affected compared with concentrations in fed rats. Hepatic phospholipids had significantly higher concentrations (mol/100 mol) of 22:6(n-3) in food-deprived rats than in fed rats. Triglycerides had significantly higher concentrations of stearic and arachidonic acids in livers of both groups of food-deprived rats compared with fed rats. The total activity of carnitine palmitoyl transferase [mmol/(min x liver)] was 48% higher in rats studied at the end of phase II than in fed rats but was similar in fed rats and in rats studied at the beginning of phase III. The total activity of fatty acyl-CoA oxidase was 73% lower only in rats studied at the beginning of phase III when compared with fed rats. Our results indicate that during food deprivation the change in the rate of protein utilization is associated with important qualitative and quantitative alterations of hepatic lipids and oxidative capacity of fatty acids. These modifications appear to be related to the change from a preferential use of lipids to a preferential utilization of proteins.

Diets rich in saturated n-9 and n-3 fatty acids differentially affect the fatty acid composition of phospholipids and function of rat platelets.

The aim of the present study was to determine whether dietary intake of monounsaturated or long chain n-3 fatty acids could be effective in lowering platelet responsiveness through modulation of platelet phospholipid composition. Rats were fed diets containing 20% fat with equal cholesterol and 13a-tocopherol contents. These diets were supplemented with saturated, oleic or n-3 fatty acids, n-3 polyunsaturated fatty acids being added either pure, as eicosapentaenoic and docosahexaenoic ethyl esters, or as MaxEPA oil. Dietary n-3 fatty acids did not affect the oxidation status of plasma lipids. Oleic acid- and saturated fatty acid-rich diets led to similar enrichment of platelet phospholipids in arachidonic acid and to comparable thromboxane A(2) generation on stimulation with collagen or thrombin. Platelets of n-3-fed groups were differently enriched in eicosapentaenoic and docosahexaenoic acids at the expense of arachidonic acid. These groups displayed similar thromboxane A(2) production, although levels were lower than those for groups fed with oleic- or saturated fatty acid-rich diets. Only the MaxEPA diet led to a reduction in platelet reactivity, measurable as a small decrease in the aggregation induced by collagen. This diet was also responsible for a high cholesteroUphospholipid ratio and low a-tocopherol content in platelets. Overall results indicated that (i) only MaxEPA reduced platelet reactivity and (ii) this effect was moderate and apparently unrelated to platelet arachidonic acid content, membrane cholesterol to phospholipid ratio or thromboxane A(2) production.

Stimulation of brown adipose tissue activity in tumor-bearing rats.

Despite anorexia, cancer development is frequently accompanied by an increase of energy expenditure. Considering the pivotal role played by brown adipose tissue (BAT) in the energy metabolism of small mammals, we investigated the functional and compositional modification in BAT of anorexic tumor-bearing (Yoshida sarcoma) and pair-fed control rats. BAT thermogenic activity (assessed by maximal mitochondrial GDP binding) was 1.8-fold greater in tumor-bearing rats than in controls, while the thermogenic capacity (assessed by measurement of uncoupling protein) was unchanged. This suggests that tumor bearing had induced an unmasking of uncoupling protein sites. BAT hypertrophy and hyperplasia, characteristic of full-fledged BAT activation, did not occur. The mitochondrial oxidative capacity of BAT (assessed by cytochrome c oxidase activity) was 1.6-fold lower in tumor-bearing than in control rats. The main compositional modification observed in BAT of tumor-bearing rats was an increase in the saturation of cardiolipin fatty acids. These results suggest that the BAT stimulation induced by tumor bearing after 10 days is almost exclusively functional and that the tissue development is limited, probably by anorexia. However, a suppressive effect of anorexia inhibition by tumor bearing cannot be excluded.