A linoleic acid enriched diet increases serum cholesterol esterification by lecithin:cholesterol acyltransferase in meal-fed rats.

Dietary fats are known to influence the fatty acid profile of plasma lipids, including phospholipids which are substrates of lecithin:cholesterol acyltransferase (LCAT; EC 2.3.1.43), an important enzyme in lipoprotein metabolism. We tested whether the dietary fatty acid profile has an effect on LCAT activity in an animal model. Rats were conditioned to eat two meals per day, which were enriched in either palmitic, oleic or linoleic acids, for 10 weeks. Serum was isolated from blood samples taken prior to the meal. The LCAT activity was determined in two ways: (1) by measuring serum cholesterol esterification rates, which are an estimate of LCAT action on endogenous lipoproteins, and (2) by measuring serum LCAT activity levels with excess exogenous substrates, an estimate of LCAT mass. Animals receiving the linoleic acid diet had lower serum concentrations of unesterified cholesterol and triglycerides, if compared with animals fed oleic acid or palmitic acid diets (p < 0.05). Serum LCAT activity levels (measured with excess exogenous substrates) were not different, but both the absolute and fractional rates of cholesterol esterification were highest on the linoleic acid rich diet (p < 0.01), showing that LCAT action on endogenous lipoproteins is improved. No differences were found in serum apolipoprotein B and A-IV concentrations between the dietary groups. Apolipoprotein A-I levels were lowest in the palmitic acid group (oleic and linoleic > palmitic; p < 0.05), and apolipoprotein E levels were highest in the palmitic acid group (palmitic > oleic and linoleic; p < 0.05). It is concluded that a linoleic acid rich diet may cause increased metabolism of serum cholesterol by LCAT in rats. This effect is not due to elevated serum concentrations of LCAT or of its apolipoprotein activators, but most likely to changes in the chemical composition of endogenous lipoprotein substrates. It remains to be established whether the serum cholesterol esterification rates measured in vitro are related to in vivo rates of reverse cholesterol transport.

The influence of a fish oil-enriched diet on the phospholipid fatty acid turnover in the rabbit red cell membrane in vivo.

The influence of the polyunsaturated fatty acid (PUFA) composition of the diet on the rate of fatty acid turnover of individual phospholipids in the erythrocyte membrane in vivo was studied. Following modification of the fatty acid composition of the membrane phospholipids by the use of a fish oil or a linoleic acid enriched diet, phospholipids--labelled in the unsaturated fatty acid at the 2-position of the glycerol moiety--were introduced into the membrane of freshly isolated rabbit erythrocytes. Thereafter, the labelled erythrocytes were reinjected into the bloodstream of the animal. It appears that, with the exception of 1-palmitoyl,2-linoleoyl phosphatidylcholine, all other phosphatidylcholines disappear faster from the erythrocytes of fish oil-fed rabbits than from the red cells of linoleic acid-fed rabbits. Another parameter, which possibly influences the turnover rates of PUFA containing phospholipids, can be peroxidation. An attempt was made to measure peroxidative damage of lipids in vivo by the introduction of 1-palmitoyl,2-cis-parinaroyl phosphatidylcholine (PnPC)--a probe to measure oxidative stress--into the membrane of freshly isolated erythrocytes, in the same way as is described for the radioactive phospholipids. The data demonstrate that the fluorescent signal from the PnPC decreases at a fast rate which is independent of the dietary conditions.

Dietary non-tocopherol antioxidants present in extra virgin olive oil increase the resistance of low density lipoproteins to oxidation in rabbits.

Consumption of a range of dietary antioxidants may be beneficial in protecting low density lipoprotein (LDL) against oxidative modification, as studies have demonstrated that antioxidants other than vitamin E may also function against oxidation of LDL in vitro. In the present study, the effect of polyphenol antioxidants on the susceptibility of LDL to copper-mediated oxidation was investigated after feeding semi-purified diets to 3 groups of New Zealand white (NZW) rabbits. All diets comprised 40% energy as fat with 17% energy as oleic acid. Dietary fatty acid compositions were identical. Oils with different polyphenol contents were used to provide the dietary source of oleic acid-refined olive oil, extra virgin olive oil and Trisun high oleic sunflower seed oil. Polyphenolic compounds (hydroxytyrosol and p-tyrosol) could only be detected in the extra virgin olive oil. Vitamin E was equalised in all diets. LDL oxidizability in vitro was determined by continuously monitoring the copper-induced formation of conjugated dienes after 6 weeks of experimental diet feeding. The lag phase before demonstrable oxidation occurred was significantly increased in the high polyphenol, extra virgin olive oil group (P < 0.05) when compared with combined results from the low polyphenol group (refined olive oil and Trisun), even though the LDL vitamin E concentration in the high polyphenol group was significantly lower. The rate of conjugated diene formation was not influenced by the presence of dietary polyphenols. Results demonstrate that antioxidants, possibly phenolic compounds which are present only in extra virgin olive oil, may contribute to the endogenous antioxidant capacity of LDL, resulting in an increased resistance to oxidation as determined in vitro.

Comparison of the effects of dietary vitamin E on in vivo and in vitro parameters of lipid peroxidation in the rabbit.

This study has investigated the effect of dietary vitamin E on markers of antioxidant status. Four groups of rabbits received diets containing 30 energy percent (en%) total fat (7.8 en% contributed by linoleic acid) for 12 weeks. D,1-alpha tocopheryl acetate was added to the diets to obtain a range of vitamin E concentrations (49, 114, 179, or 775 tocopherol equivalents per kg diet). Increased vitamin E concentrations were demonstrated in plasma lipoproteins and erythrocyte membranes following supplementation, and dietary effects on lipid peroxidation were investigated by (i) monitoring a fluorescent parinaric acid probe incorporated into erythrocyte membranes in vivo, (ii) determination of malondialdehyde and oxysterols in plasma, and (iii) investigation of the susceptibility of low density lipoprotein (LDL) to copper-induced conjugated diene formation in vitro. No effects of vitamin E were observed on parinaric acid oxidation in vivo or on the accumulation of lipid peroxidation products in plasma, but the resistance of LDL to oxidation in vitro increased significantly as vitamin E was supplemented to the diets. Our results demonstrate that under these dietary conditions (7.8 en% linoleic acid) increasing the vitamin E content of plasma and erythrocytes approximately two-fold does not reduce the level of lipid peroxidation in vivo, indicating sufficient antioxidant capacity on the lowest vitamin E diet. In contrast, LDL became more resistant to an extreme oxidative stress applied in vitro. The relevance of these assays to currently proposed mechanisms of atherosclerosis is discussed.

Positional distribution of stearic acid and oleic acid in a triacylglycerol and dietary calcium concentration determines the apparent absorption of these fatty acids in rats.

In this study the effect of the positional distribution of stearic acid (18:0) and oleic acid (18:1) in a triacylglycerol on absorption of fat, energy and nutrients was investigated in young rats. In addition the effect of dietary calcium on these variables was studied. Forty rats were fed purified diets containing either a fat blend high in 2-oleoyl-distearate or a fat blend high in 1-oleoyl-distearate. Both diets were given at low (0.3 g/100 g) and high (1.0 g/100 g) dietary calcium concentrations. Total fat absorption, expressed as the percentage of fat intake, was significantly lower in rats fed 2-oleoyl-distearate compared with 1-oleoyl-distearate at both dietary calcium concentrations. When expressed as absolute figures, the lower fat absorption in rats fed 2-oleoyl-distearate compared with 1-oleoyl-distearate only reached statistical significance at the high dietary calcium concentration. The reduced absorption of total fat was mainly caused by the lower absorption of stearic acid. The percentage of, but not absolute, absorption of oleic acid and energy were lower in rats fed 2-oleoyl-distearate. Absolute and percentage of calcium absorption were lower in rats fed 2-oleoyl-distearate compared with 1-oleoyl-distearate. Absolute and percentage of magnesium absorption were not significantly affected by the positional distribution of stearic acid and oleic acid in the triacylglycerol, but were decreased at a high dietary calcium concentration. We concluded that the lowered stearic acid absorption from 2-oleoyl-distearate compared with 1-oleoyl-distearate might have been due to the greater formation of insoluble calcium and magnesium soaps in the intestine.

Vitamin E and fatty acid intervention does not attenuate the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbits.

We investigated the effect of different interventions on aortic atherosclerosis in Watanabe rabbits. Four groups of rabbits were fed either an oleic acid-enriched diet (80% of total fat intake) with or without vitamin E supplementation (250 IU/kg) or a diet enriched in linoleic acid with or without vitamin E supplementation for 6 months. At the start of the study, plasma cholesterol concentration was 21.4 +/- 3.6 mmol/L (n = 32). The diets did not influence the mean plasma lipids and lipoprotein concentrations except for HDL cholesterol, which was increased more on the oleic acid-enriched diets than on the linoleic acid-enriched diets. Vitamin E levels in plasma and LDL were increased on the oleic acid diet and reduced on the linoleic acid diet. On the latter diet, supplementation of vitamin E was quantitatively less effective in raising plasma or LDL vitamin E levels. The susceptibility of LDL to oxidation was determined in vitro. Both oleic acid-enriched diets increased the lag time by 140% from baseline. The linoleic acid diet supplemented with vitamin E increased lag time by 59%. Linoleic acid alone, however, decreased the lag time by 30%. Similar but inverse effects were seen on LDL oxidation rate. Thus, intervention protected LDL to oxidation in the following order: oleic acid plus vitamin E > oleic acid > linoleic acid plus vitamin E > linoleic acid. Despite the differences in LDL oxidizability induced by the four experimental diets, assessment of aortic atherosclerosis at the end of the 6-month dietary study period revealed no differences among the four study groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Positional distribution of fatty acids in dietary triglycerides: effects on fasting blood lipoprotein concentrations in humans.

We examined the effect of the positional distribution of fatty acids within dietary triglycerides on serum lipoproteins. Sixty subjects consumed two diets of equal fatty acid composition for 3 wk each. In the palm oil diet 82% of palmitic acid was attached to the outer two carbon atoms of glycerol, and 18% to the middle carbon. In the diet rich in enzymatically modified palm oil these figures were 35% and 65%, respectively. On the modified-fat diet, average lipoprotein concentrations showed nonsignificant (P > 0.13) increases of 0.06 mmol/L for total, 0.03 mmol/L for HDL, and 0.04 mmol/L for LDL cholesterol compared with palm oil. The small increases in total and LDL cholesterol were statistically significant in the men (n = 23) but not in the women (n = 37). The ratio of HDL to LDL cholesterol and serum triglyceride concentrations were unchanged. Thus, a large difference in dietary fatty acid configuration had little effect on lipoprotein concentrations in humans.

In vivo turnover of phospholipids in rabbit erythrocytes.

The rate of phospholipid turnover in erythrocyte membranes in vivo has been studied using a recently developed procedure (Kuypers, F.A., Easton, E.W., van den Hoven, R., Wensing, T., Roelofsen, B., Op den Kamp, J.A.F. and van Deenen, L.L.M. (1985) Biochim. Biophys. Acta 819, 170-178). The technique is based on the application of phospholipid transfer proteins in order to introduce trace amounts of radiolabelled phospholipids in the membrane of isolated erythrocytes, followed by re-injection of the erythrocytes into the bloodstream of the animal. The most abundant species of the phosphatidylcholine (PC) class, 1-palmitoyl,2-linoleoyl PC, has, on the basis of loss of the radioactivity in its fatty acyl part, a relatively high turnover with a half-time value of 1.5 days. Other PC species studied exhibit more moderate turnover rates of about 5 days for 1-palmitoyl,2-oleoyl PC and 1-stearoyl,2-arachidonoyl PC. Dipalmitoyl PC, labelled in the polar headgroup, turns over at a slow rate with a half-time value of 9 days. From these data and the relative abundance of the various species, it can be calculated that, on a daily basis in vivo, about one third of the total PC pool in rabbit erythrocyte membranes is replaced and/or modified by de-/reacylation. The only phosphatidylethanolamine (PE) species studied so far, 1-palmitoyl,2-arachidonoyl PE, appeared to be renewed at a relatively low rate with a half-time value of 12 days. The data demonstrate that the in vivo turnover values of phospholipids in the erythrocyte membrane may depend on their polar head group structure, their localization in the membrane and, to a large extent, on their fatty acid composition.

Absorption of isomeric, palmitic acid-containing triacylglycerols resembling human milk fat in the adult rat.

The effect of the positional distribution of palmitic acid (16:0) in triacylglycerols (TAG) on 16:0 apparent absorption in adult rats was investigated. The rats were fed two diets which contained 30 energy % as fat with identical total fatty acid compositions, both containing 30% 16:0. The Betapol diet contained TAG with 73% of total 16:0 in the sn-2 position, the control diet contained TAG with 6% of total 16:0 in the sn-2 position. After six weeks on these diets, the rats were killed two or six hours after the last meal, and the small intestine was removed, cut into 10-cm segments, and the fatty acid composition of the segment's contents was determined. At both time points the amount of 16:0 in the intestinal segments starting at 40 cm from the stomach was much lower in the animals fed Betapol than in the animals fed the control diet. Overall absorption of 16:0 and stearic acid was significantly greater in the Betapol group. Absorption of oleic and linoleic acid from the small intestine was similar in both groups, although the overall absorption was significantly greater in the animals fed Betapol. Total fat absorption was significantly higher in the Betapol-fed rats than in the control-fed rats. No effect on calcium and nitrogen absorption, on plasma total cholesterol and TAG levels, and on bodyweights (growth) was seen. The data demonstrate that the positional distribution of the fatty acids in the TAG molecule affects the site of absorption in the small intestine and particularly the net absorption of saturated fatty acids.

Activated protein C accelerates clot lysis by virtue of its anticoagulant activity.

The effect of human activated protein C (APC) on t-PA dependent fibrinolysis was studied in vitro using plasma (and whole blood) clot lysis techniques. Clot lysis was monitored by measuring the release of soluble 125I-labelled fibrin degradation products from the clot over time. It was demonstrated that the stimulatory effect of APC on plasma and blood clot lysis was specific for APC and depended on the presence of its active site and Ca2+ ions. Furthermore, the effect depended on the presence of phospholipids in plasma or cells in blood. The presence of pro-urokinase, factor XIII or alpha 2-antiplasmin was not required for the expression of the profibrinolytic effect of APC. Subsequent experiments revealed that the profibrinolytic effect of APC was only observed when thrombin was formed through the coagulation pathway during the initial phase of the clot lysis experiment. It was also shown that the addition of increasing concentrations of thrombin itself could delay the t-PA dependent lysis of clots prepared from Al(OH)3 adsorbed plasma via a mechanism not yet understood. Based on these findings we propose that (a) t-PA dependent lysis of clots prepared from pooled normal plasma is delayed by thrombin generated through the coagulation system, and (b) that by its anticoagulant properties APC blocks this thrombin generation and thereby prevents the delay in clot lysis. Because in this model the profibrinolytic effect of APC is directly related to its anticoagulant properties we predicted and confirmed that other anticoagulants--like heparin--also have profibrinolytic activity. Conversely, procoagulants such as phospholipids can be antifibrinolytic.

Increased n-3 polyunsaturated fatty acid content of red blood cells from fish oil-fed rabbits increases in vitro lipid peroxidation, but decreases hemolysis.

In view of a possible relationship between fish oil, lipid peroxidation, and atherosclerosis, the in vitro lipid peroxidation susceptibility of red blood cells (RBCs) from rabbits on conventional (-FO) and fish oil-enriched diets (+FO) was investigated. The diet caused substantial increases in the RBC concentrations of n-3 polyunsaturated fatty acids (PUFAs), in combination with decreases in the concentration of oleic acid (18:1) and linoleic acid (18:2). Cumene hydroperoxide-induced oxidative stress led to increased overall fatty acid peroxidation in +FO RBCs compared with with -FO RBCs, as quantitated by GLC fatty acid analysis. However, the increased overall susceptibility to lipid peroxidation of +FO RBCs was not reflected in increased peroxidation of every individual fatty acid. This was observed for endogenous arachidonic acid (20:4) as well as, in separate experiments, for exogenously added parinaric acid (PnA). The increased cumene hydroperoxide-induced PUFA oxidation in +FO RBCs was accompanied by a lesser extent of hemolysis. To account for these observations, it is proposed that the increased n-3 PUFA content of +FO RBCs serves as an oxidizable buffer. The present data suggest that oxidation of fatty acids can occur until a critically low level of intact phospholipid in the RBC membrane is reached, after which the membrane destabilizes and hemolysis occurs. At the same time, the PUFA buffer in +FO RBCs could also prevent oxidative damage to specific membrane proteins, which could also help prevent cell lysis.

Protein C and fibrinolysis: a link between coagulation and fibrinolysis.

The effect of purified human activated protein C (APC) on fibrinolysis was studied by using in vitro clot lysis techniques. Clots were formed from citrated blood or plasma (supplemented with 125I-labeled fibrinogen) by adding thrombin and Ca(2+)-ions; lysis of the clots was achieved by the addition of tissue-type plasminogen activator before clot formation. The gradual release of labeled fibrin degradation products from the clot into the supernatant was taken as a measure for the lysis rate. It was demonstrated that the acceleration of clot lysis by APC added before clot formation depends on the presence of Protein S, Ca(2+)-ions and phospholipids. These observations suggest a role of APC as anticoagulant in clot lysis, since the cofactors for the expression of its anticoagulant and profibrinolytic effect are very similar. Indeed, we could demonstrate that the profibrinolytic effect of APC in vitro is associated with reduction of thrombin generation through the coagulation cascade by inactivation of factor VIIIa and factor Va. For instance, APC did not accelerate the lysis of factor X deficient blood clots. More generally, thrombin generation was associated with retarded fibrinolysis in vitro. Consequently anticoagulants such as APC or Heparin are profibrinolytic, whereas pro-coagulants such as phospholipids (in cell-free plasma) inhibit fibrinolysis through the generation of thrombin. Thrombin thus plays a crucial role as a link between coagulation and fibrinolysis. As thrombin is able to inhibit the lysis of blood and plasma clots, and not of purified fibrin clots, we hypothesize that thrombin inhibits lysis through an as yet unidentified mediator in plasma.

Activated protein C increases fibrin clot lysis by neutralization of plasminogen activator inhibitor--no evidence for a cofactor role of protein S.

The effect of purified human activated protein C (APC) on fibrinolysis was studied using a clot lysis system consisting of purified glu-plasminogen, tissue-type plasminogen activator, plasminogen activator inhibitor (released from endothelial cells or blood platelets), fibrinogen, 125I-fibrinogen and thrombin. All proteins were of human origin. In this system APC could increase fibrinolysis in a dose dependent way, without affecting fibrin formation or fibrin crosslinking. However, this profibrinolytic effect of APC could only be observed when plasminogen activator inhibitor (PAI-1) was present. The effect of APC was completely quenched by pretreatment of APC with anti-protein C IgG or di-isopropyl-fluorophosphate. Addition of the cofactors of APC-protein S, Ca2+-ions and phospholipid-alone or in combination did not enhance the profibrinolytic effect of APC. These observations indicate that human APC can accelerate in vitro clot lysis by the inactivation of PAI-1 activity. However, the neutralization of PAI-1 by APC is independent of the presence or absence of protein S, phospholipid and Ca2+-ions.

The interaction of activated protein C and thrombin with the plasminogen activator inhibitor released from human endothelial cells.

The effects of human activated protein C (APC) and thrombin on plasminogen activator inhibitor (PAI-1) released from cultured human umbilical endothelial cells, grown in serum-free 35S-methionine containing medium, were studied in two ways: measurement of PAI-1 activity with an amidolytic assay, and immunoprecipitation of the medium with anti-PAI-1 IgG, anti-protein C IgG or anti-thrombin IgG followed by SDS-PAGE and autoradiography. Addition of APC or thrombin to the endothelial cell conditioned medium results in a time and concentration dependent loss of PAI-1 activity and in the degradation of PAI-1 from 46 kD into a 42 kD product. After incubation of the medium with APC in the presence of cells, an additional band of 95 kD was found, which could be immunoprecipitated with both anti-PAI-1 IgG and anti-protein C IgG, indicating the formation of an APC-PAI-1 complex before degradation occurs. No complex could be detected after incubation of the medium with thrombin in the presence of endothelial cells. Blocking the active sites of APC and thrombin prevented both the formation of APC-PAI-1 complexes and the inactivation and degradation of PAI-1. After removal of the active PAI-1 from the medium, no degradation of the inactive PAI-1 by APC or thrombin could be found. It is concluded that both APC and thrombin react with the active PAI-1, resulting in inactivation and degradation of PAI-1.

The cofactor role of protein S in the acceleration of whole blood clot lysis by activated protein C in vitro.

The effect of purified human activated protein C (APC) and protein S on fibrinolysis was studied by using an in vitro blood clot lysis technique. Blood clots were formed from citrated blood (supplemented with 125I-fibrinogen) by adding thrombin and Ca2+-ions; lysis of the clots was achieved by adding tissue-type plasminogen activator. The release of labeled fibrin degradation products from the clots into the supernatant was followed in time. We clearly demonstrated that APC accelerates whole blood clot lysis in vitro. The effect of APC was completely quenched by antiprotein C IgG, pretreatment of APC with diisopropylfluorophosphate, and preincubation of the blood with antiprotein S IgG. This demonstrates that both the active site of APC and the presence of the cofactor, protein S, are essential for the expression of the profibrinolytic properties. At present, the substrate of APC involved in the regulation of fibrinolysis is not yet known. Analysis of the radiolabeled fibrin degradation products demonstrated that APC had no effect on the fibrin cross-linking capacity of factor XIII.

Differential cytotoxicity of deoxyguanosine and 8-aminoguanosine for human leukemic cell lines and normal bone marrow progenitor cells.

The inhibitory effect of deoxyguanosine (GdR) alone or in combination with the purine nucleoside phosphorylase (PNP) inhibitor, 8-aminoguanosine (AG) was tested on human T, B, cALL and myeloid leukaemia cell lines and on normal human bone marrow haemopoietic progenitor cells. GdR was found to be toxic to T-leukaemia cells. AG (100 microM) alone did not have any inhibitory effect, but when used with GdR (2.5 X 10(-5)M) a synergistic effect was seen towards T cells. Incubation with GdR and AG resulted in a marked decrease in cell viability (greater than 90 per cent in three and greater than 75 per cent in four of 5 T leukaemic cell lines tested at 72 h). This drug combination did not inhibit the growth of non-T leukaemic cells and was also non-toxic to normal bone marrow multipotent progenitor cells (CFU-GEMM) in vitro. Adenosine deaminase (ADA) acts consecutively with PNP in purine degradation. The addition of an ADA inhibitor, deoxycoformycin and deoxyadenosine, however, did not enhance the toxicity of GdR and AG for T cell leukaemia. The possibility of using GdR and AG for in vitro removal of residual T leukaemic blasts with the sparing of normal bone marrow cells, prior to autologous bone marrow transplantation should be further explored.