J.E.M. Groener

Different locations of cholesteryl ester transfer protein and phospholipid transfer protein activities in plasma

Activities of cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) were measured in plasma of four vertebrate species: man, rabbit, pig, and rat. The activities were measured in the absence and presence of antibodies raised against purified human CETP. PLTP activities were present in all four species with highest values in pig (11.7 +/- 1.2 U/ml) and human plasma (9.2 +/- 1.6 U/ml). Considerable lower activities were found in rabbit (3.5 +/- 0.6 U/ml) and rat plasma (1.6 +/- 0.7 U/ml). These activities were not affected significantly by antibody against human CETP. CETP activities could be measured in human (0.23 +/- 0.05 U/ml) and in rabbit plasma (0.19 +/- 0.03 U/ml). CETP activity in human plasma was inhibited over 97% by antibody against human CETP. Plasma was chromatographed on a Superose 6 gel filtration column. Average HDL particle sizes in the four species differed notably and decreased in the order: rat HDL greater than rabbit HDL greater than human HDL greater than pig HDL. A separation of the two lipid transfer activities was evident after gel filtration chromatography. The peak of the PLTP activity coeluted with a fraction of HDL particles with the size of human HDL2 (particle weights 300-375 kDa). CETP activity in human and rabbit plasma coeluted largely with relatively small HDL particles (particle weights 140-180 kDa). These results show that CETP and PLTP activities are located in different macromolecular complexes.

The activity of cholesteryl ester transfer protein is decreased in hypothyroidism: a possible contribution to alterations in high-density lipoproteins.

Abstract. The activity of cholesteryl ester transfer protein is instrumental in the distribution of cholesteryl ester between lipoproteins in plasma. We measured the activity of cholesteryl ester transfer protein in plasma, designated cholesteryl ester transfer activity, as the rate of cholesteryl ester transfer between exogenous radiolabelled low‐density and high‐density lipoproteins. The effect of hypothyroidism on cholesteryl ester transfer activity was investigated in 13 athyreotic patients who were studied in the hypothyroid condition and in the euthyroid state, after they had received triiodothyronine supplementation for 33 to 67 days.

Effect of lipid transfer protein on plasma lipids, apolipoproteins and metabolism of high-density lipoprotein cholesteryl ester in the rat.

The role of human plasma lipid transfer protein (LTP) in lipoprotein metabolism was studied in the rat, a species without endogenous cholesteryl ester and triacylglycerol transfer activity. Partially purified human LTP was injected intravenously into rats. The plasma activity was between 1.5- and 4-fold that of human plasma during the experiments. 6 h after the injection of LTP, a significant increase in serum apoB, and no significant changes in serum total cholesterol, free cholesterol, triacylglycerols, apoA-I, apoE, or apoA-IV were noted. Cholesterol was increased in very-low density and low-density lipoproteins (VLDL and LDL) and decreased in large-sized apoE-rich HDL. ApoA-I-containing particles with a size smaller than in normal rats were present in serum of LTP-treated rats. The mean diameter of HDL particles decreased and apoE, normally present on large-sized HDL, was present on smaller sized particles. The metabolic fate of cholesteryl ester, originally associated with HDL, was studied by injection of [3H]cholesteryl linoleyl ether-labelled apoA-I-rich HDL in the absence and in the presence of LTP. The disappearance of [3H]cholesteryl linoleyl ether, injected as part of apoA-I-rich HDL, from serum was increased in the LTP-treated rats; the t1/2 changed from 3.9 to 2.2 h, resulting in an increased accumulation of [3H]cholesteryl linoleyl ether in the liver. This can be explained by the redistribution of HDL [3H]cholesteryl linoleyl ether to VLDL and LDL in the presence of LTP, leading to the combined contribution of VLDL, LDL and HDL to the hepatic uptake. The present findings show profound effects of LTP on the chemical composition of HDL subspecies, the size of HDL and on the plasma turnover and hepatic uptake of cholesteryl esters originally present in apo A-I-rich HDL.

Cholesteryl Ester Transfer Protein Gene Polymorphism Is a Determinant of HDL Cholesterol and of the Lipoprotein Response to a Lipid-Lowering Diet in Type 1 Diabetes

The TaqlB cholesteryl ester transfer protein (CETP) gene polymorphism (B1B2) is a determinant of HDL cholesterol in nondiabetic populations. Remarkably, this gene effect appears to be modified by environmental factors. We evaluated the effect of this polymorphism on HDL cholesterol levels and on the lipoprotein response to a linoleic acid-enriched, low-cholesterol diet in patients with type 1 diabetes. In 44 consecutive type 1 diabetic patients (35 men), CETP polymorphism, apolipoprotein (apo) E genotype, serum lipoproteins, serum CETP activity (measured with an exogenous substrate assay, n = 30), clinical variables, and a diet history were documented. The 1-year response to diet was assessed in 14 type 1 diabetic patients, including 6 B1B1 and 6 B1B2 individuals. HDL cholesterol was higher in 10 B2B2 than in 14 B1B1 homozygotes (1.63 ± 0.38 vs. 1.24 ± 0.23 mmol/l, P < 0.01). HDL cholesterol, adjusted for triglycerides and smoking, was 0.19 mmol/l higher for each B2 allele present. CETP activity levels were not significantly different between CETP genotypes. Multiple regression analysis showed that VLDL + LDL cholesterol was associated with dietary polyunsaturated: saturated fatty acids ratio (P < 0.02) and total fat intake (P < 0.05) in the B1B1 homozygotes only and tended to be related to the presence of the apo E4 allele (P < 0.10). In response to diet, VLDL + LDL cholesterol fell (P < 0.05) and HDL cholesterol remained unchanged in 6 B1B1 homozygotes. In contrast, VLDL + LDL cholesterol was unaltered and HDL cholesterol decreased (P < 0.05) in 6 B1B2 heterozygotes (P < 0.05 for difference in change in VLDL + LDL/HDL cholesterol ratio). This difference in response was unrelated to the apo E genotype. Thus, the TaqlB CETP gene polymorphism is a strong determinant of HDL cholesterol in type 1 diabetes. This gene effect is unlikely to be explained by a major influence on the serum level of CETP activity, as an indirect measure of CETP mass. Our preliminary data suggest that this polymorphism may be a marker of the lipoprotein response to dietary intervention.

Induction of net mass lipid transfer reactions in plasma by wine consumption with dinner

Abstract. Moderate alcohol consumption is associated with a reduced risk of coronary heart disease. Alcohol may exert protection through its effects on the metabolism of plasma lipoproteins. In the present study we investigated the effects of moderate wine consumption with an evening dinner on lipoprotein composition and parameters of reverse cholesterol transport (plasma lipid transfer reactions and cholesterol esterification) in eight healthy middle‐aged men. Wine consumption, if compared with mineral water, resulted in increased postprandial plasma levels of triglyceride‐(TG)‐rich lipoproteins (P < 0.005 or <0.002 at two different time points) and in increased net mass transfer of cholesterylesters (CE) from high‐density lipoprotein (HDL) to apolipoprotein B‐containing lipoproteins during in vitro incubation of plasma (P < 0.001). Net mass transfer of TG (in the opposite direction) was also significantly increased by wine (P= 0.014). The concentrations of total plasma cholesterol, HDL‐cholesterol and apolipoproteins A‐I, A‐II and B did not change postprandially and were not affected significantly by wine, but the CE TG‐1 in HDL was affected postprandially and decreased by wine consumption. It is concluded that moderate wine consumption with evening dinner induces transfer reactions of CE and TG between HDL and TG‐rich lipoproteins. Due to the fact that wine raises plasma TG, it also causes changes in plasma cholesterol metabolism and lipoprotein composition, without major effects on total plasma cholesterol concentration.

Net mass transfer of cholesteryl esters from low density lipoproteins to high density lipoproteins in plasma from normolipidemic subjects.

Net mass transfer of lipids was measured in plasma from fasted, normolipidemic subjects. The plasma was incubated, and lipoproteins were subsequently separated by polyanion precipitation or density gradient ultracentrifugation. Total cholesterol, unesterified cholesterol, and triglycerides were measured in the isolated lipoprotein fractions. The rate of cholesterol esterification was measured simultaneously. All plasma samples showed an increase in high density lipoprotein (HDL) cholesteryl esters during 1-hour incubations. In most cases, this increase was higher than the cholesteryl ester formation in total plasma due to cholesterol esterification. Therefore, we concluded that a net mass transfer of cholesteryl esters occurred from the very low plus low density lipoprotein (VLDL + LDL) fractions to HDL in plasma from most of the subjects studied. Transfer of triglycerides occurred from VLDL + LDL to HDL in plasma from all subjects. The cholesteryl ester transfer (measured after 1 hour) is not related to the activity of cholesteryl ester transfer protein. Inhibition of cholesterol esterification did not change the direction of cholesteryl ester or triglyceride transfer. Ultracentrifugal separation of plasma lipoproteins revealed that both HDL and VLDL are acceptors of cholesteryl esters and that VLDL donates triglycerides to both LDL and HDL. Removal of VLDL from plasma by ultracentrifugation did not affect the cholesteryl ester transfer from LDL to HDL. We conclude that LDL may act as a donor of cholesteryl esters during incubation of normolipidemic plasma.

Delayed increase in high density lipoprotein-phospholipids after ingestion of a fat load in normolipidemic patients with coronary artery disease.

We studied the effect of a single oral fat load, supplemented with retinyl palmitate (RP), on high density lipoprotein (HDL) lipids in six normolipidemic men with coronary artery disease (CAD) and in six age- and lipid-matched controls. All subjects were selected from a study group which underwent the same protocol 2 years earlier. Post-prandial total plasma lipids, plasma RP levels, and HDL lipids were evaluated at 2-h intervals up till 10 h after the meal. In most subjects the post-prandial response of plasma triglyceride (TG) and plasma RP was identical in the first and second tests. Following the fat load, control subjects showed no change in HDL total cholesterol (TC) or HDL cholesteryl ester (CE) and showed an increase in HDL-TG. CAD subjects however showed a decrease in HDL-TC and HDL-CE and an increase in HDL-TG, similar to the increase in control subjects. In control subjects an increase in HDL phospholipid (PL) was apparent between 0 and 8 h after the fat load. By contrast, in CAD subjects the increase in HDL-PL was only found after as long as 6 h. The magnitude of the post-prandial response of HDL-PL measured during the test was significantly lower in the CAD group. The effects of the fat load on HDL free cholesterol (FC) were similar to the changes in HDL-PL. These data support the hypothesis that PL and FC released during the degradation of chylomicrons as surface remnants are taken up by HDL. This process is clearly delayed in normolipidemic CAD subjects compared with controls. The data suggest that differences in the post-prandial response to an oral fat load in normolipidemic CAD patients and control subjects are not confined to the clearance of TG-rich lipoproteins, but also involve a difference in the uptake of chylomicron surface material by HDL.

Lowering of serum cholesteryl ester transfer protein - But not lecithin:cholesterol acyltransferase-activity levels by hypocholesterolemic drugs in the rabbit

Cholesteryl ester transfer protein (CETP) and lecithin:cholesterol acyltransferase (LCAT) are important factors in the regulation of serum lipoprotein metabolism. Rabbits were fed hypocholesterolemic drugs to investigate the effect on serum CETP and LCAT activity levels. The activities were assayed using exogenous substrate assays and are an estimate of CETP and LCAT mass. Groups of eight rabbits were fed a cholesterol-free diet containing either 0.03% simvastatin or 1% cholestyramine for 6 weeks. For comparison eight rabbits were fed a cholesterol-free control diet without drugs or a diet containing 0.1% cholesterol for 6 weeks. Total serum and lipoprotein triglyceride concentrations were not different after intervention with the hypocholesterolemic drugs or the cholesterol diet. Dietary cholesterol induced higher VLDL, IDL, and LDL cholesterol, as well as serum CETP activity, as expected. Serum LCAT activity showed little change with intervention. Both simvastatin and cholestyramine tended to lead to decreased cholesterol in all lipoprotein fractions and caused a significant decrease in serum CETP activity when compared with the control diet. Both drugs also caused a significant lower LDL particle concentration, as judged from differences in LDL protein levels. Intervention with simvastatin or cholestyramine led to relatively cholesterol-poor LDL. These effects on LDL concentration and composition were opposite from the effects of cholesterol feeding. Differences in the cholesterol contents of VLDL and IDL were comparable with those in LDL. The results suggest that decreasing serum CETP activity levels by treatment with simvastatin or cholestyramine may contribute to lowering of cholesterol apo B-containing lipoproteins. The effects are additional to the well-known increase in hepatic LDL receptor activity, which is likely to be the most important factor in LDL cholesterol lowering by these drugs.

Cholesterol Esterification and Net Mass Transfer of Cholesterylesters and Triglycerides in Plasma From Healthy Subjects and Hyperlipidemic Coronary Heart Disease Patients

Plasma from a variety of mammals, including man, contains lipid transfer proteins (LTP’s) that promote the exchange/transfer of cholesterylesters (CE), triglycerides (TG) and phospholipids (PL) between the plasma lipoproteins VLDL, LDL and HDL. One plasma LTP has been purified to homogeneity’ The gene for this protein has been cloned and sequenced-*.

Lipoprotein Structure and Metabolism During Progression and Regression of Atherosclerosis in Pigs Fed With Fish Oil-Derived Fatty Acids

Evidence in favor of beneficial effects of dietary fish oil on prevention or regression of atherosclerosis has been obtained from epidemiological studies in man, as well as from experimental studies in pigs (1–5). Both human and animal studies have shown that fish oil-derived fatty acids may have several mechanisms of action, resulting in protection against the atherosclerotic proces. Effects have been described to occur on platelet aggregation (6,7), cytokine synthesis by mononuclear cells (8) and on plasma lipoproteins, especially the very low density lipoproteins (9–13). Some studies show beneficial effects on progression of atherosclerosis even in the absence of significant effects on (extremely elevated) plasma low density lipoprotein (LDL) levels (2,3,14), but a complete analysis of the chemical composition of all lipoprotein classes is lacking. The aim of the present study is to evaluate the concentration and chemical composition of plasma lipoproteins during progression and regression of atherosclerosis, induced by dietary interventions. Plasma cholesterol metabolism by lecithin:cholesterol acyltransferase (LCAT) is also emphasized. Lecithimcholesterol acyltransferase (LCAT) plays a major role in the formation of plasma cholesterylesters. Its activity is crucial for normal plasma cholesterol turnover and a specific function in reverse cholesterol transport (e.g. transport of cholesterol out of the arterial wall to the liver) has been proposed (15).