apoC-III In VLDL Research Articles

Effects of eicosapentaenoic acid on plasma lipoprotein subfractions and activities of lecithin: cholesterol acyltransferase and lipid transfer protein

The effects of 12 weeks eicosapentaenoic acid (EPA) administration (2.7 g/day) on plasma lipoprotein subfraction levels and on activities of lecithin: cholesterol acyltransferase (LCAT) and lipid transfer protein (LTP) were investigated. Plasma VLDL-C, VLDL-TG, VLDL-PL, VLDL-apo B, VLDL-apo C-II and VLDL-apo C-III levels were decreased by 32.8% (P less than 0.05), 31.2% (P less than 0.01), 31.5% (P less than 0.05), 32.5% (P less than 0.05), 34.7% (P less than 0.05) and 34.1% (P less than 0.05), respectively. EPA did not change plasma IDL-TC, IDL-TG, IDL-PL and IDL-apo B levels. Plasma large, light LDL (LDL1)-TC, LDL1-PL and LDL1-apo B levels were decreased by EPA by 18.7% (P less than 0.02), 19.1% (P less than 0.01) and 23.3% (P less than 0.01) while LDL1-TG level was not changed. Plasma small, heavy LDL (LDL2)-TC level was increased by 25.7% (P less than 0.02) while LDL2-TG, LDL2-PL and LDL2-apo B levels were not altered. Plasma HDL2-TC, HDL2-TG, HDL2-PL and HDL2-apo A-I levels stayed unchanged by EPA treatment. EPA did not affect plasma HDL3-TC, HDL3-PL and HDL3-apo A-I levels but decreased HDL3-TG level significantly (P less than 0.02). LCAT activity was not altered by EPA. LTP activity was increased by 24.8% at 4 weeks (P less than 0.02) and by 32.1% (P less than 0.001) at 12 weeks EPA treatment. We conclude that EPA reduces plasma large, light LDL levels as well as plasma VLDL amounts and stimulates LTP activities.

Independent regulation of plasma apolipoprotein C-II and C-III concentrations in very low density and high density lipoproteins: implications for the regulation of the catabolism of these lipoproteins.

Apolipoproteins C-II (apoC-II) and C-III (apoC-III) are distributed among all the major lipoprotein classes, particularly very low density (VLDL) and high density lipoproteins (HDL). We have determined concentrations of apoC-II and apoC-III in VLDL and HDL in subjects with a wide range of VLDL triglyceride and HDL cholesterol levels, and correlated these levels with fractional catabolic rates (FCR) of VLDL triglyceride and HDL apolipoprotein A-I (apoA-I). Both apoC-II and apoC-III levels increased in VLDL as VLDL apolipoprotein B (apoB) and triglyceride levels rose. The rate of rise of VLDL apoC-III, however, was approximately 3 times greater than that of apoC-II, and positive correlations were present between the ratio of VLDL apoC-III/apoC-II and both VLDL apoB (r = 0.59; P less than 0.01) and VLDL triglyceride (r = 0.70; P less than 0.005) levels. Univariate analysis demonstrated that the FCR for VLDL triglyceride was inversely related to the ratio of apoC-III/apoC-II in VLDL (r = -0.58; P less than 0.05), although this relationship was not significant in a multivariate analysis. In HDL, concentrations of apoC-III and apoA-I were correlated (r = 0.73; P less than 0.005) while no correlation was observed between apoC-II and apoA-I levels. Univariate analyses of HDL variables revealed inverse correlations between the concentration of apoC-III and the FCR for apoA-I (r = -0.67; P less than 0.005) and between the ratio of apoC-III/apoA-I and the FCR for apoA-I (r = -0.66; P less than 0.005). Multivariate analysis confirmed the latter relationship.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for non-equilibrating pools of apolipoprotein C-III in plasma lipoproteins.

Using immunoaffinity chromatography to isolate apoC-III from radiolabeled lipoproteins for direct determination of specific radioactivity, we have studied the metabolism of human apoC-III in VLDL and in HDL following the bolus injection of 125I-labeled VLDL. Transfer of apoC-III radioactivity from VLDL to HDL was detected in the plasma sample drawn 5 min after injection of the tracer. However, the specific radioactivity of apoC-III in VLDL was found to be higher than that in HDL, with this difference being maintained throughout the sampling period (48-72 hr). The ratios of the respective specific activities ranged from 1.2 to 1.9 in six subjects studied (two normolipidemics and four hypertriglyceridemics). When 125I-labeled HDL was injected as the tracer, however, the higher apoC-III specific radioactivity was associated with the HDL fraction. This lack of complete equilibration of apoC-III between VLDL and HDL in vivo was further characterized by in vitro studies using either 125I-labeled VLDL or 125I-labeled HDL. All incubations were carried out for 3 hr at 37 degrees C followed by 16 hr at 4 degrees C and the apoC-III specific activity in each lipoprotein fraction was directly determined after immunoaffinity chromatography. In a study of plasma from a mildly hypertriglyceridemic subject in which 125I-labeled VLDL was incubated with unlabeled HDL, apoC-III specific activities in VLDL remained 30% greater than that in HDL. When 125I-labeled HDL (from the same subject) was incubated with unlabeled VLDL of apoC-III, final specific activity in VLDL was less than 10% of that of HDL apoC-III. Differences in specific activities were also demonstrated when radiolabeled purified apoC-III was exchanged onto VLDL prior to its incubation with HDL. A consistent difference in apoC-III specific activities in VLDL and HDL was observed after isolation of the particles either by molecular sieve chromatography or by ultracentrifugation. These studies demonstrated that, while the exchange of apoC-III between VLDL and HDL may be very rapid, this equilibration is not complete. Pools of apoC-III that do not participate in the equilibration process are present in both the VLDL and HDL fractions and could account for 30-60% of the total apoC-III mass in each lipoprotein fraction.

Immunonephelometric quantitation of the apolipoprotein C-III in human plasma

A quantitative assay, based on endpoint immunonephelometry, was developed for human apolipoprotein C-III (Apo C-III) in plasma and lipoprotein fractions. The standard curve was constructed either with purified Apo C-III 2 as a primary standard or with plasma as a secondary standard. It was linear between 50 and 400 ng Apo C-III per sample, corresponding to 1 μl undiluted plasma. The intra- and interassay coefficients of variation (CV values) were 2.2 and 6.3%, respectively. The Apo C-III immunoreactivity was not influenced by detergents, denaturants nor by delipidation. The use of a non-ionic detergent (Apovax, 0.1 g/1) avoided the need for organic solvent extraction for plasma containing up to 4 g of triglycerides/1 by reducing the sample turbidity. As measured in 126 normolipidemic subjects, the plasma Apo C-III concentration was 0.118 ± 0.028 g/l (mean ± sd. Apo C-III concentrations were only slightly elevated in patients with Fredrickson type IIa hyperlipoproteinaemia. The Apo C-III levels were nearly 3 times higher in type I, IIb, III and IV patients, while subjects with type V hyperlipaemia had about a 5-fold increase in Apo C-III compared to the healthy. The plasma Apo C-III values were strongly correlated with the plasma triglyceride concentrations ( r = 0.80, n = 201). The Apo C-III distribution among the various lipoprotein fractions showed a higher proportion of Apo C-III in VLDL in hypertriglyceridaemic subjects compared to normolipaemic subjects.

Direct measurement of apoprotein C-III specific activity in 125I-labeled very low density lipoproteins using immunoaffinity chromatography.

We have developed a technique for isolating apoprotein C-III by immunoaffinity chromatography, allowing the measurement of its specific radioactivity in lipoprotein fractions from small plasma samples. IgG specific for apoC-III was purified from goat antisera and bound to Sepharose. One ml of this gel (5 mg of IgG) bound 80-90 micrograms of apoC-III. The specific activity of apoC-III was determined by application of delipidated very low density lipoproteins to 1-ml columns and analysis of the protein eluted at pH 2.5 for mass and radio-activity. The coefficient fo variation for apoC-III specific activity determination from 125I-labeled VLDL was 4.3%. Minimal contamination of the eluates by apoproteins B, E, and C-II was confirmed by radioimmunoassay (0.3-1.2%). Following the injection of autologous 125I-labeled VLDL, specific activity decay curves for VLDL apoC-III were biexponential, with the clearance of apoC-III being slower in hypertriglyceridemic subjects. These affinity columns can be used repeatedly and yield reproducible results. This technique should be useful for simultaneous studies of the turnover of several apoproteins in the same individual following a single injection of labeled autologous lipoprotein.

慢性透析患者におけるIIbおよびIV型高脂血症に対する Pantethine の効果

Pantethine (600mg/day) was administered perorally to hemodialysis patients with type IIb and IV hyperlipidemia for three months. Various lipoprotein parameters before and after the drug administration were determined. VLDL (d< 1.006g/ml) and intermediate density lipoproteins (IDL)(d=1.006-1.019g/ml) were isolated by preparative ultracentrifugation. High density lipoproteins (HDL) were separated by magnesium dextransulfate precipitation method. The measurement of sialic acid in VLDL was performed according to Warren's thiobarbituric acid assay. Lipid levels in VLDL, IDL and HDL were determined by routine methods, and protein concentration in VLDL and IDL by Lowry's method. The percent distribution of apo C-II and apo C-III in VLDL was estimated by Kane's polyacrylamide gel electrophoresis, and apo B levels in VLDL were determined by single radial immunodiffusion method.Consequently, the following results were summarized.1) Sialic acid content in VLDL of hemodialysis patients increased not only in the levels but also in the ratio of sialic acid to protein, while pantethine did not affect these changes. With respect to apoproteins containing sialic acid, apo B levels significantly decreased but the percentage of apo C-III tended to increase after pantethine administration.2) Pantethine normalized an increase in the total amounts of VLDL and IDL of hemodialysis patients.3) Pantethine increased HDL cholesterol and phospholipid levels which usually decreased in hemodialysis patients.These results led us to a conclusion that pantethine had no effect on VLDL sialic acid levels but increased HDL in parallel to the normalization of the deranged VLDL and IDL metabolism, regardless of a decrease in the apo C-II/C-III ratio of VLDL, in hemodialysis patients.

The Degree of Sialylation of ApoC-III Is Altered by Diet

ApoC-III, an apolipoprotein that contains from 0 to 3 or more moles of sialic acid per mole of protein, plays important roles in lipoprotein metabolism. A high carbohydrate diet alters the relative proportions of the variously sialylated isoforms of ApoC-III in the plasma VLDL of man and of rat. Hepatic perfusion experiments in the rat indicate that the diet produces its affects on plasma VLDL by altering the hepatic output of ApoC-III. Diet is a relatively long-term stimulus to VLDL secretion. Our aim was to ascertain whether the changes in ApoC-III sialylation patterns could be reproduced by an acute stimulus to increased hepatic VLDL secretion, i.e., the perfusion of rat livers with media containing high levels of free fatty acid (FFA). Male Sprague-Dawley rats (∼250 g) were fed either Purina rat meal or a high carbohydrate formula for 3 wk. Livers were perfused with Krebs-Ringers bicarbonate, 3% bovine serum albumin containing either no added FFA, or 200 mg of added oleate. ApoB and ApoC-III3 were measured by radioimmunoassay and lipids by enzymatic assays. Lipoproteins were isolated by preparative or zonal ultracentrifugation. The relative proportions of the various isoforms of ApoC-III were quantified by isoelectric focusing. The carbohydrate diet increased the plasma levels of triglycerides and ApoC-III3 ∼ twofold. The diet also increased the proportions of ApoC-III0 relative to total ApoC-III from 33 to 42%, in plasma VLDL (P &amp;lt; 0.02). Perfusates of livers of carbohydrate-fed rats accumulated VLDL-TG and ApoC-III3 at increased rates (93 versus 211, P &amp;lt; 0.001, and 12.7 versus 23.5, P &amp;lt; 0.05, μg/g/h, respectively) and the relative proportions of perfusate VLDL ApoC-lll were altered: ApoC-III0 rose from 37% to 59% and ApoC-III3 fell from 60% to 38% (P &amp;lt; 0.02 for each). Perfusion of livers of control (meal-fed) animals with 200 mg oleate yielded twofold increases in the rates of accumulation of VLDL-TG over meal fed non-FFA perfused controls and changes in gross VLDL composition that resembled those produced by carbohydrate feeding, but ApoC-III3 levels were not increased and the relative proportions of ApoC-III0, ApoC-III, 2 and ApoC-III3 were not altered. Perfusion of livers of carbohydrate-fed rats with oleate produced fivefold and 2.5-fold increases in the rates of accumulation of VLDL-TG and ApoC-III3, respectively. The proportion of ApoC-III isoforms were identical with those seen in the livers of carbohydrate-fed rats perfused without added oleate. Thus, the changes in ApoC-lll sialylation were related more to diet than to the presence of added FFA, i.e., an acute stimulus did not reproduce the changes seen with diet. These data indicate that the sialylation of ApoC-III is under metabolic control.

Alterations in levels and interrelations of plasma apolipoproteins induced by diet

Diets high in carbohydrates increase the rates of hepatic secretion of VLDL in man. VLDL particles are secreted in greater numbers; they also tend to contain more triglycerides and to be larger in size. Comparable changes in intestinal secretion of chylomicrons and VLDL accompany fat absorption. We studied the metabolism of the ApoC apoproteins under these circumstances because in vitro studies suggest that these apoproteins may play important roles in the catabolism of VLDL. ApoA-I and ApoB were also examined because of their importance in HDL and VLDL structure, respectively. The fasting plasmas of 16 normolipemic young adults were examined before and after they had been fed high carbohydrate diets for 4–5 days. ApoA-I and ApoB were measured by radio-immunoassay. The relative proportions of ApoC-II and ApoC-III in VLDL were determined by disc gel electrophoresis. Lipids in plasma and in individual lipoproteins were determined chemically. Lipoproteins were isolated by ultracentrifugal and precipitation methods. VLDL-TG and VLDL-chol and VLDL-protein rose by factors of 2.4, 1.67, and 1.88, respectively. (Final value divided by initial value.) Since TG rose more than the others, VLDL became enriched by TG. LDL-chol fell by a factor of 0.78 while LDL-TG and LDL-ApoB remained constant, i.e., LDL became relatively enriched in TG and ApoB. HDL-TG rose by 1.42 and HDL-chol fell by 0.74, i.e., HDL too became TG enriched. Plasma ApoA-I fell by 0.84. Thus, there were alterations in both the levels and compositions of all lipoproteins. The proportion of ApoC-II relative to ApoC-III in VLDL increased in each subject on the carbohydrate diet (mean rise was 1.55-fold). The increases in ApoC were detectable after as little as 48 hr of diet, and the same results were obtained in two people who were each tested twice on two separate occasions several months apart. Thus, the changes in ApoC were reproducible. Proportions of ApoC-II were greater in S F > 400 than in S F 20–400 subfractions of VLDL on ad lib diets. Proportions of ApoC-II rose in both density subfractions on carbohydrate diets. The changes in both subfractions suggest that rises of ApoC-II in the total VLDL (S F > 20) fraction were not due solely to the accumulation of S F > 400 particles of unaltered ApoC composition in postdiet plasma. The changes following carbohydrate could have been due to secretion of altered lipoproteins or due to diet induced alterations in apoprotein catabolism. Three subjects were given 150 gm of corn oil to drink. Lipoproteins were analyzed, as above, at 0 and 3–12 hr after the drink. In spite of several-fold rises in the S F > 20 TG and proteins, the relative proportions of ApoC-II and ApoC-III remained constant. Thus, an acute dietary fat load and 2–4 days of carbohydrate diets provoked different responses. The fluctuations in the levels and relative proportions of ApoC support the notion that the ApoC group may have importance in vivo in the metabolism of VLDL.