Normolipidemic Individuals Research Articles

Low level quantification of cholesteryl ester transfer protein in plasma subfractions and cell culture media by monoclonal antibody-based immunoassay.

Sensitive immunoradiometric (IRMA) and ELISA assays for cholesteryl ester transfer protein (CETP) have been developed using two different monoclonal antibodies (MAbs). The MAbs were prepared against human plasma CETP and demonstrated specificity by their inhibition of cholesteryl ester transfer activity and by immunoblots of crude plasma fractions and whole media from transfected CHO cells. For these sandwich-type assays, one MAb, 2F8, is used for capture, and the second MAb, 2E7, is iodinated (IRMA) or conjugated with alkaline phosphatase (ELISA) and used for detection. Both assays are linear and provide sensitivities much greater than previously reported. The IRMA allows for the accurate quantification of CETP in the range of 0.5-20 ng/assay (5-200 ng/ml), the ELISA 0.05-5 ng/assay (0.5-50 ng/ml). Using the IRMA, the mean plasma CETP concentration in 44 normolipidemic individuals was determined to be 2.10 +/- 0.36 micrograms/ml; 2.05 +/- 0.33 for males (n = 25) and 2.16 +/- 0.40 for females (n = 19). Values ranged from 1.28 to 2.97 micrograms/ml and CETP mass correlated well with cholesteryl ester transfer activity (r = 0.913, n = 23). The distribution of CETP in human plasma was examined both by gel permeation fast protein liquid chromatography (FPLC) and by native gel electrophoresis. For FPLC using agarose resins, a low ionic strength, isotonic buffer system resulted in near total recoveries of CETP, and demonstrated a peak for CETP mass centered at molecular masses of 150 to 180 kilodaltons, larger than that for free monomeric CETP. Native acrylamide gel electrophoresis of plasma from six individuals, followed by 2F8/2E7 sandwich immunoblotting, showed CETP migrating within a size range of 170-220 kilodaltons. This result is consistent with suggestions that plasma CETP is associated with small-sized HDL. Agarose gel electrophoresis showed plasma CETP, as well as purified recombinant CETP, to be prebeta migrating. For determining the concentration of CETP in the media of cultured HepG2 cells, advantage was taken of the high sensitivity of the ELISA. CETP levels were found to increase linearly over the 100-h culture period, reaching 8.0 +/- 0.4 ng/ml (18.0 +/- 1.3 ng/mg cell protein). These sensitive, direct immunoassays for CETP mass should be valuable aids for examining the behavior of CETP in plasma and other complex systems, as well as for studying the synthesis and secretion of CETP by different cells and tissues.

Apolipoprotein A-II production rate is a major factor regulating the distribution of apolipoprotein A-I among HDL subclasses LpA-I and LpA-I:A-II in normolipidemic humans.

HDLs are heterogeneous in their apolipoprotein composition. Apolipoprotein (apo) A-I and apoA-II are the major proteins found in HDL and form the two major HDL subclasses: those that contain only apoA-I (LpA-I) and those that contain both apoA-I and apoA-II (LpA-I:A-II). Substantial evidence indicates that these two subclasses differ in their in vivo metabolism and effect on atherosclerosis, with LpA-I the more specifically protective subfraction against atherosclerosis. The purpose of this study was to investigate the effect of apoA-I and apoA-II production and catabolism on plasma LpA-I and LpA-I:A-II levels. Fifty normolipidemic subjects (those with HDL cholesterol levels in the top and bottom tenth percentiles were excluded) underwent kinetic studies with radiolabeled apoA-I and apoA-II, and the kinetic parameters of apoA-I and apoA-II were correlated with LpA-I and LpA-I:A-II levels. ApoA-I levels were strongly correlated with apoA-I residence times and less strongly correlated with apoA-I production rates. In contrast, apoA-II levels were correlated only with apoA-II production rates and not with apoA-II residence times. Levels of apoA-I in LpA-I were correlated with apoA-I residence times, whereas levels of apoA-I in LpA-I:A-II were correlated primarily with apoA-II production rates. The fraction of apoA-I in LpA-I was highly inversely correlated with apoA-II production rate (r = -.67, P < .001). In multiple regression analysis, apoA-II production rate was the most significant independent variable determining percent apoA-I in LpA-I among all the kinetic parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma cholesteryl ester transfer in patients with non-insulin dependent diabetes mellitus

Plasma newly synthesised cholesteryl ester transfer (NCET) rates from high density lipoproteins (HDL) to very low density lipoproteins (VLDL) and low density lipoproteins (LDL) were measured in 26 patients with non-insulin dependent diabetes mellitus (NIDDM), 26 healthy subjects with closely matching plasma triglyceride (TG) levels and 10 normolipidaemic healthy individuals. In addition, insulin mediated glucose uptake was measured in the NIDDM patients and the normolipidaemic subjects. Rates of NCET were significantly ( P < 0.05) elevated in NIDDM patients compared with healthy normolipidaemic individuals but were similar to rates in healthy subjects with closely matching TG levels. In all groups of subjects plasma NCET was significantly ( P < 0.001) correlated with plasma TG concentration. In NIDDM patients correlations between NCET and plasma glucose ( r = 0.489, P = 0.011) independently of plasma TG levels, and glycated haemoglobin levels ( r = 0.430, P = 0.028) were also recorded. Insulin mediated glucose uptake was unrelated to plasma NCET rates in the study. These data suggest that in NIDDM patients under good diabetic control elevated plasma NCET rates are mainly due to hypertriglyceridaemia and a specific and possibly stimulatory effect of diabetes on these rates may be seen only in patients with poorly controlled diabetes.

Associations of allelic differences at the A-I/C-III/A-IV gene cluster with carotid artery intima-media thickness and plasma lipid transport in hypercholesterolemic-hypertriglyceridemic humans.

Individuals with elevated levels of plasma cholesterol and triglyceride may be at higher risk for coronary artery disease than those with isolated elevations of either cholesterol or triglyceride. Sequence variation in the A-I/C-III/A-IV gene cluster has been implicated in the etiology of some disorders associated with premature atherosclerosis and/or hypertriglyceridemias with or without elevations of cholesterol. This led to the hypothesis that allelic variation at this gene locus alters plasma lipid transport and affects susceptibility for atherosclerosis. The study population, from the Atherosclerosis Risk in Communities (ARIC) Study, consisted of 50 normolipidemic individuals, 48 subjects with elevated plasma cholesterol, 47 subjects with elevated plasma triglyceride, and 123 subjects with both elevated plasma cholesterol and triglyceride who were used to evaluate associations between an Xmn I polymorphic site 2.5 kilobase pairs (kbp) upstream of the structural gene for apolipoprotein (apo) A-I, intimal-medial thickening of the extracranial carotid arteries, and several plasma lipid factors. The relative allele frequencies of the 8.3-kbp allele and the 6.6-kbp allele were .86 and .14, respectively, in the entire study population and did not differ among the lipid phenotypes. In the group with elevated plasma cholesterol and triglyceride, subjects possessing the 6.6-kbp allele exhibited a greater carotid artery intimal-medial thickness (P = .034) and higher plasma levels of apoA-I, high-density lipoprotein (HDL) cholesterol, and HDL3 cholesterol (P < .02) than subjects homozygous for the 8.3-kbp allele. In contrast, subjects with the 6.6-kbp allele displayed lower mean ratios of apolipoproteins C-II to C-III, C-II to A-IV and E to A-IV in plasma (P < .05) and a lower mean ratio of apolipoprotein C-II to C-III in the triglyceride-rich lipoproteins (P = .026). Sequence variation in or near the genes encoding apolipoproteins A-I, C-III, and A-IV may therefore identify a group of hypercholesterolemic-hypertriglyceridemic persons who are at higher risk for atherosclerosis than others with the same lipoprotein phenotype.

Preferential cholesteryl ester acceptors among the LDL subspecies of subjects with familial hypercholesterolemia.

Elevated cholesteryl ester transfer protein-mediated transfer of cholesteryl ester (CE) from high-density lipoprotein (HDL) to low-density lipoprotein (LDL) may contribute to the atherogenicity of LDL in subjects with familial hypercholesterolemia (FH). To identify the major CE acceptors among LDL subspecies, we investigated the qualitative and quantitative features of CE transfer and exchange to LDL on incubation of plasma under physiological conditions. LDL subspecies were fractionated by density-gradient ultra-centrifugation. Both mass transfer and exchange of HDL CE to and with very-low-density lipoprotein plus intermediate-density lipoprotein and LDL were linear for the first 6 hours of incubation. Thereafter mass transfer ceased, but exchange continued at a comparable rate. The rate of CE mass transfer to apolipoprotein B-containing lipoproteins was significantly enhanced in heterozygous FH subjects compared with normolipidemic individuals (91.6 +/- 28.2 versus 52.9 +/- 19.6 micrograms CE/h per milliliter plasma, FH versus normal subjects, P < .02). In FH subjects the predominant LDL subspecies (LDL 3 and 4, d = 1.029 to 1.050 g/mL) accounted for 59.7 +/- 9.2% of the total CE transferred to LDL from HDL. By contrast, expression of CE mass transfer relative to the mass of each lipoprotein acceptor showed the triglyceride (TG)-rich (10.7% to 17.3%), light LDL subspecies (LDL 1 and 2, d = 1.019 to 1.029 g/mL) to represent the preferential CE acceptors (LDL 1 and 2, 94.8 to 136.5 micrograms CE/mg LDL mass; LDL 3 through 5 [d = 1.029 to 1.063 g/mL], 47.1 to 64.1 micrograms CE/mg LDL mass).(ABSTRACT TRUNCATED AT 250 WORDS)

Screening for familial defective apolipoprotein B-100 with improved U937 monocyte proliferation assay

Frostegård et al. (J Lipid Res 1990;31:37-44) demonstrated that the proliferation of the human monocyte cell line U937 is critically dependent on the uptake of low-density lipoprotein (LDL) via the apo B, E (LDL) receptor, a characteristic that was used to detect patients with familial defective apolipoprotein B-100 (FDB). Here we applied this principle to develop a simple and reproducible assay for the detection of patients with functionally defective LDL. We added serum to U937 cells in cholesterol-free incubation medium and determined the increase in cell number after a 72-h incubation at 37 degrees C by using an electronic cell counter. Sera from 10 normolipidemic individuals and from 34 patients with type IIa hyperlipoproteinemia stimulated the growth of U937 cells in proportion to the exogenous cholesterol concentration (r = 0.83, P < 0.001) and the LDL-cholesterol concentration (r = 0.81, P < 0.001). However, sera from 16 patients with FDB stimulated less cell proliferation than did sera from patients with type IIa hyperlipoproteinemia with equal LDL-cholesterol concentrations. With a 15% reduction in growth as the cutoff value, this test had a sensitivity and specificity for diagnosis of FDB of 87.5% and 100%, respectively. The improved U937 monocyte proliferation assay can be used for screening hypercholesterolemic patients to detect individuals with functionally defective LDL.

Apoprotein B structure and receptor recognition of triglyceride-rich low density lipoprotein (LDL) is modified in small LDL but not in triglyceride-rich LDL of normal size.

We compared the effect of lipid composition and particle size of triglyceride-rich low density lipoprotein (LDL) upon apoprotein B conformation and binding to the LDL receptor. Three groups of triglyceride-rich LDL were studied: (a) LDL isolated from chronic hypertriglyceridemic individuals (HTG-LDL); (b) normal LDL made triglyceride-rich by in vitro incubation with triglyceride emulsion and the neutral lipid transfer protein (R-LDL); and (c) LDL from normolipidemic individuals made acutely hypertriglyceridemic by intravenous infusion of 10% Intralipid (IV-LDL). HTG-LDL was small and dense, whereas R-LDL and IV-LDL had normal size. HTG-LDL, but not R-LDL or IV-LDL, exhibited decreased binding to the LDL receptor on human skin fibroblasts in studies at 4 degrees C and reduced degradation at 37 degrees C. Apoprotein B conformation was assessed by circular dichroism and by analyzing the immunoreactivity of different monoclonal antibodies. HTG-LDL but not R-LDL or IV-LDL showed a change in the CD spectra and a consistent decrease in the immunoreactivity of monoclonal antibody 3F5 (2.5-fold) which recognizes an epitope adjacent to the receptor binding domain of apoprotein B. These findings suggest that in triglyceride-rich LDL, the relative content of neutral lipid in the core of LDL in the absence of changes in the size of the particle does not significantly affect apoprotein B conformation or its affinity for the LDL receptor.

Variability in cholesterol content and physical properties of lipoproteins containing apolipoprotein B-100

The primary objective of this study was to determine the variability in cholesterol carrying capacity of low density lipoproteins (LDLs) and other apolipoprotein B (apo B)-containing lipoproteins in normolipidemic men. One hundred and fifty-nine normolipidemic men, ages 21 to 73 years, were enrolled. In addition to determining plasma lipids and lipopropoteins, three primary measurements were made: ratios of cholesterol to apo B in LDL; the electrophoretic pattern of LDL, i.e. pattern A, AB, or B; and levels of cholesterol in all lipoproteins other than high density lipoproteins (nonHDL-cholesterol) along with total apo B. First, the data revealed that about 85% of the variability of LDL-cholesterol levels can be accounted for by LDL-apo B levels, whereas the remaining 15% can be explained by differences in LDL-cholesterol/apo B ratios. Second, LDL electrophoretic pattern A was the predominant pattern in young adult men, but in older men the pattern shifted increasingly to AB and B. And third, there was a high correlation between nonHDL-cholesterol levels and total apo B levels, which suggests that nonHDL-cholesterol can be used as a relatively accurate surrogate for total apo B levels in normolipidemic individuals.

Determination of lipid transfer inhibitor protein activity in human lipoprotein-deficient plasma.

Lipid transfer protein (LTP) activity is modulated by a distinct plasma protein termed lipid transfer inhibitor protein (LTIP). The objective of this study was to establish an assay for LTIP that could be used to quantify its activity in lipoprotein-deficient plasma. A straightforward heating protocol (56 degrees C for 60 minutes) was found to inactivate more than 90% of LTIP activity. The responses of individual lipoprotein-deficient plasma samples to this heating procedure were unique. Among normolipidemic donors, inactivation of LTIP caused a 230% to 600% increase in LTP activity. Essentially all measurable transfer activity in native and heated samples was inhibited by an antibody to LTP. Whole-plasma samples from these donors were spiked with radiolabeled lipoproteins to measure the rates of lipid transfer among the major lipoprotein classes. In general, plasma lipid transfer rates were negatively correlated with LTIP activity in these samples. However, the decrease in lipid transfers from very-low-density lipoprotein (VLDL) to low-density lipoprotein (LDL) and from LDL to VLDL was from 2.4- to 5.1-fold greater than in the transfers from VLDL to high-density lipoprotein (HDL) or from HDL to VLDL. In these samples, the molecular weight of HDL2 was negatively correlated with LTIP activity. Thus, LTIP activities among normolipidemic individuals were observed to vary severalfold; compared with other lipoprotein transfers, higher LTIP activities were associated with a relative reduction in LDL-VLDL lipid transfer events.

Investigations into the haemorheological significance of postprandial and fasting hypertriglyceridaemia.

Two study designs were conceived to evaluate the rheological significance of hypertriglyceridaemia. We first investigated the course of serum- (SV) and plasma viscosity (PV) and erythrocyte aggregation in serum (SEA) and plasma (PEA) of healthy normolipidaemic individuals over 4 h after a fatty rich meal, in native material and after removal of triglyceride rich lipoproteins by centrifugation. Secondly, blood from patients with untreated hypertriglyceridaemia was investigated under fasting conditions. PEA and SEA increased in parallel with postprandial triglycerides (+135 mg dl-1), but the effect on PEA was more pronounced (+0.8 abs% increase; 2 h after the meal) as compared to SEA (+0.4 abs% increase). PV and SV increased in parallel to the same extent (+0.05 mPas). In the triglyceride poor infranatant no significant changes occurred. In fasting plasma PEA and PV were significantly lower (1.1 abs% and PV 0.04 mPas respectively) in infranatant than in native plasma, while only small differences in triglyceride (mostly VLDL) were observed. This phenomenon was barely detectable in serum samples. We conclude that triglyceride rich lipoproteins have a profound influence on haemorheological parameters, and that fibrinogen in particular, potentiates the effect of large fasting VLDL on plasma viscosity and erythrocyte aggregation.

The effect of the apolipoprotein E polymorphism on lipid levels in patients with familial defective apolipoprotein B-100.

Serum lipid concentrations of patients with familial defective apolipoprotein B-100 (FDB) show a high interindividual variability although the underlying defect is caused by a single point mutation. On the other hand, several genetic factors modulating serum cholesterol levels are known, such as DNA polymorphisms of the apolipoprotein B or the apolipoprotein E (apo E) gene. To assess the effect of the apo E polymorphism on serum cholesterol, lipid levels of FDB patients (n = 36) were compared with those of a normolipidemic control group (n = 272) according to their apo E genotype. For the FDB group mean values of low-density lipoprotein (LDL) cholesterol (mg/dl) were 225.7 +/- 53.7 for E3/2 genotype (n = 3), 234.2 +/- 48.3 for E3/3 genotype (n = 20), and 252.4 +/- 73.8 for E4/3 genotype (n = 13). Means of triglycerides (mg/dl) were 121.0 +/- 21.2, 114.8 +/- 60.7, and 110.0 +/- 62.8 for the respective apo E genotypes. The calculated average effect of the apo E alleles on LDL cholesterol levels was -6.0% for allele e2 and +3.7% for e4 relative to the whole FDB group. The effect on triglyceride levels was +7.5% for e2 and -3.6% for e4. The control group showed a similar variation in LDL cholesterol depending on the different apo E genotypes. About 6% of the total variation in LDL cholesterol can be accounted for by the apo E locus in normolipidemic and hypercholesterolemic individuals alike.

Effect of hepatic lipase on LDL in normal men and those with coronary artery disease.

Hepatic triglyceride lipase (HL) is thought to play a role in the formation of low density lipoproteins (LDLs) from small very low density lipoproteins (VLDLs) and intermediate density lipoproteins (IDLs). To analyze the possible physiological role of HL in determining LDL buoyancy, size, and chemical composition, HL activity and LDL were studied in 21 patients with coronary artery disease (CAD) and 23 normolipidemic subjects. In both groups, LDL buoyancy and size were inversely associated with HL activity levels. The effect of HL on LDL size was comparable in CAD patients and in normolipidemic subjects. HL appeared to influence LDL lipid composition primarily by affecting the surface lipid components. The free cholesterol content of LDL particles was highly correlated with HL activity in both CAD and normolipidemic individuals. The free cholesterol to phospholipid ratio in LDL particles correlated with HL in both CAD and normolipidemic subjects. When the individuals were separated according to their LDL subclass patterns, pattern B subjects had significantly higher HL than pattern A subjects in both CAD and normolipidemic groups. The analysis of the cholesterol distribution profiles across the lipoprotein density gradient confirmed that LDL buoyancy is affected by HL. These data support the hypothesis that HL modulates the physical and compositional properties of LDL and contributes to the expression of the LDL subclass phenotype, suggesting a physiological role for HL in LDL metabolism.

Mechanism of plasma cholesteryl ester transfer in hypertriglyceridemia.

Plasma net cholesteryl ester (CE) transfer and optimum cholesteryl ester transfer protein (CETP) activity were determined in primary hypertriglyceridemic (n = 11) and normolipidemic (n = 15) individuals. The hypertriglyceridemic group demonstrated threefold greater net CE transfer leading to enhanced accumulation of CE in VLDL. This increased net transfer was not accompanied by a change in CETP activity. In normolipidemia, but not in hypertriglyceridemia, net CE transfer correlated with VLDL triglyceride (r = 0.92, P less than 0.001). In contrast, net CE transfer in hypertriglyceridemia, but not in normolipidemia, correlated with CETP activity (r = 0.73, P less than 0.01). Correction of hypertriglyceridemia with bezafibrate reduced net CE transfer towards normal and restored the correlation with VLDL triglyceride (r = 0.90, P less than 0.005) while suppressing the correlation with CETP activity. That net CE transfer depends on VLDL concentration was confirmed by an increase of net CE transfer in normolipidemic plasma supplemented with purified VLDL. Supplementation of purified CETP to normolipidemic plasma did not stimulate net CE transfer. In contrast, net CE transfer was enhanced by addition of CETP to both plasma supplemented with VLDL and hypertriglyceridemic plasma. Thus, in normal subjects, VLDL concentration determines the rate of net CE transfer. CETP becomes rate limiting as VLDL concentration increases, i.e., in hypertriglyceridemia.

Apolipoprotein E polymorphism affects plasma levels of lipoprotein(a)

In a group of 303 healthy Caucasian adults of both sexes we studied the influence of the apolipoprotein E (apo E) polymorphism on plasma levels of Lipoprotein(a) (Lp(a)). The APOE ∗2 allele was found to decrease the mean plasma Lp(a) level by 24.8%, whereas the APOE ∗4 allele increased the mean Lp(a) level by 25.7%. These effects were parallel to the effect of apo E polymorphism on plasma cholesterol and low density lipoprotein (LDL)-cholesterol. For the Lp(a) levels, the genetic variance associated with the APOE locus contributed about 4% to the total phenotypic variance. For plasma cholesterol and LDL-cholesterol this contribution was 4.5 and 6.3%, respectively. We also found a significant positive correlation between LDL-cholesterol and Lp(a) levels. Since the apo E polymorphism effects LDL-receptor activity, we conclude that, at least in healthy normolipidemic individuals, plasma levels of Lp(a) are modulated by the LDL-receptor activity.

Varying susceptibility of different low density lipoproteins to oxidative modification.

The oxidative modification of low density lipoprotein (LDL) may provide a crucial link between plasma LDL and the atherosclerotic lesion. The studies presented herein define time-dependent modifications of LDL constituents caused by CuSO4-catalyzed oxidation. Measurement of the cholesterol content of oxidized LDL by the cholesterol esterase-oxidase assay was found to be inaccurate. The enzymatic assay detected oxysterols as well as cholesterol and thus substantially overestimated the actual cholesterol content. Alteration of electrophoretic mobility and conversion of sterols into oxysterols increased in a parallel, time-dependent manner. Lipid peroxidation, judged by the thiobarbituric acid-reacting substances assay, increased early to maximal values but was not linearly related to either electrophoretic mobility or to oxysterol formation. Neither electrophoretic mobility nor oxysterol formation varied much between repeated oxidative modifications of any given LDL preparation but varied markedly among LDLs from different normolipidemic individuals, suggesting that LDL particles contain some factor conferring susceptibility or resistance to oxidation. Indeed, LDL preparations from different individuals have varying susceptibilities to oxidative modification as evidenced by the three indexes used. The major oxysterol generated was 7-ketocholesterol. Macrophage modification of LDL also resulted in the generation of oxysterols. Thus, measurement of oxysterols may afford an additional index of the oxidative modification of LDL. Since incubation of macrophages with oxidized LDL but not native LDL resulted in the accumulation of oxysterols, this could account for some of the toxic and metabolic effects of oxidized LDL on cells.

Variation in the low density lipoprotein receptor gene is associated with differences in plasma low density lipoprotein cholesterol levels in young and old normal individuals from Italy.

We have used four restriction fragment length polymorphisms (RFLPs) of the human low density lipoprotein receptor (LDL-R) gene, detected by the restriction enzymes Ava II, Pvu II, and ApaLI (5' and 3'), to study the effect of variation at this gene locus in determining plasma cholesterol and LDL cholesterol levels. Two hundred eighty-nine normolipidemic individuals were studied from the Liguria region of Italy. The Pvu II RFLP was significantly associated with differences in plasma total and LDL cholesterol levels, explaining 9.6% of the sample variance in LDL cholesterol levels. The other RFLPs, which are in strong linkage disequilibrium with the Pvu II RFLP, were associated with smaller effects on LDL cholesterol. The Pvu II allele, distinguished by the presence of the variable cutting site (P2 allele), was associated with lower levels of total and LDL cholesterol, and the frequency of the P2 allele was significantly reduced in individuals with LDL cholesterol levels higher than the 75th percentile. The frequency of the P2 allele was significantly higher in the group of individuals over 65 years old, and in this group the P2 allele was also associated with a similar reduction in LDL cholesterol levels. Because of linkage disequilibrium, only four RFLP haplotypes were common in this sample. Of these, only the haplotype P2A2VI (relative frequency, 0.269) was associated with a reduction in LDL cholesterol (average excess, -11.5 mg/dl).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of n-3 and n-6 fatty acid-enriched diets on plasma lipoproteins and apolipoproteins in heterozygous familial hypercholesterolemia.

Polyunsaturated fatty acids in vegetable (n-6) and marine (n-3) oils have been shown to reduce cholesterol levels in normolipidemic individuals. However, there is relatively little information available on the lipoprotein responses to dietary n-6 and n-3 fatty acids in individuals with genetic forms of hyperlipidemia at risk for premature cardiovascular disease. We studied five subjects with heterozygous familial hypercholesterolemia (FH), as well as five normal controls, on three rigidly controlled diets differing primarily in their fatty acid composition. FH subjects reduced their total plasma cholesterol by 34% during the n-3 diet and by 26% with the n-6 diet (both p less than 0.001) when compared with values while on a butter diet. In addition, low density lipoprotein (LDL) cholesterol fell 31% and 29% (both p less than 0.001), and apolipoprotein B (apo B) levels dropped 28% and 27% (both p less than 0.01) during the n-3 and n-6 diets, respectively. A significant reduction of total and LDL cholesterol as well as of apo B was also noted in normal controls during n-3 and n-6 diets. Total plasma triglyceride and high density lipoprotein cholesterol fell significantly during n-3 diets in normal and FH subjects. Thus, FH and normal subjects respond in a similar fashion to diets low in saturated fatty acids and rich in n-3 and n-6, with decreased LDL cholesterol and apo B concentrations.

デキストラン硫酸-セルロースカラム(DS)によるリポ蛋白, Lp(a), RLP(Remnant like particles)の吸着能について

LDL-apheresis with a dextran sulfate-cellulose adsorption column (Liposorber) is a popular method for treating hypercholesterolemia. In the present study, we analyzed various lipoprotein particles which were adsorbed during the treatment of hypercholesterolemia using a Liposorber, and in an in vitro column operation.LDL apheresis with a 3-liter plasma processing capacity was conducted on patients with hypercholesterolemia. In addition, 3ml each of sera from normolipidemic individuals and patients before or after LDL apheresis were allowed to pass through a dextran sulfate-cellulose column with a bed volume of 7ml; or were incubated with the absorbent body to evaluate the concentration of lipids and apoproteins, the composition of lipoprotein particles, size, and the effects on Lp (a) and RLP. The particle size was determined under electron microscopy; Lp (a), using a kit by Biopool; and RLP, with an anti-apo B-100 monoclonal antibody adsorption column.Compared with the data before LDL apheresis (100%), the samples were examined during the processing of 1.5-liter plasma (a mid-stage) and during the processing of 3-liter plasma (postapheresis). The results were: plasma chol., 51% and 30%; plasma TG, 40% and 27%; plasma PL, 57% and 37%; apo B, 78% and 44%; apo E, 41% and 28%; and apo A-I, 73% and 68%. Both Lp (a) and RLP showed a marked reduction through the process [Lp (a); (before) 207.6mg/dl (100%)—(midstage) 110mg/dl (53%)—(post-apheresis) 79mg/dl (38%); RLP; (before) 59.4mg/dl (100%)—(midstage) 12.9mg/dl (21%)—(post-apheresis) 10.5mg/dl (17%)]. With the in vitro column operation, more than 90% of the Lp (a) and RLP were removed from the sera. Furthermore, the sizes of the particles adsorbed in the column appeared to be more heterogeneous and larger at the start than at the completion of the apheretic procedure. The apoprotein composition of the bound fraction of chylomicron (SF>400) was determined by SDS PAGE. The fraction of chylomicron bound to the absorbent body contained apo B-48.It was concluded that the dextran sulfatecellulose column provides superior LDL-C adsorption removal; and is simultaneously effective in adsorbing Lp (a), RLP (presumably remnant particles), and apo B-48-containing particles.

Characterization of apolipoprotein B-containing lipoproteins separated by preparative free flow isotachophoresis.

Preparative free flow isotachophoresis (ITP) was used for the fractionation of apoB-containing lipoproteins (d less than 1.063 g/ml) from fasting and postprandial sera derived from normolipidemic individuals. According to their net electric mobility, four major particle groups (I-IV) have been recognized. The fast-migrating particles in group I, which correspond predominantly to very low density lipoproteins (VLDL), are rich in triglycerides, free cholesterol, phosphatidylcholine, and apoE and C apolipoproteins. This group expresses nonspecific binding to fibroblasts but binds to HepG2 cells with high affinity (KD = 3.6 micrograms/ml, Bmax = 37 ng) to a single class of binding sites. The particles migrating in group II, which are related to intermediate density lipoproteins (IDL), are richer in cholesteryl esters and apoB than those in group I. They interact specifically with a single site on fibroblasts (KD = 7.8 micrograms/ml, Bmax = 54 ng) while on HepG2 cells two binding sites, one with a higher (KD = 3.5 micrograms/ml, Bmax = 22 ng) and one with a lower affinity component (KD = 16.9 micrograms/ml, Bmax = 53 ng), are involved. The particles migrating in groups III and IV correspond to low density lipoproteins (LDL). The protein moiety of both fractions consists almost exclusively of apoB. Group III represents cholesteryl ester-rich LDL particles, while the particles in group IV contain smaller amounts of cholesteryl esters. The lipoproteins of both groups are ligands for apoB,E-receptors. However, the particles in group IV interact with fibroblasts with the highest affinity (KD = 2.3 micrograms/ml, Bmax = 58 ng) and with the biphasic HepG2 cell binding sites with the lowest affinity of all analyzed groups (KD1 = 11.2 micrograms/ml, Bmax1 = 58 ng, KD2 = 68 micrograms/ml, Bmax2 = 170 ng). When apoB-containing lipoproteins were isolated from postprandial sera of the same individuals, significant changes in the lipid composition were observed only in particle groups I and II, where the triglyceride and phospholipid content was enhanced. Group I particles from postprandial serum bind to HepG2 cells with a higher affinity (KD = 2.5 micrograms/ml) than group I particles from fasting serum. Postprandial group II particles bind with the same affinity to the biphasic HepG2 cell receptor as fasting group II particles, while the affinities of postprandial group III (KD1 = 4.1 micrograms/ml, KD1 = 47 micrograms/ml) and group IV particles (KD1 = 3.9 micrograms/ml, KD2 = 38 micrograms/ml) to the high affinity binding site of the biphasic receptor are enhanced.(ABSTRACT TRUNCATED AT 400 WORDS)

Lipid peroxides, glutathione peroxidase, high density lipoprotein subfractions and apolipoproteins in young adults

Lipid peroxides have been implicated in the initiation of atherogenesis. In this study of 23 young healthy subjects, high levels of serum lipid peroxides were associated with low serum HDL2 cholesterol concentrations and low HDL2/HDL3 cholesterol and apolipoprotein A-I/A-II ratios. Low levels of LDL cholesterol and apolipoprotein B, and low apolipoprotein B/A-I ratios were typical of subjects with high serum glutathione peroxidase activities. Serum lipid peroxide levels varied widely in subjects without abnormal cholesterol, HDL cholesterol or LDL cholesterol concentrations. This variation may play a role in the frequent occurrence of coronary artery disease in normolipidaemic individuals.