Heterogeneity of human lipoprotein Lp[a]: cytochemical and biochemical studies on the interaction of two Lp[a] species with the LDL receptor

Human Lp[a] can be fractionated into two species with different affinities for lysine-Sepharose. Forty to 81% of the total Lp[a] in the density fraction 1.055-1.15 g/ml from five individuals was retained by this affinity column. The remaining unretained Lp[a] species with no apparently functional lysine binding site was similar to the retained species in its electrophoretic mobility, lipid, protein, and apolipoprotein composition, and the heterogeneity was not related to apo[a] size polymorphism. Interaction of the two species with the low density lipoprotein (LDL) receptor was studied in human fibroblasts. Using gold-labeled lipoproteins and an immunochemical procedure, both Lp[a] species could be located in clusters on the cell surface, but the extent of labeling was far lower than that seen with LDL. Both Lp[a] variants were less effective than LDL in 1) down-regulation of LDL-receptor activity; 2) suppression of cellular sterol synthesis; and 3) stimulation of cholesteryl ester formation in human fibroblasts. Although degradation of both species of Lp[a] by the perfused rat liver was stimulated after estrogen induction of hepatic LDL-receptor activity, the stimulation amounted to only a quarter of that seen with LDL. The heterogeneity of Lp[a] with respect to the ability to bind epsilon-aminocarboxylic acid will need to be considered in studying the physiological role of this lipoprotein. Both Lp[a] species exhibited a similar interaction with the LDL-receptor in vitro, and confirmed previous investigations that Lp[a] is only a poor ligand for the LDL-receptor.     

Interaction of LDL, Lp[a], and reduced Lp[a] with monoclonal antibodies against apoB

Five monoclonal antibodies (2A, 9A, 6B, L3, L7) produced in mice against human apolipoprotein B were investigated by competitive and inhibitive electroimmunoassay (EIA) for their reactivity with low density lipoprotein (LDL), lipoprotein[a] (Lp[a]), and reduced Lp[a]. All of the antibodies reacted with apoB of the different lipoproteins indicated by very similar slopes of the binding curves. None of them gave a positive reaction with apolipoprotein[a]. The amount of apoB required for 50% inhibition of antibody binding varied for the different antibodies and lipoproteins. Antibody 9A showed almost the same affinity for LDL, Lp[a], and reduced Lp[a]. Antibodies 2A and 6B bound about twofold better to LDL and reduced Lp[a] than to untreated Lp[a]. Antibodies L3 and L7 needed nearly threefold higher amounts of Lp[a]-apoB for 50% inhibition of antibody binding than of apoB of LDL and reduced Lp[a]. The amount of apoB required for 50% inhibition of antibody binding was somewhat higher in inhibitive assay than in competitive assay. We suggest that apo[a] covers certain epitopes of apoB in native Lp[a] leading to a reduced reaction with the monoclonal antibodies. However, it could also be that the binding of the [a]antigen to apoB via disulfide bridges causes profound conformational changes of the apoB region exposed to the surface.     

Hereditary and dietary effects on apolipoprotein[a] isoforms and Lp[a] in baboons

Baboons possess Lp[a] that is similar to human Lp[a], including the presence of the unique protein, apo[a]. Baboon apo[a] occurred in at least nine isoforms distinguishable by size. Isoforms were resolved by 3-12% polyacrylamide gradient gel electrophoretic separation of serum proteins, and were detected with baboon apo[a]-specific antibodies. Thirty one different apo[a] isoform phenotypes were detected in a population of 165 unrelated baboons. Identical isoform phenotypes were observed in different samples from individual baboons, and isoform phenotypes were unaffected by changes in diet. In one experiment, 16 baboons were fed a series of five diets differing in amounts of cholesterol and saturated or unsaturated fats. There was no significant effect of diet on serum Lp[a] levels. In another group of baboons (n = 70) controlled for age and dietary history, enrichment of the diet with cholesterol and saturated fat caused a small, but significant (P less than 0.005), increase (means = 0.6 mg/dl) in serum Lp[a] concentration. Analysis of two large sire families suggested that apo[a] isoform patterns and serum Lp[a] concentrations were inherited. Putative parental alleles responsible for specific isoform bands appeared to segregate randomly. Heritability (h2) of serum Lp[a] concentration was estimated to be 0.95 +/- 0.04. We conclude that apo[a] isoform phenotypes and serum Lp[a] concentrations are inherited, and that Lp[a] concentrations are only slightly influenced by diet.     

Influence of amount and type of dietary fat on plasma cholesterol concentrations in goats

Adult, non-pregnant, dry goats were fed diets differing in the amount and type of fat. To modulate the type of dietary fat, rations containing either palm oil or olive oil were used, the amount of fat being 86 g/kg dry matter (DM). To modulate the amount of dietary fat, the oils were replaced by an isoenergetic amount of native corn starch so that the dietary fat concentration dropped to 26 g/kg DM. The high fat diets, when compared with the low fat diet, significantly raised plasma total cholesterol by 91%, high-density lipoprotein (HDL) cholesterol by 100%, triglycerides by 47% and phospholipids by 57%. On an average, dietary palm oil versus olive oil significantly increased plasma total cholesterol by 6%, HDL cholesterol by 9% and phospholipids by 4%, but left plasma triglycerides unchanged. This study shows that plasma lipid concentrations in goats respond to the amount and type of fat in the diet.     

Contribution of the apo[a] phenotype to plasma Lp[a] concentrations shows considerable ethnic variation.

Apolipoprotein[a] polymorphism has been investigated by sodium dodecyl sulfate polyacrylamide (5.37%) gel electrophoresis and immunoblotting using a standardized sample load in four ethnic groups: German, Ghanaian, Chinese, and San (Kalahari Bushmen). A total of 10 different apparent molecular weight (Mr) polymorphs, designated 1 to 10 with increasing Mr, were detected in greater than 99% of all individuals tested (German, 99%; Ghanaian, 99%; Chinese, 100%; San 100%). A null allele is therefore at most an infrequent variant in all populations. Polymorphs 6-10 were common to all four populations, while polymorphs 1-5 appeared to be relatively rare variants not universally detected in each group in the present study. The Chinese had the highest proportion of double-band phenotypes and the observed frequencies were not significantly different from those expected according to simple Mendelian inheritance, whereas the observed apo[a] phenotype distributions of the other three groups did not concur with those expected for Hardy Weinberg equilibrium. The German and Ghanaian groups displayed similar distributions of apo[a] phenotypes while the Chinese and San had significantly higher frequencies of polymorphs 9 and 10. Mean plasma Lp[a] concentrations in Ghanaians (36.2 +/- 31.5 mg/dl) were almost 2-fold greater than in Germans (18.7 +/- 23.1 mg/dl) and ca 1.65-fold greater than in either Chinese (22.9 +/- 18.3 mg/dl) or San (21.1 +/- 19.3 mg/dl). A strong inverse correlation was observed between apo[a] Mr and plasma Lp[a] concentration in Germans but this was much less pronounced in Ghanaians. While the mean plasma Lp[a] levels associated with polymorphs 1-6 were similar in both Germans (43.4 +/- 30.0 mg/dl) and Ghanaians (49.2 +/- 37.6 mg/dl), those Ghanaians with any combination of the polymorphs 9 and 10 had an almost 3-fold greater mean plasma Lp[a] level (20.6 +/- 11.3 mg/dl) than their German counterparts (7.8 +/- 5.7 mg/dl). It is therefore apparent that: 1) differences in apo[a] allele frequencies are not primarily responsible for differences in Lp[a] levels between populations; and 2) the greatest ethnic variation is observed in plasma Lp[a] concentrations associated with the high molecular weight apo[a] polymorphs.     

Chronic dietary fat and cholesterol inhibit the normal postprandial stimulation of plasma cholesterol metabolism

The response of parameters of plasma cholesterol metabolism was studied in baboons adapted either to a low-fat, low-cholesterol diet or a high-fat, high-cholesterol diet. Animals adapted to the low-fat diet responded to a single low-fat or high-fat meal, as do normal humans, by a stimulation of cholesterol transport from blood cells to plasma, a stimulation of esterification of cholesterol, and a stimulation of cholesteryl ester transfer to very low and low density lipoproteins. While fasting rates of esterification and transfer increased as a result of diet-induced hypercholesterolemia, the postprandial response was reversed, so that postprandial metabolism was characterized by a movement of cholesterol from plasma to blood cells, an inhibition of cholesterol esterification, and a net transfer of cholesteryl esters from VLDL and LDL to HDL. These data indicate that the effects of postprandial lipemia on plasma cholesterol metabolism critically depend upon fasting plasma cholesterol levels.     

Effect of dietary cholesterol on plasma cholesterol concentration in subjects following reduced fat,high fibre diet.

One hundred and sixty eight subjects participated in a randomised crossover study to determine whether halving or doubling the present dietary cholesterol intake from eggs had any influence on blood cholesterol concentration in people following current dietary recommendations. During the first eight weeks all participants were advised to follow a reduced fat diet (26% total energy for hyperlipidaemic patients, 35% total energy for normolipidaemic volunteers) with an increased ratio of polyunsaturated to saturated fatty acids. This background diet was continued throughout the 16 week experimental period, during which participants ate either two or seven eggs a week. A small but significant increase in total cholesterol was seen after four weeks in the group eating seven eggs a week compared with that in the group eating two eggs a week, but this was no longer apparent after eight weeks. Previous studies suggesting that dietary cholesterol has a greater effect on the serum cholesterol concentration either have been carried out against a background of a higher fat intake or have contrasted extreme cholesterol intakes. A further reduction in dietary cholesterol seems to be unnecessary in those people who have already reduced their intake of saturated fat and increased the ratio of polyunsaturated to saturated fatty acids and fibre rich carbohydrate.     

Addition of dietary fat to cholesterol in the diets of LDL receptor knockout mice: effects on plasma insulin, lipoproteins, and atherosclerosis

The factors underlying cardiovascular risk in patients with diabetes have not been clearly elucidated. Efforts to study this in mice have been hindered because the usual atherogenic diets that contain fat and cholesterol also lead to obesity and insulin resistance. We compared plasma glucose, insulin, and atherosclerotic lesion formation in LDL receptor knockout (Ldlr(-/-)) mice fed diets with varying fat and cholesterol content that induced similar lipoprotein profiles. Ldlr(-/-) mice fed a high-fat diet developed obesity, mild hyperglycemia, hyperinsulinemia, and hypertriglyceridemia. Quantitative and qualitative assessments of atherosclerosis were unchanged in diabetic Ldlr(-/-) mice fed a high-fat diet compared with lean nondiabetic control mice after 20 weeks of diet. Although one group of mice fed diets for 40 weeks had larger lesions at the aortic root, this was associated with a more atherogenic lipoprotein profile. The presence of a human aldose reductase transgene had no effect on atherosclerosis in fat-fed Ldlr(-/-) mice with mild diabetes. Our data suggest that when lipoprotein profiles are similar, addition of fat to a cholesterol-rich diet does not increase atherosclerotic lesion formation in Ldlr(-/-) mice.     

Radioimmunoassay of human plasma Lp(a) lipoprotein.

A quantitative immunodiffusion assay demonstrated Lp(a) lipoprotein in 91% (911 of 1000) of subjects. In order to quantitate Lp(a) in all plasma, a sensitive and specific double antibody radioimmunoassay was developed. The between-assay coefficient of variation was 8%. Lp(a) levels by radioimmunoassay were highly correlated with those obtained by the less sensitive radial immunodiffusion method (r = 0.98, n = 51). All but one of the 89 Lp(a) "negative" subjects by immunodiffusion had detectable levels of Lp(a) by radioimmunoassay. The one subject without detectable Lp(a) had abetalipoproteinemia (without detectable apolipoprotein B by radioimmunoassay). Furthermore, Lp(a) was detected in all three non-human primates examined: patas monkey, baboon, and pig-tail monkey. Quantitation of Lp(a) levels in 90 male myocardial infarction (MI) survivors and their spouses showed that the distribution of Lp(a) levels of MI survivors was significantly higher above the 50th percentile cut-point (P < 0.02) and exceeded that of the spouses. Furthermore, the Lp(a) distribution at and above the 50th percentile for the MI survivors who had an MI at age <50 (n = 36) was shifted to values higher than those having an MI at age >50. Thus, high levels of Lp(a) may be associated with premature coronary disease. We conclude that Lp(a) is present in all individuals with apolipoprotein B and that apolipoprotein B appears necessary for the plasma transport of the Lp(a) lipoprotein. Consistent with this hypothesis, quantitative immunochemical precipitation of (125)I-Lp(a) indicated that essentially all individual molecules of six purified Lp(a) preparations contain both the Lp(a) antigen and apolipoprotein B.     

Physicochemical transfer of [3H]cholesterol from plasma lipoproteins to cultured human fibroblasts.

The transfer of free cholesterol from [3H]cholesterol-labelled plasma lipoproteins to cultured human lung fibroblasts was studied in a serum-free medium. The uptake of [3H]cholesterol depended upon time of incubation, concentration of lipoprotein in the medium, and temperature. Modified (reduced and methylated) low-density lipoprotein (LDL), which did not enter the cells by the receptor pathway, gave a somewhat lower transfer rate than unmodified LDL, but if the transfer values for native LDL were corrected for the receptor-mediated uptake of cholesterol the difference was eliminated. The initial rates of transfer of [3H]cholesterol from LDL and high-density lipoprotein (HDL) were of the same order of magnitude (0.67 +/- 0.05 and 0.75 +/- 0.06 nmol of cholesterol/h per mg of cell protein, respectively) while that from very-low-density lipoprotein (VLDL) was much lower (0.23 +/- 0.02 nmol of cholesterol/h per mg) (means +/- S.D., n = 5). The activation energy for transfer of cholesterol from reduced, methylated LDL to fibroblasts was determined to be 57.5 kJ/mol. If albumin was added to the incubation medium the transfer of [3H]cholesterol was enhanced, while that of [14C]dipalmitoyl phosphatidylcholine was decreased compared with the protein-free system. The results demonstrate that, in spite of its low water solubility, free cholesterol can move from lipoproteins to cellular membranes, probably by aqueous diffusion. We propose that physicochemical transfer of free cholesterol may be a significant mechanism for net uptake of the sterol into the artery during atherogenesis.     

Distinct regulation of plasma LDL cholesterol by eicosapentaenoic acid and docosahexaenoic acid in high fat diet-fed hamsters: Participation of cholesterol ester transfer protein and LDL receptor

Despite established anti-atherogenic action, previous reports have shown that fish oils or n-3 poly-unsaturated fatty acid (PUFA) increase plasma LDL-C in animals and humans. However, which component of n-3 PUFAs and what mechanisms contribute to this increase are unclear. We investigated the effects of the major components of n-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on plasma LDL-C in high fat diet-fed hamsters. While LDL-C increased significantly with n-3 PUFA oil and DHA, EPA had no effect on LDL-C. Interestingly, a positive correlation was found between plasma cholesterol ester transfer protein (CETP) activity and LDL-C. Only DHA increased plasma CETP activity and significantly decreased LDL receptor expression in the liver. Our data suggest that DHA, not EPA, is a major factor in the LDL-C increasing effect of n-3 PUFA oil. These differential effects on LDL-C may arise from differences in plasma CETP activity and LDL receptor expression.     

Association of human mitochondrial DNA variants with plasma LDL levels

Increasing evidence supports the relationship between mitochondrial DNA variability and differences in energy metabolism, which may have pathophenotypic consequences. MtDNA pathological mutation has been also described to be associated with hypercholesterolemia. The target of this work consisted in studying the possible existence of an association between the mitochondrial DNA variability and plasma cholesterol levels. For this, two populations of 61 sedentary and 83 sportsmen were used to estimate the association of the lipidemic levels with the mitochondrial DNA variant harboured by them. Triglycerides, HDL-c, LDL-c and cholesterol/HDL-c were essayed, and mitochondrial DNA polymorphisms were assessed by HVR I sequencing and PCR/RFLP analysis. Major Caucasian mtDNA clades (HV, JT, U and IWX) did not associate with lipidemic levels in the sedentary population. However, in the case of a more disciplined population in term of nutritional habits and life style as sportsmen are, a significantly higher and lower level of LDL-c was associated with HV and JT clade, respectively. This observation could have relevant significance for metabolic distress diseases affecting plasma cholesterol levels.