Characterization of the basis of lipoprotein [a] lysine-binding heterogeneity.
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Although elevated plasma concentrations of lipoprotein [a] (Lp[a]) are considered to be a risk factor for atherosclerosis, the mechanisms by which Lp[a] mediates its pathogenic effects have not been conclusively determined. The apolipoprotein [a] (apo[a]) component of Lp[a] confers unique structural properties to this lipoprotein, including the ability to bind to lysine residues in biological substrates. It has been shown, however, that only a fraction of plasma Lp[a] (Lp[a]-Lys(+)) binds to lysine-Sepharose in vitro. The nature of the non-lysine-binding Lp[a] fraction in plasma (Lp[a]-Lys(-)) is currently unknown. In the present study, the Lp[a]-Lys(+) fraction was determined in the plasma of six unrelated individuals; the Lp[a]-Lys(+) fraction in these plasma samples ranged from approximately 37 to approximately 48%. Interestingly, purification of the Lp[a] by density gradient ultracentrifugation followed by gel filtration and ion-exchange chromatography resulted in progressive increases in the Lp[a]-Lys(+) fraction. Addition of either purified low density lipoprotein (LDL) or fibronectin to the purified Lp[a] at a 1:1 molar ratio reduced the Lp[a]-Lys(+) fraction (maximal decrease of 34 and 20%, respectively) whereas addition of both fibronectin and LDL to the purified Lp[a] resulted in a further decrease (45% maximally) in this fraction. Similar results were obtained by using a recombinant expression system for apo[a]: addition of a 4-fold molar excess of either LDL or fibronectin to conditioned medium containing metabolically labeled recombinant apo[a] reduced the Lys(+) fraction by 49 and 23%, respectively. Taken together, our data suggest that the lysine-binding heterogeneity of plasma Lp[a] is not primarily an intrinsic property of the lipoprotein, but rather results in large part from its ability to noncovalently associate with abundant plasma components such as LDL and fibronectin. These interactions appear to mask the lysine-binding site in apo[a] kringle IV type 10, which mediates the interaction of Lp[a] with lysine-Sepharose. The contribution of these interactions to the function of Lp[a] in vivo remains to be investigated. (+info)
Apolipoprotein A4-1/2 polymorphism and response of serum lipids to dietary cholesterol in humans.
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The response of serum lipids to dietary changes is to some extent an innate characteristic. One candidate genetic factor that may affect the response of serum lipids to a change in cholesterol intake is variation in the apolipoprotein A4 gene, known as the APOA4-1/2 or apoA-IVGln360His polymorphism. However, previous studies showed inconsistent results. We therefore fed 10 men and 23 women with the APOA4-1/1 genotype and 4 men and 13 women with the APOA4-1/2 or -2/2 genotype (carriers of the APOA4-2 allele) two diets high in saturated fat, one containing cholesterol at 12.4 mg/MJ, 136.4 mg/day, and one containing cholesterol at 86.2 mg/MJ, 948.2 mg/day. Each diet was supplied for 29 days in crossover design. The mean response of serum low density lipoprotein cholesterol was 0.44 mmol/l (17 mg/dl) in both subjects with the APOA4-1/1 genotype and in subjects with the APOA4-2 allele [95% confidence interval of difference in response, -0.20 to 0.19 mmol/l (-8 to 7 mg/dl)]. The mean response of high density lipoprotein cholesterol was also similar, 0.10 mmol/l (4 mg/dl), in the two APOA-4 genotype groups [95% confidence interval of difference in response, -0.07 to 0.08 mmol/l (-3 to 3 mg/dl)]. Thus, the APOA4-1/2 polymorphism did not affect the response of serum lipids to a change in the intake of cholesterol in this group of healthy Dutch subjects who consumed a background diet high in saturated fat. Knowledge of the APOA4-1/2 polymorphism is probably not a generally applicable tool for the identification of subjects who respond to a change in cholesterol intake. (+info)
Expression of human apolipoprotein A-I/C-III/A-IV gene cluster in mice induces hyperlipidemia but reduces atherogenesis.
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The apolipoprotein (apo)A-I/C-III/A-IV gene cluster is involved in lipid metabolism and atherosclerosis. Overexpression of apoC-III in mice causes hypertriglyceridemia and induces atherogenesis, whereas overexpression of apoA-I or apoA-IV increases cholesterol in plasma high density lipoprotein (HDL) and protects against atherosclerosis. Each gene has been studied alone in transgenic mice but not in combination as the entire cluster. To determine which phenotype is produced by the expression of the entire gene cluster, transgenic mice were generated with a 33-kb human DNA fragment. The results showed that the transgene contained the necessary elements to direct hepatic and intestinal expression of the 3 genes. In the pooled data, plasma concentrations were 257+/-9, 7.1+/-0.5, and 1.0+/-0.2 mg/dL for human apoA-I, apoC-III, and apoA-IV, respectively (mean+/-SEM). Concentrations of these apolipoproteins were higher in males than in females. Human apoA-I and apoC-III concentrations were positively correlated, suggesting that they are coregulated. Transgenic mice exhibited gross hypertriglyceridemia and accumulation of apoB(48)-containing triglyceride-rich lipoproteins. Plasma triglyceride and cholesterol concentrations were correlated positively with human apoC-III concentration, and HDL cholesterol was correlated with apoA-I concentration. In an apoE-deficient background, despite being markedly hypertriglyceridemic, cluster transgenic animals compared with nontransgenic animals showed a 61% reduction in atherosclerosis. This suggests that apoA-I and/or apoA-IV can protect against atherosclerosis even in the presence of severe hyperlipidemia. These mice provide a new model for studies of the regulation of the 3 human genes in combination. (+info)
Replication of linkage of familial combined hyperlipidemia to chromosome 1q with additional heterogeneous effect of apolipoprotein A-I/C-III/A-IV locus. The NHLBI Family Heart Study.
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Familial combined hyperlipidemia (FCHL), the most common familial dyslipidemia, is implicated in up to 20% of cases of premature coronary heart disease. Although underlying mutations for FCHL have yet to be identified, several candidate genes/regions have been identified. A positive linkage to chromosome 1q markers has been reported, with the highest lod score of 5.93 occurring at a location between D1S104 and D1S1677. Using the same diagnostic criteria, the Family Heart Study (FHS) has defined 71 FCHL families, comprising 170 cases, for a total of 137 possible affected sibling pairs. The FCHL criteria require elevation in serum low density lipoprotein cholesterol and triglyceride levels within the family, with at least 2 affected first-degree relatives. Markers D1S104 and D1S1677 were typed, and significant allele sharing was found in FCHL sibships (multipoint lod score with use of the model from the Finnish study was 2.52, and multipoint nonparametric score was 2.48; P=0.007), replicating linkage in this chromosome 1 region. In addition, previously reported linkage of FCHL to apolipoprotein A-I/C-III/A-IV has been investigated in FHS families. FHS results revealed positive but nonsignificant allele sharing among FCHL sibships with apolipoprotein A-I/C-III/A-IV by use of marker D11S4127 (nonparametric linkage score 1.11, P=0.13). Two-locus analyses of D1S104 and D11S4127 suggested possible heterogeneity rather than epistasis, with a maximum 2-locus lod score of 3.05. A nonparametric 2-locus analysis revealed significant improvement in the 2-locus versus single-locus scores. Finally, no linkage was found with markers near the lipoprotein lipase gene region. (+info)
Apo(a)-kringle IV-type 6: expression in Escherichia coli, purification and in vitro refolding.
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Lipoprotein (a) [Lp(a)] belongs to the class of highly thrombo-atherogenic lipoproteins. The assembly of Lp(a) from LDL and the specific apo(a) glycoprotein takes place extracellularly in a two-step process. First, an unstable complex is formed between LDL and apo(a) due to the interaction of the unique kringle (K) IV-type 6 (T6) in apo(a) with amino groups on LDL, and in the second step this complex is stabilized by a disulfide bond between apo(a) KIV-T9 and apoB(100). In order to understand this process better, we overexpressed and purified apo(a) KIV-T6 in Escherichia coli. Recombinant KIV-T6 was expressed as a His-tag fusion protein under control of the T7 promoter in BL21 (DE3) strain. After one-step purification by affinity chromatography the yield was 7 mg/l of bacterial suspension. Expressed fusion apo(a) KIV-T6 was insoluble in physiological buffers and it also lacked the characteristic kringle structure. After refolding using a specific procedure, high-resolution (1)H-NMR spectroscopy revealed kringle structure-specific signals. Refolded KIV-T6 bound to Lys-Sepharose with a significantly lower affinity than recombinant apo(a) (EC(50) with epsilon-ACA 0.47 mM versus 2-11 mM). In competition experiments a 1000-fold molar excess of KIV-T6 was needed to reach 60% inhibition of Lp(a) assembly. (+info)
Plasma lipid profiles in adults after prenatal exposure to the Dutch famine.
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BACKGROUND: Small body size at birth has been reported to be associated with an atherogenic lipid profile in humans, and animal experiments have shown that undernutrition during pregnancy permanently alters cholesterol metabolism in the offspring. There is no direct evidence in humans that maternal malnutrition during pregnancy affects the lipid profiles of the offspring. OBJECTIVES: We assessed the effects of maternal malnutrition during specific periods of gestation on plasma lipid profiles in persons aged approximately 50 y. DESIGN: This was a follow-up study of men and women born at term as singletons in a university hospital in Amsterdam between 1 November 1943 and 28 February 1947 around the time of a severe famine. RESULTS: Persons exposed to famine in early gestation had a more atherogenic lipid profile than did those who were not exposed to famine in utero. Their LDL-HDL cholesterol ratios were significantly higher (by 13.9%; 95% CI: 2.6-26.4%). Additionally, their plasma HDL-cholesterol and apolipoprotein A concentrations tended to be lower, and their plasma total cholesterol, LDL-cholesterol, and apolipoprotein B concentrations tended to be higher, although these differences were not statistically significant. The effect of famine was independent of size at birth and adult obesity. CONCLUSIONS: An atherogenic lipid profile might be linked to a transition from poor maternal nutrition in early gestation to adequate nutrition later on. This suggests that maternal malnutrition during early gestation may program lipid metabolism without affecting size at birth. (+info)
Effect of apolipoprotein A-IV genotype and dietary fat on cholesterol absorption in humans.
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We investigated the effect of the A-IV-2 allele, which encodes a Q360H substitution in apolipoprotein (apo) A-IV, and dietary fat on cholesterol absorption in humans. In three separate studies we compared fractional intestinal cholesterol absorption between groups of subjects heterozygous for the A-IV-2 allele (1/2) and homozygous for the common allele (1/1) receiving high cholesterol ( approximately 800 mg/day) diets with different fatty acid compositions. All subjects had the apoE 3/3 genotype. There was no difference in cholesterol absorption between the two genotype groups receiving a high saturated fat diet (33% of total energy as fat; 18% saturated, 3% polyunsaturated, 12% monounsaturated) or a low fat diet (22% of total energy as fat; 7% saturated, 7% polyunsaturated, 8% monounsaturated) diet. However, on a high polyunsaturated fat diet (32% of total energy as fat; 7% saturated, 13% polyunsaturated, 12% monounsaturated) mean fractional cholesterol absorption was 56. 7% +/- 1.9 in 1/1 subjects versus 47.5% +/- 2.1 in 1/2 subjects (P = 0.004). A post hoc analysis of the effect of the apoA-IV T347S polymorphism across all diets revealed a Q360H x T347S interaction on cholesterol absorption, and suggested that the A-IV-2 allele lowers cholesterol only in subjects with the 347 T/T genotype. We conclude that a complex interaction between apoA-IV genotype and dietary fatty acid composition modulates fractional intestinal cholesterol absorption in humans. (+info)
High levels of Lp(a) with a small apo(a) isoform are associated with coronary artery disease in African American and white men.
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Elevated levels of lipoprotein(a) [Lp(a)] and the presence of small isoforms of apolipoprotein(a) [apo(a)] have been associated with coronary artery disease (CAD) in whites but not in African Americans. Because of marked race/ethnicity differences in the distribution of Lp(a) levels across apo(a) sizes, we tested the hypothesis that apo(a) isoform size determines the association between Lp(a) and CAD. We related Lp(a) levels, apo(a) isoforms, and the levels of Lp(a) associated with different apo(a) isoforms to the presence of CAD (>/=50% stenosis) in 576 white and African American men and women. Only in white men were Lp(a) levels significantly higher among patients with CAD than in those without CAD (28.4 versus 16.5 mg/dL, respectively; P:=0.004), and only in this group was the presence of small apo(a) isoforms (<22 kringle 4 repeats) associated with CAD (P:=0.043). Elevated Lp(a) levels (>/=30 mg/dL) were found in 26% of whites and 68% of African Americans, and of those, 80% of whites but only 26% of African Americans had a small apo(a) isoform. Elevated Lp(a) levels with small apo(a) isoforms were significantly associated with CAD (P:<0.01) in African American and white men but not in women. This association remained significant after adjusting for age, diabetes mellitus, smoking, hypertension, HDL cholesterol, LDL cholesterol, and triglycerides. We conclude that elevated levels of Lp(a) with small apo(a) isoforms independently predict risk for CAD in African American and white men. Our study, by determining the predictive power of Lp(a) levels combined with apo(a) isoform size, provides an explanation for the apparent lack of association of either measure alone with CAD in African Americans. Furthermore, our results suggest that small apo(a) size confers atherogenicity to Lp(a). (+info)