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To investigate the structure-function relationship of human apolipoprotein A-IV (apoA-IV), several deletion mutants of this protein were constructed by sequentially removing pairs of 22-residue repeats, potentially having an amphipathic alpha-helical conformation. The mutants, produced as recombinant poly-histidine-tagged apolipoproteins (t-apo) in Escherichia coli, assembled with phosphatidylcholine (i.e. dimyristoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine, or egg lecithin) as did native apoA-IV. Lecithin:cholesterol acyltransferase (LCAT) cofactor function, measured as cholesterol esterification occurring when t-apo-phosphatidylcholine-cholesterol complexes were incubated with purified enzyme, decreased significantly when pairs of repeats between residues 117 and 248 were deleted and most markedly when residues 117-160 were deleted. LCAT cofactor activity decreased by 90 and 75%, respectively, when egg lecithin or palmitoyloleoylphosphatidylcholine was used to form the particles with the delta aa 117-160 mutant. Thus, on the basis of deletion scanning of t-apo, residues 117-160 seem to be involved in the LCAT cofactor function of apoA-IV.

In this paper we analyse the structural organization of human apolipoprotein E (apoE) at the surface of triglyceride (TG)-rich lipoproteins, in relation to the metabolic pathway of these particles. ApoE acts as a receptor-binding ligand at the surface of chylomicrons and VLDL (very low density lipoproteins). The degree of exposure of apoE at the surface of lipoproteins and its affinity for the receptor both determine the uptake and catabolism of these lipoproteins. ApoE and/or apoB100, the major apolipoprotein constituent of LDL, contribute to the interaction of lipoproteins with five different cellular receptors: 1) the low density lipoprotein (LDL) receptor; 2) the LDL receptor-related protein (LRP); 3) the macrophage receptor for hypertriglyceridemic VLDL; 4) the scavenger receptor; 5) the VLDL receptor. The degree of exposure of apoE at the surface of normo- and hyperlipidemic VLDL can modulate their uptake by the LDL receptor. Normolipidemic VLDL are poorly recognized by the LDL receptor whereas hypertriglyceridemic VLDL are cleared more efficiently through this pathway. On the other hand, the extent of apoE self-association, which is dependent upon the degree of hydrolysis of the TG-rich particles, can control their interaction with the LDL-receptor related protein. The lateral organization of apoE at the surface of TG-rich particles, its interaction with other apoproteins and its extent of self-association might therefore be important factors in the clearance of these lipoproteins. Finally, structural defects of apoE might result in an impaired interaction of apoE-containing lipoproteins with these receptors and lead to the development of atherogenic dyslipidemias.

Two international surveys for Lp(a) measurements were organized from 1989 to 1991. The results of the first survey led to the conclusion that the lack of a common primary standard was the main cause of the large inter-laboratory variation observed. No major effects of techniques or antisera were observed. The same findings were confirmed during the second survey, which was extended to include more samples and a larger number of participants. During the second survey, no consistent effect due to freezing or lyophilization could be demonstrated, although there was a trend towards lower Lp(a) values in lyophilized samples. The inter- and intra-laboratory coefficients of variation did not vary significantly for the different Lp(a) phenotypes, and variability was comparable for lyophilized, liquid and frozen materials. Large intra-assay coefficients of variation were observed during both surveys. Results obtained in different laboratories using the same commercial reagents and standards also showed a large variation. These initial results demonstrate that the lack of a primary standard and poor assay precision are the main factors responsible for the high inter-laboratory variation observed during these surveys.

To determine possible associations between lipoprotein(a) [Lp(a)] and the severity of coronary artery lesions, we measured lipid, apolipoprotein, and Lp(a) in a large population of Belgian patients (n = 1054) undergoing coronary angiography. In both women and men, univariate analysis demonstrated significant differences in the Lp(a) concentrations according to the severity of the coronary stenosis. However, after adjustment for possible confounding factors, many of these differences were attenuated, indicating that other variables that differentiate patients from control subjects also influence Lp(a) distribution. Differences in lipid, apolipoprotein, and Lp(a) concentrations between male and female patients are discussed.

We present a six-year follow-up of a boy with a novel type of hypolipoproteinemia, with clinical and biochemical features distinct from classical hypoalphalipoproteinemias. There were abnormally low concentrations of total and high-density lipoprotein (HDL) cholesterol, apolipoprotein (apo) B, apo A-I, and apo A-II, and the phospholipids were decreased. The most striking abnormality was an extra fraction containing mainly phospholipids and apo A-I in the HDL3 subfraction. This fraction is reminiscent of concentric 20- to 50-nm-diameter lamellar phospholipid liposomes. Plasma lecithin:cholesterol acyltransferase activity was strongly decreased. We noted a persisting polyclonal hypergammaglobulinemia, hematological abnormalities (hemolytic anemia and thrombocytopenia), and a progressive splenomegaly. After the five-year follow-up, the patient had recurrent severe infections; moderate hematuria and proteinuria developed gradually. Treatment with corticosteroids and immunoglobulins improved thrombocytopenia and hypolipoproteinemia. These clinical and biochemical findings differ from those in the known primary and secondary hypo-alpha-lipoproteinemia syndromes. Although investigation of the relatives suggests a familial predisposition for hypo-alpha-lipoproteinemia, the subject's condition can be regarded as acquired.

We have examined the associations between levels of plasma apolipoprotein (apo) AI, apo CIII and apo AIV and genetic variation in the apo AI-CIII-AIV gene cluster in 162 boys and young men from Belgium aged from 7 to 23 years. Genotypes were determined for six restriction enzymes XmnI, PstI, SstI, PvuIIA-CIII, PvuIIB-AIV and XbaI, and for the G to A substitution at -75 bp in the 5' region of the apo AI gene. The polymorphism most strongly associated with apo AI levels was the G to A substitution (P = 0.025, R2 x 100 = 3.6%) confirming previous observations. The polymorphism most strongly associated with apo CIII levels was that of PvuIIA-CIII (P = 0.023, R2 x 100 = 2.9%) in the apo CIII gene. This novel association must be interpreted with caution until it has been confirmed in an independent sample. The polymorphism associated with the largest effect on apo AIV levels was that detected with XbaI in the apo AIV gene, but this association was not statistically significant. Previously reported associations between the SstI polymorphism and triglyceride levels, and between the PstI polymorphism and apo AI levels, were weakly detected in the present sample. Our results show that variation associated with some of the polymorphisms in the apo AI-CIII-AIV cluster makes a small, but statistically significant, contribution to the determination of apo AI and apo CIII levels in this sample of young men and boys. These observations may, in part, explain reported associations between polymorphisms in this gene cluster, differences in plasma lipid and lipoprotein levels, and prevalence of coronary artery disease.

We studied the molecular basis of familial Type I hyperlipoproteinemia in two brothers of Turkish descent who had normal plasma apolipoprotein C-II levels and undetectable plasma post-heparin lipoprotein lipase (LPL) activity. We cloned the cDNAs of LPL mRNA from adipose tissue biopsies obtained from these individuals by the polymerase chain reaction and directional cloning into M13 vectors. Direct sequencing of pools of greater than 2000 cDNA clones indicates that their LPL mRNA contains two mutations: a missense mutation changing codon 156 from GAU to GGU predicting an Asp156----Gly substitution and a nonsense mutation changing the codon for Ser447 from UCA to UGA, a stop codon, predicting a truncated LPL protein that contains 446 instead of 448 amino acid residues. Both patients were homozygous for both mutations. Analysis of genomic DNAs of the patients and their family members by the polymerase chain reaction, restriction enzyme digestion (the GAT----GGT mutation abolishes a TaqI restriction site), and allele-specific oligonucleotide hybridization confirms that the patients were homozygous for these mutations at the chromosomal level, and the clinically unaffected parents and sibling were true obligate heterozygotes for both mutations. In order to examine the functional significance of the mutations in this family, we expressed wild type and mutant LPLs in vitro using a eukaryotic expression vector. Five types of LPL proteins were produced in COS cells by transient transfection: (i) wild type LPL, (ii) Asp156----Gly mutant, (iii) Ser447----Ter mutant, (iv) Gly448----Ter mutant, and (v) Asp156----Gly/Ser447----Ter double mutant. Both LPL immunoreactive mass and enzyme activity were determined in the culture media and intracellularly. Immunoreactive LPLs were produced in all cases. The mutant LPLs, Asp156----Gly and Asp156----Gly/Ser447----Ter, were devoid of enzyme activity, indicating that the Asp156----Gly mutation is the underlying defect for the LPL deficiency in the two patients. The two mutant LPLs missing a single residue (Gly448) or a dipeptide (Ser447-Gly448) from its carboxyl terminus had normal enzyme activity. Thus, despite its conservation among all mammalian LPLs examined to date, the carboxyl terminus of LPL is not essential for enzyme activity. We further screened 224 unrelated normal Caucasians for the Ser447----Ter mutation and found 36 individuals who were heterozygous and one individual who was homozygous for this mutation, indicating that it is a sequence polymorphism of no functional significance. Human LPL shows high homology to hepatic triglyceride lipase and pancreatic lipase.(ABSTRACT TRUNCATED AT 400 WORDS)

A number of immunological techniques--radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), electroimmunoassay, radial immunodiffusion, and a variety of immunoprecipitin assays--have been used to quantify apolipoproteins in plasma. This paper outlines their technical details and discusses their major advantages and drawbacks. The most sensitive procedures, RIAs and ELISAS, are best suited to quantifying those apoproteins found in low concentration in plasma. Immunoturbidimetric assays, on the other hand, which are readily automated, are being widely used to quantify apolipoproteins A-I and B. Apolipoprotein quantification is complicated by the interaction of the proteins with lipids, which can often mask their antigenic determinants. This problem may be circumvented by pretreatment of the samples, by selection of appropriate standards, or by the use of polyclonal or monoclonal antibodies that interact with permanently exposed epitopes on the lipoproteins' surfaces. Our proposed methods for measurement of the individual apolipoproteins give consideration to these approaches.

The Dutch (E22Q) and Flemish (A21G) mutations in the betaAPP region of the amyloid precursor protein (APP) are associated with familial forms of Alzheimer dementia. However, patients with these mutations express substantially different clinical phenotypes. Therefore, secondary structure and cytotoxic effects of the three Abeta(12-42) variants [wild-type (WT), Dutch and Flemish] were tested. At a concentration of 5 microM the aggregation of these peptides followed the order: Abeta(1-42) WTAbeta(12-42) WTAbeta(12-42) FlemishAbeta(12-42) Dutch. The stability of the secondary structure of these peptides upon decreasing the trifluoroethanol (TFE) concentration in the buffer was followed by circular dichroism measurements. WT peptides progressively lost their alpha-helical structure; this change occurred faster for both the Flemish and Dutch peptides, and at higher percentages of TFE in the buffer, and was accompanied by an increase in beta-sheet and random coil content. Apoptosis was induced in neuronal cells by the Abeta(12-42) WT and Flemish peptides at concentrations as low as 1-5 microM, as evidenced by propidium iodide (PI) staining, DNA laddering and caspase-3 activity measurements. Even when longer incubation times and higher peptide concentrations were applied the N-truncated Dutch peptide did not induce apoptosis. Apoptosis induced by the full length Abeta(1-42) peptide was weaker than that induced by its N-truncated variant. These data suggest that N-truncation enhanced the cytotoxic effects of Abeta WT and Flemish peptides, which may play a role in the accelerated progression of dementia.

We investigated the lipoprotein distribution and composition in cerebrospinal fluid (CSF) in a group of patients with Alzheimer's disease (AD) or affected by other types of dementia in comparison to non-demented controls. We found slightly decreased apolipoprotein (apo)E and cholesterol concentrations in CSF of AD patients and moderately increased apoA-I concentrations, while in patients suffering from other types of dementia the apoA-I CSF concentration was increased. ApoA-IV concentrations varied widely in human CSF, but were not associated with any clinical condition. HDL(2)-like apoE-containing lipoproteins represent the major lipoprotein fraction. In CSF of normal controls, only a minor HDL(3)-like apoA-I-containing lipoprotein fraction was observed; this fraction was more prevalent in AD patients. ApoA-II was recovered mostly in the HDL(3) density range, while apoA-IV was not associated with lipoproteins but appeared in a lipid-free form, co-localizing with LCAT immunoreactivity. Bi-dimensional analysis demonstrated pre-beta and alpha apoA-I-containing particles; apoE and apoA-II were detected only in alpha-migrating particles. ApoA-IV distributed both to pre-beta and gamma-migrating particles; the LCAT signal was co-localized in this gamma-migrating fraction. Enzymatically active LCAT was present in human CSF as well as PLTP activity and mass; no CETP mass was detected. In CSF from AD patients, LCAT activity was 50% lower than in CSF from normal controls. CSF lipoproteins induced a significant cholesterol efflux from cultured rat astrocytes, suggesting that they play an active role in maintaining the cholesterol homeostasis in brain cells.

The goal of the present study was to assess whether the effect of the apolipoprotein E polymorphism on postprandial lipemia explained part of the risk attributable to familial history of coronary heart disease. Cases (n = 407) were students, aged between 18 and 28 years, whose fathers had a proven myocardial infarction before the age of 55 years. Age-matched controls (n = 415) were recruited from the corresponding student registers. Blood was obtained after an overnight fast and at 2, 3, 4 and 6 h after ingestion of a fatty meal for triglyceride measurements. Apolipoprotein E phenotype was associated with postprandial triglyceride variability in both cases and controls. However, the apolipoprotein E-dependent triglyceride response was not significantly heterogeneous between cases and controls. In the pooled data, postprandial triglyceride levels were higher in carriers of the E2 and, to a lesser extent, of the E4 isoform, than in E3/3 homozygotes, independently of fasting triglyceride levels. At 6 h, triglyceride levels were increased by 21.2% (P0.01) in E2 carriers and 11.5% (P = 0.053) in E4 carriers by comparison to E3/3 subjects. These effects were not significantly different between regions. In conclusion, the effects of the apolipoprotein E polymorphism on postprandial triglyceridemia are similar across regions of Europe, and homogeneous in healthy young subjects with and without a family history of early myocardial infarction. This suggests that the influence of apolipoprotein E on myocardial infarction risk may be acting through mechanisms other than through effects on postprandial triglyceridemia.

The assembly of atherogenic lipoproteins requires the formation in the endoplasmic reticulum of a complex between apolipoprotein (apo)B, a microsomal triglyceride transfer protein (MTP) and protein disulphide isomerase (PDI). Here we show by molecular modelling and mutagenesis that the globular amino-terminal regions of apoB and MTP are closely related in structure to the ancient egg yolk storage protein, vitellogenin (VTG). In the MTP complex, conserved structural motifs that form the reciprocal homodimerization interfaces in VTG are re-utilized by MTP to form a stable heterodimer with PDI, which anchors MTP at the site of apoB translocation, and to associate with apoB and initiate lipid transfer. The structural and functional evolution of the VTGs provides a unifying scheme for the invertebrate origins of the major vertebrate lipid transport system.

Oral fat tolerance tests (FTTs) have been widely used as a tool to investigate post-prandial lipaemia. However, there is no consensus regarding the type and amount of fat used in the tests.We compared three commonly used FTTs, each containing 63 g of fat: a mixed meal, a liquid cream meal and a liquid soybean oil meal. The study group consisted of 10 healthy normolipidaemic men. We measured triglycerides (TGs), retinyl esters (REs), apolipoprotein E (apoE), apolipoprotein B-48 (apoB-48) and apolipoprotein B-100 (apoB-100) in plasma and in triglyceride-rich lipoprotein (TRL) fractions separated by density-gradient ultracentrifugation at baseline and 3, 4, 6, and 8 h after the FTTs.We observed similar TGs, apoE, apoB-48 and apoB-100 responses after all three FTTs, despite the different fatty acid composition of the meals. In contrast, the commonly used marker for exogenous particles, RE, differed clearly when polyunsaturated (soybean oil) and saturated fat (cream or mixed meal) were used. The RE response in plasma (P0.005, repeated measures ANOVA), in chylomicrons (P0.013) and in very low-density lipoprotein (VLDL) 1 (P0.017), as well as the RE area under the incremental curve in plasma and chylomicron fractions, were markedly increased after the soybean oil meal compared with the mixed meal and cream meal tests. The peak of RE response occurred parallel to the responses of other markers (i.e. TG or apoB-48) of post-prandial TRL during soybean oil meal. In contrast, RE peak concentration was delayed after saturated fat-containing meals. After soybean oil, FTT plasma cholesterol concentration was lower and the chylomicron cholesterol concentration was higher compared with mixed or cream meals, but no differences were detected in post-prandial high-density lipoprotein (HDL)-cholesterol concentration.When the amount of fat is similar, post-prandial responses of TG, apoE, apoB-48, apoB-100 and HDL-cholesterol were comparable after different FTTs.

To elucidate how the apolipoprotein (apo) E polymorphism and modifiable factors interact in explaining plasma lipid and apolipoprotein levels, we studied 1448 young adults (18 to 26 years old), participating in the European Atherosclerosis Research Study (EARS). Venous blood was collected after an overnight fast. Modifiable factors, eg, body mass index (BMI), waist-to-hip ratio (WHR), tobacco and alcohol consumption, and physical activity, were determined by using standardized protocols. Associations of modifiable factors with apoE levels were homogeneous across apoE phenotypes. In contrast, correlations of BMI with total cholesterol and apoB levels, as well as correlations between WHR and apoB, were significantly (P.05 to P.01) stronger in E2 carriers than in subjects with other phenotypes. Total cholesterol and apoB levels were comparable in E2 carriers in the upper tertile of BMI or WHR to those in E3/3 subjects, suggesting that the lowering effect of the E2 allele was no longer present. The inverse association between the plasma cholesteryl linoleate-to-oleate ratio, a marker for the dietary polyunsaturated-to-saturated fatty acid ratio, and triglycerides was also stronger in E2 carriers (-0.33 versus -0.17 in E3/3 and -0.24 in E4 carriers). Associations with other modifiable factors were notably consistent across apoE phenotypes. Gender and modifiable factors explained three times more (31%) of the interindividual variation in apoB levels in E2 carriers than in E3/3 subjects (9%) or E4 carriers (14%), mainly due to a larger variance explained by BMI. Our results suggest that the apoE polymorphism acts in a relatively uniform manner, independently of lifestyle. However, the associations of adiposity to total cholesterol and apoB levels appear to be stronger in apoE2 carriers.

To model the common structural unit in the system of reverse cholesterol transport, we studied the composition, structure, and physicochemical properties of complexes generated between dipalmitoylphosphatidylcholine (DPPC) or palmitoyllinoleoylphosphatidylcholine (PLPC) and apoE3 in the absence and in the presence of cholesterol (Chol); the data were compared with similar experiments using apoA-I, the major proteins of high-density lipoproteins. The conformation and organization of lipid-binding domains of apoE3 within the complexes were calculated by computer modeling. The transition temperatures of DPPC within discoidal complexes with mean diameters of 116 A (GGE) or 148 A (EM) were higher for complexes versus liposomes both in the absence and in the presence of Chol. Association of apoE3 with DPPC resulted in a more structured state of the apolipoprotein molecule versus the soluble apolipoprotein; this state was characterized by parallel orientation of alpha-helixes of apoE3 and DPPC acyl chains. Substrate efficiency of the apoE3-PLPC-Chol complexes in the lecithin-cholesterol acyltransferase (LCAT) reaction expressed as Vmax/Km was 0.5 mole cholesteryl esters/h per 1 microM. The transformation of discoidal apoE3-DPPC-Chol complexes into spherical particles was induced by LCAT and accumulation of cholesteryl esters was approximately 62% of the total cholesterol. Parallel orientation of phospholipid acyl chains with helical segments disappeared in these particles. Discoidal apoE3-DPPC complexes incorporated unesterified cholesterol released from Chol-loaded J774 macrophages. The data support the concept that association of apoE3 and apoA-I with phospholipids is qualitatively similar due to similar orientation of helical repeats in the C-terminal domains of apoE3 and apoA-I.

In the European Atherosclerosis Research Study, genetic and environmental markers of risk of premature coronary heart disease were compared in offspring of men with and without myocardial infarction before the age of 55 years. Cases were 682 students with a paternal history of myocardial infarction, and control subjects were 1312 students without such a history. The students were enrolled in 14 universities in five European regions (Finland, Great Britain, and northern, middle, and southern Europe). Lipoprotein(a) [Lp(a)] concentrations were skewed towards lower concentrations in both cases (median, 7.3 mg/dL; 95% confidence interval, 6.3 to 8.1 mg/dL) and control subjects (median, 6.6 mg/dL; 95% confidence interval, 6.1 to 7.2 mg/dL) (P = .37). Significantly more northern European male cases than control subjects had Lp(a) levels exceeding 30 mg/dL (P = .040), but this did not pertain to females (P = .29), and overall, there was no difference between cases (16.5%) and control subjects (15.5%) in the frequency of Lp(a) concentrations above 30 mg/dL (P = .63). As expected, there was a significant (P.01) inverse relationship between apo(a) molecular size and Lp(a) concentration. In Great Britain there was a significant difference in phenotype distribution between cases and control subjects (P = .035), due mainly to a high frequency of the apo(a) S2 isoform in cases. A similar but statistically insignificant tendency was seen in northern Europeans. In the three other regions, however, the distribution of apo(a) phenotypes among cases and controls was similar, and in the study population overall, the distribution of apo(a) phenotypes did not differ significantly (P = .74) between cases and control subjects.

To investigate the structure-function relationship of human apolipoprotein A-IV (apoA-IV), several deletion mutants of this protein were constructed by sequentially removing pairs of 22-residue repeats, potentially having an amphipathic alpha-helical conformation. The mutants, produced as recombinant poly-histidine-tagged apolipoproteins (t-apo) in Escherichia coli, assembled with phosphatidylcholine (i.e. dimyristoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine, or egg lecithin) as did native apoA-IV. Lecithin:cholesterol acyltransferase (LCAT) cofactor function, measured as cholesterol esterification occurring when t-apo-phosphatidylcholine-cholesterol complexes were incubated with purified enzyme, decreased significantly when pairs of repeats between residues 117 and 248 were deleted and most markedly when residues 117-160 were deleted. LCAT cofactor activity decreased by 90 and 75%, respectively, when egg lecithin or palmitoyloleoylphosphatidylcholine was used to form the particles with the delta aa 117-160 mutant. Thus, on the basis of deletion scanning of t-apo, residues 117-160 seem to be involved in the LCAT cofactor function of apoA-IV.

In this paper we analyse the structural organization of human apolipoprotein E (apoE) at the surface of triglyceride (TG)-rich lipoproteins, in relation to the metabolic pathway of these particles. ApoE acts as a receptor-binding ligand at the surface of chylomicrons and VLDL (very low density lipoproteins). The degree of exposure of apoE at the surface of lipoproteins and its affinity for the receptor both determine the uptake and catabolism of these lipoproteins. ApoE and/or apoB100, the major apolipoprotein constituent of LDL, contribute to the interaction of lipoproteins with five different cellular receptors: 1) the low density lipoprotein (LDL) receptor; 2) the LDL receptor-related protein (LRP); 3) the macrophage receptor for hypertriglyceridemic VLDL; 4) the scavenger receptor; 5) the VLDL receptor. The degree of exposure of apoE at the surface of normo- and hyperlipidemic VLDL can modulate their uptake by the LDL receptor. Normolipidemic VLDL are poorly recognized by the LDL receptor whereas hypertriglyceridemic VLDL are cleared more efficiently through this pathway. On the other hand, the extent of apoE self-association, which is dependent upon the degree of hydrolysis of the TG-rich particles, can control their interaction with the LDL-receptor related protein. The lateral organization of apoE at the surface of TG-rich particles, its interaction with other apoproteins and its extent of self-association might therefore be important factors in the clearance of these lipoproteins. Finally, structural defects of apoE might result in an impaired interaction of apoE-containing lipoproteins with these receptors and lead to the development of atherogenic dyslipidemias.

As a part of the EARS study we assessed the role of the common apo A-IV polymorphism in determining the hereditary predisposition to cardiovascular disease. The study population consisted of 1261 controls and 629 cases (students whose father had MI before 55 years) from five different European regions. The apo A-IV 1-1 phenotype accounted for 85% of the individuals. One per cent of subjects were homozygous for the apo A-IV2 allele. There was significant regional variation in the apo A-IV allele frequencies from North to South in Europe, with the lowest A-IV2 frequency in Finland. The distribution of the apo A-IV phenotypes was similar in cases and controls, as was the regional variation. The apo A-IV polymorphism did not affect HDL cholesterol. There was no correlation between apo A-IV alleles and the plasma concentration of apo A-IV. The plasma concentration of apo A-IV was lower in females than in males; furthermore, there was a significant difference in apo A-IV concentrations between oral contraceptive users and nonusers: users had the lowest values. As no strongly significant genetic difference could be demonstrated between plasma lipid concentration in cases and controls, and as the apo A-IV polymorphism did not significantly influence plasma lipid concentration, we conclude that the apo A-IV gene is not a major determinant of the risk for MI and/or CHD.

The European Atherosclerosis Research Study (EARS) is a multicenter collaborative project within the European community. Its main objective is to study in young individuals the biological expression of a paternal history of premature coronary heart disease and to analyze the relative contribution of genetic and environmental factors to this expression. This study was carried out in 14 centers in 11 European countries, where the offspring of fathers who suffered a documented myocardial infarction before the age of 55 years (cases) were compared with age- and sex-matched control subjects. Plasma apolipoproteins A-I, B, A-II, A-IV, and E and lipoprotein (Lp) A-I lipoparticles were measured in this student population. Comparison of the values between cases and control subjects showed significantly higher apo B levels in cases compared with control subjects, and these differences were homogeneous throughout Europe. Regional differences were observed for apo E levels with an increasing north-south gradient, which was inversely related to that observed for triglycerides. A stepwise regression analysis including the lipid and apolipoprotein variables showed that apo B and triglycerides were the strongest discriminators between offspring of fathers with premature coronary heart disease and control subjects.

A clinical trial was conducted in three centres to assess the effects of long-term use of the injectable contraceptive depot-medroxyprogesterone acetate (DMPA) on lipid metabolism. Fifty women who had used DMPA at a dose of 150 mg every three months for 3 to 9 years were recruited in Bangkok, Christchurch and Mexico City. They were compared to a control group of 120 IUD users. Total cholesterol, LDL-cholesterol, HDL-cholesterol, total triglycerides, apolipoproteins AI, AII and B were measured throughout one injection interval. Significant findings differed between centres. Compared to their own centre controls, DMPA users in Bangkok had higher LDL-cholesterol levels; those in Christchurch had lower HDL-cholesterol, apolipoprotein (apo) AI and apo AI/B ratio and higher apo B levels; those in Mexico City had a lower apo AI/B ratio. Further changes were observed during the injection interval, some of which were correlated to changes in serum MPA levels. It is concluded that long-term use of DMPA induces moderate changes in lipid metabolism which are unfavourable in terms of risk for atherosclerosis. This should be borne in mind when weighing the overall risks and benefits of this contraceptive method for a potential user.

To determine possible associations between lipoprotein(a) [Lp(a)] and the severity of coronary artery lesions, we measured lipid, apolipoprotein, and Lp(a) in a large population of Belgian patients (n = 1054) undergoing coronary angiography. In both women and men, univariate analysis demonstrated significant differences in the Lp(a) concentrations according to the severity of the coronary stenosis. However, after adjustment for possible confounding factors, many of these differences were attenuated, indicating that other variables that differentiate patients from control subjects also influence Lp(a) distribution. Differences in lipid, apolipoprotein, and Lp(a) concentrations between male and female patients are discussed.

We studied the molecular basis of familial Type I hyperlipoproteinemia in two brothers of Turkish descent who had normal plasma apolipoprotein C-II levels and undetectable plasma post-heparin lipoprotein lipase (LPL) activity. We cloned the cDNAs of LPL mRNA from adipose tissue biopsies obtained from these individuals by the polymerase chain reaction and directional cloning into M13 vectors. Direct sequencing of pools of greater than 2000 cDNA clones indicates that their LPL mRNA contains two mutations: a missense mutation changing codon 156 from GAU to GGU predicting an Asp156----Gly substitution and a nonsense mutation changing the codon for Ser447 from UCA to UGA, a stop codon, predicting a truncated LPL protein that contains 446 instead of 448 amino acid residues. Both patients were homozygous for both mutations. Analysis of genomic DNAs of the patients and their family members by the polymerase chain reaction, restriction enzyme digestion (the GAT----GGT mutation abolishes a TaqI restriction site), and allele-specific oligonucleotide hybridization confirms that the patients were homozygous for these mutations at the chromosomal level, and the clinically unaffected parents and sibling were true obligate heterozygotes for both mutations. In order to examine the functional significance of the mutations in this family, we expressed wild type and mutant LPLs in vitro using a eukaryotic expression vector. Five types of LPL proteins were produced in COS cells by transient transfection: (i) wild type LPL, (ii) Asp156----Gly mutant, (iii) Ser447----Ter mutant, (iv) Gly448----Ter mutant, and (v) Asp156----Gly/Ser447----Ter double mutant. Both LPL immunoreactive mass and enzyme activity were determined in the culture media and intracellularly. Immunoreactive LPLs were produced in all cases. The mutant LPLs, Asp156----Gly and Asp156----Gly/Ser447----Ter, were devoid of enzyme activity, indicating that the Asp156----Gly mutation is the underlying defect for the LPL deficiency in the two patients. The two mutant LPLs missing a single residue (Gly448) or a dipeptide (Ser447-Gly448) from its carboxyl terminus had normal enzyme activity. Thus, despite its conservation among all mammalian LPLs examined to date, the carboxyl terminus of LPL is not essential for enzyme activity. We further screened 224 unrelated normal Caucasians for the Ser447----Ter mutation and found 36 individuals who were heterozygous and one individual who was homozygous for this mutation, indicating that it is a sequence polymorphism of no functional significance. Human LPL shows high homology to hepatic triglyceride lipase and pancreatic lipase.(ABSTRACT TRUNCATED AT 400 WORDS)

Lipoprotein(a) [Lp(a)] is considered an additional, independent, and largely genetically determined risk factor for the development of premature coronary heart disease. Analogous with increased Lp(a) concentrations that represent an additional risk factor in adults, above-normal concentrations of Lp(a) can be detected in five- to seven-day-old newborns. We describe a simple enzyme-linked immunosorbent assay for measuring Lp(a) in dried blood spots collected by heel-prick in five- to seven-day-old infants. Lp(a) could be quantitatively recovered from blood spots. We chose a cutoff value of 100 mg/L for identifying the newborns at risk, based on the Lp(a) distribution in 180 such infants.

Apolipoprotein measurements have been applied to the estimation of coronary risk in patients and also to the characterization of various kinds of dyslipidemia, where apolipoprotein profiles are altered in a significant way. The concentration of apolipoprotein B is increased in various types of primary and secondary dyslipidemia including: Type II, III and V primary dyslipidemia, chronic renal failure and Type I, insulin-dependent diabetes. Apo AI concentrations are decreased in Type I, II B and IV dyslipidemia, as well as in liver disease. Apo E is increased in Type III and V dyslipidemia.

The standardization programs for the immunological assays of apolipoproteins have temptatively addressed several of the problems linked with this type of assays. Such problems include: lack of primary standards with certified values, method-to-method variation, lack of comparability for sample treatment, etc.

A number of immunological techniques--radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), electroimmunoassay, radial immunodiffusion, and a variety of immunoprecipitin assays--have been used to quantify apolipoproteins in plasma. This paper outlines their technical details and discusses their major advantages and drawbacks. The most sensitive procedures, RIAs and ELISAS, are best suited to quantifying those apoproteins found in low concentration in plasma. Immunoturbidimetric assays, on the other hand, which are readily automated, are being widely used to quantify apolipoproteins A-I and B. Apolipoprotein quantification is complicated by the interaction of the proteins with lipids, which can often mask their antigenic determinants. This problem may be circumvented by pretreatment of the samples, by selection of appropriate standards, or by the use of polyclonal or monoclonal antibodies that interact with permanently exposed epitopes on the lipoproteins' surfaces. Our proposed methods for measurement of the individual apolipoproteins give consideration to these approaches.

The effect of endurance training on plasma lipoproteins was investigated in 27 healthy sedentary men between the ages of 20 and 55 years. During the first 4 months of the study, 13 of them (group A) participated in a training program (3 hours/wk), whereas the others served as control subjects (group B). At the end of this period the control subjects also underwent a 4-month training program. In both groups the training significantly increased physical working capacity at a heart rate of 130 bpm (PWC130), whereas it decreased the resting heart rate (p less than 0.05). Concomitantly with this improvement in cardiorespiratory fitness, a significant increase in the high-density lipoprotein (HDL) cholesterol concentration was observed (p less than 0.01); this was due to an increase in both HDL2 beta and HDL2 alpha + 3 cholesterol concentrations. The plasma total and HDL-apoprotein AI and apoprotein AII concentrations were not significantly affected by the training. Significant decreases in plasma triglyceride (p less than 0.05), very-low-density lipoprotein cholesterol (p less than 0.05), and low-density lipoprotein (LDL) cholesterol (p less than 0.001) concentrations were also observed, but only in group B, which showed a much greater increase in PWC130 at the end of the training period than group A; the decrease in the LDL cholesterol concentration in this group was accompanied by a slight decrease in the LDL-apoprotein B concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum cholesterol and HDL cholesterol levels were measured in an urban and a rural population of the People's Republic of China and compared with cholesterol values obtained in Belgium and in the Republic of Korea, with use of the same methodology. Total cholesterol levels were markedly lower in the People's Republic of China than in Belgium and generally lower than in Korea, both in male and female subjects. However, the differences in HDL cholesterol levels among the three populations were small in males and only significantly higher in Belgium in the age classes below 34 years. In women of all age groups, HDL cholesterol values were significantly higher in Belgium than in China and Korea. Total cholesterol levels below 100 mg/dl were found in the People's Republic of China in about 2% of the participants. Apolipoprotein B was significantly lower, and the apolipoprotein A1/B ratio was significantly higher, in China and Korea compared to Belgium.

We developed a quantitative assay for apolipoprotein AI (apo AI) in human serum, using a "sandwich"-type enzyme-linked immunosorbent assay. Diluted serum samples were pipetted into the wells of polystyrene microtiter plates that had been previously coated with purified rabbit anti-human apo AI antibodies. After incubation for 2 h and washing, antibodies conjugated to horseradish peroxidase (EC 1.11.1.7) were added and incubated for 2 h; after further washing, the bound enzyme was assayed by oxidation of o-phenylenediamine. Assay conditions were optimized for the incubation time and the amounts of coating antibodies and conjugate. Assay sensitivity is about 0.5 ng of apo AI, with a working range of 1 to 14 ng, similar to that of radioimmunoassays for human apo AI. The standard curves for apo AI in serum or HDL and for purified apo AI were parallel. Delipidation, heat treatment, or addition of detergents did not affect the amount of immunoassayable apo AI in human serum. The intra- and interassay CVs were 4 and 8%, respectively. Results for 100 serum samples compared well with those by immunonephelometry (r = 0.94).

Apolipoprotein B, the major protein component of very-low-density (VLDL) and low-density (LDL) lipoproteins in serum, is being widely measured in serum, to help assess risk of cardiovascular disease. Standardization has proven difficult because of the physical and immunochemical heterogeneity of LDL and VLDL, the masking of antigenic determinants of LDL and VLDL, the insolubility of apolipoprotein B, the instability of lipoprotein reference materials, and the multiplicity of analytical methods. Here we have examined the literature and collected current information from lipoprotein scientists to determine the problems--and the ways investigators have attempted to solve them--in preparation, storage, and use of standards and anti-apolipoprotein B sera, analytical procedures and reagents, and the nature of apolipoprotein B in serum. Standardization appears possible with development of stable reference materials that are applicable to the various analytical methods, controlled immunochemical reaction conditions, and establishment of a selected interim reference method for use as a point of reference.

A method for the direct high-performance liquid chromatographic (HPLC) determination of free cholesterol and the individual cholesteryl esters in cell culture experiments is described. The murine macrophage-like J774 cell line was loaded with cholesterol by incubation with low-density lipoproteins. After extraction of the cellular lipids with hexane-isopropanol (3:2, v/v), the cholesteryl esters were identified and quantified by isocratic HPLC. Unesterified cholesterol and its esters were eluted with acetonitrile-isopropanol (50:50, v/v) on a Zorbax ODS column within 25 min and detected at 210 nm. Cholesteryl heptadecanoate was used as an internal standard. The detection response is linear in the analytical range of interest; the overall coefficients of variation are less than 8% and the detection limit is between 50 and 150 ng. The results demonstrate that HPLC is suitable for the determination of cellular cholesteryl ester profiles and could usefully contribute to the understanding of the mechanism of foam cell formation during the development of atherosclerosis. This method can also be applied to all experimental systems involving the study of cholesteryl esters.