Studies on the Lp(a)-lipoprotein of human serum

Summary Lp(a)-lipoprotein of human serum, when isolated by ultracentrifugation and gelfiltration on Sepharose 4 B, may disaggregate into several components either spontaneously or by mild treatment with detergents:1. A lipoprotein with characteristics of LDL₂; 2. a lipid-free protein complex, which reacts immunologically with anti-Lp(a)-sera; 3. albumin and 4. two other protein components, which were also observed in aged LDL₂-preparations and have not been characterized further. The Lp(a)-positive protein moiety could be separated from all other components by chromatography on hydroxyl-apatite.It is concluded that the Lp(a)-lipoprotein represents a complex protein composed of LDL₂-lipoprotein, the Lp(a)-protein moiety, and possibly albumin.

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Zusammenfassung Das durch Ultrazentrifugation und Gelfiltration and Sepharose 4 B isolierte Lp(a)-Lipoprotein des menschlichen Serums zerfällt spontan oder nach milder Behandlung mit Detergentien in mehrere Komponenten:1. Ein Lipoprotein mit den Eigenschaften der LDL₂; 2. einen lipidfreien Proteinkomplex, der immunologisch mit Anti-Lp(a)-Antiseren reagiert; 3. Albumin und 4. zwei weitere Proteinkomponenten, die auch in gealterten LDL₂-Präparationen beobachtet wurden, die aber hier nicht weiter charakterisiert wurden. Der Lp(a)-positive Proteinanteil konnte von allen anderen Bestandteilen durch Chromatographie an Hydroxylapatit abgetrennt werden.Es wird geschlossen, daß das Lp(a)-Lipoprotein ein komplexes Protein darstellt, das aus LDL₂-Lipoprotein, dem Lp(a)-Protein und möglicherweise Albumin zusammengesetzt ist.

     

Lp(a) interactions.

In this report, we have summarized our recent studies on lipoprotein(a) (Lp(a)) and its interactions with apolipoprotein B-containing lipoproteins (ApoB-Lp). These findings implicate the kringle-4-like domains of Apo(a) in the binding of Lp(a) to other ApoB-Lp and point to proline as important in this interaction. Other studies have indicated that Lp(a) interacts with the subendothelial extracellular matrix (ECM) and that Lp(a) is inversely related to plasma triglycerides. Since Apo(a) also has an affinity for ApoB-Lp, enhanced binding of Apo(a) to the arterial wall could increase the accumulation of LDL in the matrix and thus promote the development of cardiovascular disease.     

Lp(a) lipoprotein level and longevity.

Lp(a) lipoprotein forms a distinct class of serum lipoproteins. Its unique immunochemical properties are caused by the Lp(a) polypeptide chain which is attached to apolipoprotein B (apoB) by a disulfide bridge. The level of Lp(a) lipoprotein is under strict genetic control. It is well established that a high level of Lp(a) lipoprotein is a genetic risk factor for atherosclerotic disease, particularly coronary heart disease (CHD). Since cardiovascular disease is one of the major causes of death there should be a shortage of people with genetic determinants of cardiovascular disease in people who are very old and still have adequate physical and mental capacities. The authors have studied Lp(a) lipoprotein levels in 102 persons who were 83 years or older when blood samples were drawn. This study group was a subpopulation of a series comprising 456 persons who had been 80 years or older at intake in an intervention study of old people living at home. Only those without physical or mental incapacities were included in the present study. There was a striking shortage of persons with an Lp(a) lipoprotein level higher than the 75th percentile of the general population in this series of people who had achieved successful ageing. The highest value observed among the old people corresponds to the 88th percentile of the general population. It is highly unlikely that the present observations reflect chance events or fall in Lp(a) lipoprotein levels in people who had higher levels at a younger age. The most likely explanation of our finding is that a sizeable fraction of people with high Lp(a) lipoprotein levels die before reaching a very high age.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Studies on the structure of the Lp(a) lipoprotein: Isolation and partial characterization of the Lp(a)-specific antigen

Lp(a), a polymorphic lipoprotein of human serum, has been isolated from strongly positive normal sera. The described procedure utilizes a serum pool of several liters. The purified lipoprotein has been investigated in its intact and totally delipidized form. Protein constituents could be characterized as apolipoprotein-B, apolipoprotein-AIII, and the Lp(a)-specific, dissociable polypeptide. Albumin could only be found in some of the preparations. The Lp(a)-specific polypeptide could be separated and characterized by its amino acid composition.     

Genetics of the quantitative Lp(a) lipoprotein trait

The Lp(a) lipoprotein is a complex particle composed of a low density lipoprotein (LDL)-like lipoprotein and the disulfide bonded Lp(a) glycoprotein. The complex represents a quantitative genetic trait. SDS gel electrophoresis under reducing conditions of sera followed by immunoblotting with affinity-purified polyclonal anti-Lp(a) demonstrated inter- and intra-individual size heterogeneity of the glycoprotein with apparent Mr in the range 400-700kDa. According to their relative mobilities compared to apo B-100 the Lp(a) patterns were categorized into phenotypes F, B, S1, S2, S3 und S4 and into the respective double-band phenotypes. This size heterogeneity seems to be controlled by multiple alleles designated LpF, LpB, LpS1, LpS2, LpS3, LpS4 and a null allele (LpO) at a single locus. Phenotype frequencies observed in 441 unrelated subjects were in good agreement with those expected from the genetic hypothesis. Comparison of Lp(a) lipoprotein concentrations in the different phenotypes revealed a highly significant association of phenotypes B, S1 and S2 with high, and phenotypes S3 und S4 with intermediate Lp(a) concentrations. A third mode is represented by the null phenotype were no Lp(a) band is detected upon immunoblotting and Lp(a) lipoprotein is low or absent. We conclude that the same gene locus is involved in determining Lp(a) glycoprotein phenotype and Lp(a) lipoprotein concentrations in plasma. This major gene seems to be the Lp(a) glycoprotein structural gene locus.     

Genetics of the quantitative Lp(a) lipoprotein trait

Summary Lp(a) glycoprotein exhibits an apparent size polymorphism that is associated with genetically controlled Lp(a) lipoprotein concentrations in plasma (Utermann et al. 1988). We have tested the hypothesis that this polymorphism is genetically controlled by studying 15 matings with a total of 44 offspring. This confirmed our conclusion that Lp(a) types are controlled by a series of codominant alleles Lp^F, Lp^B, Lp^S1, Lp^S2, Lp^S3 and Lp^S4 and by a null allele Lp^o. Together with the data from the accompanying paper this indicates that the structural gene for the Lp(a) protein is the major gene locus determining Lp(a) lipoprotein concentrations in plasma.     

Genetics of the quantitative Lp(a) lipoprotein trait

Summary Apolipoprotein(a) [apo(a)] is a large serum glycoprotein with several genetically determined isoforms differing in their apparent molecular weight. We determined the effects of the apo(a) isoforms on total cholesterol, high-density lipo-protein (HDL)-cholesterol, lipoprotein(a), and triglyceride levels in a sample of 473 unrelated Tyrolean adults. Average lipoprotein(a) and total cholesterol levels were significantly different among apo(a) types. These significant differences were found among the 13 apo(a) isoform patterns observed in this sample and among several logical subsets of the isoform patterns (e.g. considering only the single band types). The data suggest that the effects of apo(a) alleles on Lp(a) levels are additive. The effects of apo(a) on total cholesterol levels cannot be entirely explained by the cholesterol fraction estimated to be contained in the lipoprotein(a) particle. We estimate that the apo(a) glycoprotein polymorphism accounts for 41.9% and 9.6% of the variability in lipoprotein(a) and total cholesterol levels, respectively. This is the strongest effect of a single polymorphic gene on plasma lipid and lipoprotein levels reported so far.     

Hypochlorite oxidation causes cross-lingking of Lp(a)

When purified low density lipoprotein (LDL) or lipoprotein(a) (Lp[a]) was oxidized in vitro using concentrations of hypochlorite (50–500 μM) which might be achieved by activated neutrophils in vivo, high molecular weight species were observed on SDS polyacrylamide gels. The reaction was concentration-, temperature- and time-dependent. The high molecular weight apoprotein complexes were resistant to heating in SDS and DTT, suggesting covalent, but non-disulfide bond, cross-linking. Negligible amounts of lower molecular weight degradation produts were formed. Bityrosine formation, measured by fluorescence and HPLC analysis, was found to increase with the amount of hypochlorite added. However, the molar concentration of bityrosine could not account for cross-linking, even if it was assumed that every bityrosine was intermolecular. Hypochlorite-oxidized Lp(a) and LDL were both effective as ligands for loading mouse peritoneal macrophages in vitro. We conclude that hypochlorite produced in inflammatory reactions might be important in the generation of atherogenic forms of lipoproteins.     

Electroelution of lipoprotein(a) [Lp(a)] from native polyacrylamide gels: a new, simple method to purify Lp(a)

Lipoprotein(a), Lp(a), is an atherogenic lipoprotein consisting of an LDL like core particle and a covalently linked glycoprotein of variable size. Lp(a), isolated from serum always contains LDL and HDL(2) as contaminants since Lp(a) floats in the density range 1.05-1.12 g/ml which overlaps that of LDL and HDL(2). Purified Lp(a) is increasingly needed as a standard to overcome various problems in the standardization of Lp(a) measurements and for in vitro biological studies. Problems inherent to the purification of Lp(a) include the aggregation of Lp(a) with LDL, overlapping size distribution and the inability of some fractions to bind to affinity columns. Here, we describe the development of a new method to purify Lp(a) from contaminating LDL and HDL(2) particles. Lp(a) was isolated from serum by sequential ultracentrifugation, resolved by native polyacrylamide gel electrophoresis and the gel segments were electroeluted to obtain pure Lp(a). l-Proline was added to the sample to a final concentration of 0.1 M to prevent the aggregation of Lp(a) with LDL.     

Quantitative genetic studies of the human plasma Lp(a) lipoprotein

Lp(a) lipoprotein [Lp(a)] was quantified in 1251 adults, including 300 mother-father-offspring triplets, by a sensitive radial immunodiffusion assay. Lp(a) was not correlated with age, sex, or cholesterol or glyceride concentrations. Significant correlations were found between mother-offspring (r=0.34), father-offspring (r=0.40), and midparent-offspring (r=0.52), whereas no correlation was found between husband-wife pairs (r=0.02). Analysis of triplets separated on the basis of midparent Lp(a) concentrations indicated a resemblance of midparent to offspring regardless of midparent concentration. Bimodality was not detected in any of the offspring distributions. These data are compatible with the hypothesis that the observed quantitative Lp(a) variation is determined by a polygenic model of inheritance. The possibility of major gene effects is not ruled out.     

Linkage between the loci for the Lp(a) lipoprotein (LP) and plasminogen (PLG)

Summary A locus, LP, that determines quantitative variation of Lp(a) lipoprotein phenotypes is linked to the plasminogen (PLG) locus (peak lod score =12.73). This linkage relationship assigns a locus with alleles that have an affect on risk for coronary artery disease to the long arm of chromosome 6.     

The Apo(a) gene is the major determinant of variation in plasma Lp(a) levels in African Americans.

The distributions of plasma lipoprotein(a), or Lp(a), levels differ significantly among ethnic groups. Individuals of African descent have a two- to threefold higher mean plasma level of Lp(a) than either Caucasians or Orientals. In Caucasians, variation in the plasma Lp(a) levels has been shown to be largely determined by sequence differences at the apo(a) locus, but little is known about either the genetic architecture of plasma Lp(a) levels in Africans or why they have higher levels of plasma Lp(a). In this paper we analyze the plasma Lp(a) levels of 257 sibling pairs from 49 independent African American families. The plasma Lp(a) levels were much more similar in the sibling pairs who inherited both apo(a) alleles identical by descent (IBD) (r = .85) than in those that shared one (r = .48) or no (r = .22) parental apo(a) alleles in common. On the basis of these findings, it was estimated that 78% of the variation in plasma Lp(a) levels in African Americans is attributable to polymorphism at either the apo(a) locus or sequences closely linked to it. Thus, the apo(a) locus is the major determinant of variation in plasma Lp(a) levels in African Americans, as well as in Caucasians. No molecular evidence was found for a common "high-expressing" apo(a) allele in the African Americans. We propose that the higher plasma levels of Lp(a) in Africans are likely due to a yet-to-be-identified trans-acting factor(s) that causes an increase in the rate of secretion of apo(a) or a decrease in its catabolism.     

Disk-elektrophoretischer Nachweis des Lp(a)-Proteins in Lipoproteinfraktionen

Zusammenfassung Die Untersuchung der Serumlipoproteinfraktion HDL₂ (1,063< <1,125 g/ml) von 35 Personen mit Hilfe der Polyacrylamid-Gelelektrophorese zeigte, daß auch bei Anwendung dieser empfindlichen Technik eine eindeutige Einteilung in Lp(a+)- und Lp(a-)-Typen nicht möglich ist. Von den 20 Seren, die durch Agar-Gel-Doppeldiffusion als Lp(a-) bestimmt wurden, zeigten 19 in ihrer HDL₂-Fraktion in der für das Lp(a)-Lipoprotein typischen Position eine Bande. Dieser Befund bestätigt die Annahme, daß der Faktor Lp(a) als ein quantitatives genetisches Merkmal angesehen werden muß.Die -Lipoproteine des HDL₂-Bereiches erwiesen sich als disk-elektrophoretisch heterogen.

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Determination of the Lp(a)-protein by disc-electrophoresis of lipoprotein-fractions

Summary Gelelectrophoretic studies of the serum lipoprotein fraction HDL₂ (1.063< <1.125 g/ml)_of 35 individuals showed, that using this technique no clear classification into Lp(a+) and Lp(a-)-types could be made. 19 out of 20 sera typed Lp(a-) by double diffusion contained in their HDL₂-fraction a lipoprotein with the electrophoretic mobility characteristic of the Lp(a)-lipoprotein. This observation confirmes previous suggestions, that Lp(a) indeed is a quantitative genetic trait.The -lipoproteins of the HDL₂-fraction are heterogeneous in gelelectrophoresis.

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Untersuchung mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Recombinant adenovirus vector mediated expression of lipoprotein (a) [Lp(a)] in rabbit plasma.

Lipoprotein (a) [Lp(a)] is a heterodimer of apolipoprotein (a) [apo(a)] and apolipoprotein B-100 (apoB-100) of low density lipoprotein linked by a disulfide bond. Apo(a) and apoB-100 are synthesized by the liver and covalently associate or couple to form Lp(a) extracellularly. Elevated plasma Lp(a) is an independent risk factor for vascular injury disorders such as restenosis after balloon angioplasty and accelerated graft atherosclerosis following heart transplantation. Lp(a) is not expressed in laboratory animals making studies of its pathophysiology difficult. To overcome this problem, we explored the possibility of generating Lp(a) in rabbit plasma using replication-deficient adenovirus vector mediated gene delivery. Rabbits were chosen because of their large vessels and unlike mouse or rat, rabbit apoB-100 could interact with apo(a) to generate Lp(a). The recombinant (r) adenovirus vector construct used encoded a 200 kDa apo(a) [Ad-apo(a)]. Ad-apo(a) injection into the rabbit marginal vein caused the appearance of plasma rLp(a). Injection of a r adenovirus vector expressing the bacterial LacZ gene (Ad-LacZ) or PBS (vehicle) did not result in detectable plasma rLp(a). These are the first results to demonstrate plasma expression of rLp(a) in rabbits using adenovirus vector mediated gene transfer. Therefore, this system may be suitable for investigating Lp(a)'s role in the development of vascular injury diseases in a rabbit model.     

Protein composition of Lp(a) lipoprotein from human plasma

The apolipoprotein composition of purified human Lp(a) lipoprotein was investigated by SDS--polyacrylamide gel electrophoresis and immunochemically. The lipoprotein contains two different polypeptides. One is identical by its app. Mr of approximately 250 000 and immunologically with apolipoprotein B of LDL (B-100). The other polypeptide has a higher app. Mr (approximately 350 000) and stains strongly with the periodate-Schiff's reagent. This high-Mr glycoprotein contains the specific Lp(a) immunoreactivity but does not react with antibodies against apo B. Apo B and Lp(a)-protein seem to be linked by disulfide bonds in the native lipoprotein. The unreduced detergent delipidized protein moiety from Lp(a) lipoprotein shows a single band of Mr approximately 700 000 in SDS--polyacrylamide gel electrophoresis and the immunoprecipitates formed against anti-Lp(a) and anti-apo B by the unreduced protein show a reaction of immunological identity.