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.     

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.     

Characterization of five mouse monoclonal antibodies to apolipoprotein[a] from human Lp[a]: evidence for weak plasminogen reactivity

We describe the development of five murine monoclonal antibodies (14A12, 39A1, 53A9, 73A7, and 128A6) specific to human apolipoprotein[a] (Mr approximately 570,000), and their characterization by a number of procedures including cotitration, competition and inhibition enzyme-linked immunosorbent assays (ELISA), immunoblotting of native lipoproteins and of SDS-solubilized apolipoproteins electrophoresed in polyacrylamide gels, and dot immunobinding assays. The patterns of immunoreactivity of these antibodies were similar. Each reacted in ELISA assays and upon electroimmunoblotting with purified apo[a], with apo[a] liberated by reduction of Lp[a], and with delipidated Lp[a] solubilized in SDS, but by contrast, they reacted with native Lp[a] to a significant degree only upon electroimmunoblotting. No reactivity was seen with LDL-apoB-100 or with other apolipoproteins. The cross-reactivity of these antibodies with the homologous protein, plasminogen, was examined by comparison of the amount of plasminogen or apo[a] required for 50% inhibition of antibody binding to apo[a], and by an ELISA assay. The inhibition assay showed reactivity with plasminogen to be 37- to 50-fold lower than with apo[a], while dot immunobinding showed the lower limit of detection of plasminogen and of apo[a] to be approximately 320 and 31 micrograms, respectively. In an ELISA sandwich assay based on monoclonal antibodies LHLP-1, 14A12, and 53A9, the lower limit of Lp[a] detection (approximately 1 ng/ml protein) was about 100-fold less than that of plasminogen. Chemical modification of apo[a] revealed a significant contribution of arginine residues to the epitopes of 14A12, 39A1, and 53A9. Modification of cysteine residues with iodoacetamide was without effect, thereby distinguishing these antibodies from LHLP-1. Each antibody reacted with the six major size forms of apo[a] (Mr approximately 450,000-750,000) in immunoblots of human sera electrophoresed in SDS-polyacrylamide gels. Marked heterogeneity in apo[a] phenotype was detected and both single and double band phenotypes were observed in a randomized study. Cotitration and competition binding studies showed varying degrees of interaction between all five epitopes, with the exception of 128A6 which appeared to be independent of 39A1 and 53A9 (and vice versa). These data suggest that our five monoclonal antibodies recognize epitopes on apolipoprotein[a] that are exposed and accessible on the native Lp[a] particle. We conclude that our monoclonal antibodies recognize a specific region of apo[a], and that this region undergoes a conformational change upon adsorption of Lp[a] to plastic thereby diminishing epitope recognition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Relationship between apo[a] isoforms and Lp[a] density in subjects with different apo[a] phenotype: a study before and after a fatty meal

Plasma Lp[a] levels and apo[a] isoform distribution among lipoproteins isolated by density gradient ultracentrifugation were studied in subjects with one-band or two-band apo[a] phenotypes as assessed by gradient gel electrophoresis before and after an oral fat load. There were no significant differences in the ultracentrifugal profile between fasting plasma and postprandial plasma that was freed of triglyceride-rich particles (TRP). One-band phenotypes exhibited a single symmetrical peak in the density gradient, whereas two-band phenotypes exhibited a multi-modal distribution. Low molecular weight apo[a] isoforms were preferentially associated with low density Lp[a] whereas high molecular weight apo[a] isoforms were found with high density Lp[a] particles. Feeding a high fat meal caused no significant increase in the total plasma level of Lp[a]. However, the isolated TRP contained the apoB-100-apo[a] complex in a quantity that represented only about 1% of its total amount in the fasting plasma. In all cases the apo[a] isoforms present in TRP were also present in the fasting plasma; however, in the two-band apo[a] phenotypes the ratio of the slow over the fast migrating band was in all cases about eightfold higher in TRP than in the fasting plasma. These observations indicate that postprandially a small percentage of apoB-100-apo[a] associates with TRP and suggest that this complex may derive from de novo synthesis rather than from a pre-existing Lp[a] plasma pool. The liver would be the source of the complex due to the presence in the latter of apoB-100.     

Differences in apolipoprotein(a) polymorphism in West Greenland Eskimos and Caucasian Danes

Summary Previous studies in Greenland suggest that death rates from ischemic heart disease [IHD] are lower in Eskimos than in Danes and other Caucasian populations. This has been explained by a high intake of n-3 polyunsaturated fatty acids with beneficial effects on blood lipids and hemostasis. In other populations, lipoprotein(a) [Lp(a)] is associated with IHD, plasma concentrations of Lp(a) being genetically determined to a major extent. We have compared Lp(a) concentrations and apo(a) phenotypes in 120 Greenlandic Eskimos with those in 466 Danish men. The median Lp(a) concentration in Eskimos (8.7mg/dl;[95% CI 6.5–10.7]) was not significantly different from that in Danes (6.3mg/dl; [95% CI 5.2–7.0]), whereas the 90th percentile was significantly higher among Danes: 46.36mg/dl; [95% Cl 43.0–54.3] vs. 27.6mg/dl [95% CI 20.7–36.9]. In 20% of the Danes, but in only 8% of the Eskimos (P = 0.009), the concentration of Lp(a) exceeded 30mg/dl. The difference is probably explained by a low frequency of the low molecular weight apo(a) phenotypes among Eskimos, since the apo(a) isoforms F and B were absent, and the S1 and S2 types were present in only 3.3% of Eskimos. In contrast, these apo(a) isoforms were present in 26.6% of the Danes in either single-band or double-band phenotypes. The pattern of apo(a) polymorphism found in this study could provide part of a genetic explanation for the putative low rates of IHD in Eskimo populations.     

Isolation, quantitation, and characterization of a stable complex formed by Lp[a] binding to triglyceride-rich lipoproteins.

Lipoprotein [a] (Lp[a]) is a cholesterol-rich lipoprotein resembling LDL to which a large polymorphic glycoprotein, apolipoprotein [a] (apo[a]), is covalently coupled. Lp[a] usually exists as a free-standing particle in normolipidemic subjects; however, it can associate noncovalently with triglyceride-rich lipoproteins in hypertriglyceridemic (HTG) subjects. In this study, 10-78% of the Lp[a] present in five HTG subjects was found in the triglyceride-rich lipoprotein (TRL) fraction. The Lp[a]-TRL complex was resistant to dissociation by ultracentrifugation (UCF) alone, but was quantitatively dissociated by UCF in the presence of 100 mM proline. Of this dissociated Lp[a], 70-88% was in the form of a lipoprotein resembling conventional Lp[a]. Incubation of Lp[a]-depleted TRL with native Lp[a] resulted in a reconstituted Lp[a]-TRL complex that closely resembled the native isolates in all examined properties. Complex formation was inhibited by several compounds in the order proline > tranexamate > epsilon-aminocaproate > arginine > lysine. Neither plasminogen nor LDL inhibited binding of Lp[a] to TRL. We observed the preferential binding of Lp[a] containing higher apparent molecular weight apo[a] polymorphs to TRL both in native and reconstituted Lp[a]-TRL complexes. A disproportionate amount of Lp[a] was bound to the larger TRL particles. Although most apo[a] bound to TRL was in the form of conventional Lp[a] particles, lipid-free recombinant apo[a] was observed to bind TRL.These results provide unequivocal evidence of the existence of an Lp[a]-TRL complex under pathophysiologic conditions. The metabolic fate of the Lp[a]-TRL complex, which is more abundant in hypertriglyceridemia, may be different from that of conventional Lp[a], and may contribute uniquely to the progression or severity of cardiovascular disease.     

Interaction of lipoprotein Lp[a] with the B/E-receptor: a study using isolated bovine adrenal cortex and human fibroblast receptors

The ability of different lipoprotein Lp[a] preparations to compete with LDL-binding to the B/E-receptor was investigated by ligand blot and filter assays. Lp[a] was purified from donors with various genetic polymorphic forms by affinity chromatography using lysine-Sepharose or specific immunoadsorbers. These preparations were free of "LDL-like" material. Part of Lp[a] was reduced and freed from specific apo[a] antigen yielding "Lpa-." 125I-labeled low density lipoproteins (LDL) were incubated with B/E-receptor preparations from bovine adrenal cortex or from human skin fibroblasts, and the competition with unlabeled LDL, Lp[a], Lpa-, apo[a], and apoE-free HDL was studied by a ligand blot or filter assay technique. The following results were obtained. 1) LDL and Lpa- were equally potent in displacing 125I-labeled from B/E-receptor in the ligand blot and the filter assay. Lpa + ( = Lp[a]) also displaced LDL but to a much lesser degree: 50% displacement was observed with LDL and Lpa- at a 1-fold excess, whereas a 7.5-fold excess was required of Lpa +. 2) Apo[a], as well as apoE-free HDL, did not compete with LDL binding. 3) Competition experiments using B/E-receptors from bovine adrenal cortex or from human skin fibroblasts were comparable. 4) There was no difference in the behavior of Lp[a] isolated from the two affinity chromatography methods. 5) Lp[a] of different genetic variants behaved virtually identically. The results are discussed from the point of view of the in vivo metabolism of Lp[a].     

Quantification of apo[a] and apoB in human atherosclerotic lesions.

Lipoprotein[a] or Lp[a] is a cholesterol-rich plasma lipoprotein that is associated with increased risk for cardiovascular disease. To better understand this association we determined the amount of apo[a] and apoB as possible estimates for Lp[a] and low density lipoprotein (LDL) accumulation in atherosclerotic lesions and in plasma, from patients undergoing vascular surgery, using specific radioimmunoassays for apolipoprotein[a] and apolipoprotein B. Apo[a] and apoB were operationally divided into a loosely bound fraction obtained by extracting minced samples of plaque with phosphate-buffered saline (PBS), and a tightly bound fraction obtained by extracting the residual tissue with 6 M guanidine-HCl (GuHCl). We found that 83% of all apo[a] but only 32% of all apoB in lesions was in the tightly bound fraction. When normalized for corresponding plasma levels, apo[a] accumulation in plaques was more than twice that of apoB. All fractions of tissue apo[a], loosely bound, tightly bound, and total, correlated significantly with plasma apo[a]. However, no significant correlations were found between any of the tissue fractions and plasma apoB. If all apo[a] and apoB had been associated with intact Lp[a] or LDL particles, the calculated mass of tightly bound Lp[a] would actually have exceeded that of tightly bound LDL in five cases with plasma Lp[a] levels above 5 mg apo[a] protein/dl. When PBS and GuHCl extracts of lesions were subjected to one-dimensional electrophoresis, the major band stained for lipid and immunoblotted positively for apo[a] and apoB, suggesting the presence of some intact Lp[a] in these extracts. These results suggest that Lp[a] accumulates preferentially to LDL in plaques, and that plaque apo[a] is directly associated with plasma apo[a] levels and is in a form that is less easily removable than most of the apoB. This preferential accumulation of apo[a] as a tightly bound fraction in lesions, could be responsible for the independent association of Lp[a] with cardiovascular disease in humans.     

Apolipoprotein [a] genotype influences isoform dominance pattern differently in African Americans and Caucasians

Plasma lipoprotein [a] (Lp[a]) concentrations are inversely associated with, and largely determined by, apolipoprotein [a] (apo[a]) gene size, a highly polymorphic trait. We studied if, within an individual, the smaller apo[a] isoform always dominated, whether there was interaction between the two alleles, and whether these features differed between Caucasians and African Americans. We determined apo[a] gene sizes, apo[a] protein sizes and relative amounts, and plasma Lp[a] levels in 430 individuals (263 Caucasians and 167 African Americans). Of the 397 heterozygotes with at least one detectable apo[a] isoform (238 Caucasians and 159 African Americans), the larger allele dominated in 28% of Caucasians and 23% of African Americans, while the smaller allele dominated in 56% of Caucasians and 45% of African Americans. In Caucasians, dominance of the smaller allele increased with Lp[a] levels, from 44% at Lp[a] < or = 30 nM to 81% at Lp[a] >100 nM (P < 0.0001). Dominance by the smaller allele increased with increasing size of the larger allele in both groups but with the smaller allele only in African Americans. There was no interaction between apo[a] alleles within genotypes; one apo[a] isoform level was not associated with the other isoform level, and isoform levels were not affected by the difference in size. More of the dominance pattern was explained by Lp[a] level and apo[a] genotype in African Americans than in Caucasians (29% vs. 13%). Thus, genotype influences isoform-specific Lp[a] levels and dominance patterns differently in African Americans and in Caucasians.     

Enzyme-linked immunoassay for Lp[a]

Based on our findings that rabbit antisera raised against human Lp[a] or apo[a] have the potential to cross-react with plasminogen, and in some cases have nearly equal affinities for plasminogen and Lp[a], we have developed an assay for plasma Lp[a] based on a "sandwich" ELISA that is insensitive to the presence of plasminogen. This was accomplished through the use of anti-apo[a] as a capture antibody and quantitation of the bound Lp[a], i.e., the apoB-100-apo[a] complex, with an anti-apoB antibody. Although apo[a] is heterogeneous in size, all Lp[a] particles tested, either in pure form or contained in whole plasma, gave parallel dose-response curves and were immunologically equivalent. However, when purified Lp[a] particles with different apo[a] isoforms were studied, those having larger isoforms were, on a weight basis, less reactive than those having a smaller size. Nearly equivalent reactivity was observed when protein concentration was expressed on a molar basis. The distribution of Lp[a] in a population of 84 subjects was skewed with one-third of the individuals having less than 1 mg/dl Lp[a] protein. All subjects tested had measurable concentrations of Lp[a] with a lower limit of detection of 0.030 mg/dl Lp[a] protein. The mean level was 3.2 mg/dl with a range of 0.045 to 13.3 mg/dl. These studies demonstrate the successful development of an ELISA for Lp[a] protein that is insensitive to the presence of plasminogen; that heterogeneity of Lp[a] and apo[a] are an important source of variation in the assay; and the need for an appropriate Lp[a] standard in order to minimize this variation.     

A quantitative assay for the non-covalent association between apolipoprotein[a] and apolipoprotein B: an alternative measure of Lp[a] assembly

Increasing evidence suggests that the assembly of lipoprotein[a] (Lp[a]) proceeds in two steps. In the first step, non-covalent interactions between apolipoprotein[a] (apo[a]) and apolipoprotein B (apoB) of low density lipoprotein (LDL) form a dissociable apo[a]:LDL complex. In the second step, a covalent disulfide linkage forms the stable Lp[a] particle. Several methods are currently used to study the assembly of Lp[a], however, these methods are laborious, time-consuming, and not suitable for a high throughput screening. We report here the development of a rapid and simple assay based on the binding of labeled LDL to a Lp[a]/apo[a] substrate which is immobilized on the surface of a microtiter plate. Quantification of bound LDL provides a measure of the extent of complex formation. Labeled LDL bound to both Lp[a] and apo[a] substrates with similar affinity. Plasma lipoproteins containing apoB as well as free apo[a] were capable of competing with LDL binding. The binding of LDL to Lp[a]/apo[a] was inhibited by L-proline and lysine analogs, which are known to inhibit the non-covalent association between apo[a] and apoB. Using this method we have found that nicotinic acid and captopril are able to inhibit the association of apo[a] with apoB. This method is compatible with automation and can be applied to a high throughput screening of inhibitors of Lp[a] formation.     

ApoE genotype affects allele-specific apo[a] levels for large apo[a] sizes in African Americans: the Harlem-Basset Study

The genetic variability of apolipoprotein E (apoE) influences plasma lipoprotein levels, and allele frequencies differ between African Americans and Caucasians. As African Americans have higher lipoprotein [a] (Lp[a]) levels than Caucasians, we investigated the effects of the apoE gene on allele-specific apolipoprotein [a] (apo[a]) levels across ethnicity. We determined apo[a] sizes, allele-specific apo[a] levels (i.e., levels associated with alleles defined by size), and the apoE gene polymorphism in 231 African Americans and 336 Caucasians. African Americans, but not Caucasians, with the apo E2 genotype had lower levels of Lp[a] compared with those with the apo E4 genotype (9.6 vs. 11.2 nmol/l; P = 0.034, expressed as square root levels). Distribution of apo[a] alleles across apoE genotypes were similar between African Americans and Caucasians. Among African Americans with large apo[a], the allele-specific apo[a] level was significantly lower among epsilon2 carriers compared with epsilon3 or epsilon4 carriers (5.4 vs. 6.6 and 7.4 nmol/l, respectively; P < 0.005, expressed as square root levels). In contrast, there was no significant difference in allele-specific apo[a] levels across apoE genotypes among Caucasians. For large apo[a] sizes, apoE genotype contributed to the observed African American-Caucasian differences in allele-specific apo[a] levels.     

A structural assessment of the apo[a] protein of human lipoprotein[a].

Apolipoprotein[a], the highly glycosylated, hydrophilic apoprotein of lipoprotein[a] (Lp[a]), is generally considered to be a multimeric homologue of plasminogen, and to exhibit atherogenic/thrombogenic properties. The cDNA-inferred amino acid sequence of apo[a] indicates that apo[a], like plasminogen and some zymogens, is composed of a kringle domain and a serine protease domain. To gain insight into possible positive functions of Lp[a], we have examined the apo[a] primary structure by comparing its sequence with those of other proteins involved in coagulation and fibrinolysis, and its secondary structure by using a combination of structure prediction algorithms. The kringle domain encompasses 11 distinct types of repeating units, 9 of which contain 114 residues. These units, called kringles, are similar but not identical to each other or to PGK4. Each apo[a] kringle type was compared with kringles which have been shown to bind lysine and fibrin, and with bovine prothrombin kringle 1. Apo[a] kringles are linked by serine/threonine- and proline-rich stretches similar to regions in immunoglobulins, adhesion molecules, glycoprotein Ib-alpha subunit, and kininogen. In comparing the protease domains of apo[a] and plasmin, apo[a] contains a region between positions 4470 and 4492 where 8 substitutions, 9 deletions, and 1 insertion are apparent. Our analysis suggests that apo[a] kringle-type 10 has a high probability of binding to lysine in the same way as PGK4. In the only human apo[a] polymorph sequenced to date, position 4308 is occupied by serine, whereas the homologous position in plasmin is occupied by arginine and is an important site for proteolytic cleavage and activation. An alternative site for the proteolytic activation of human apo[a] is proposed.     

High-level lipoprotein [a] expression in transgenic mice: evidence for oxidized phospholipids in lipoprotein [a] but not in low density lipoproteins

Efforts to elucidate the role of lipoprotein [a] (Lp[a]) in atherogenesis have been hampered by the lack of an animal model with high plasma Lp[a] levels. We produced two lines of transgenic mice expressing apolipoprotein [a] (apo[a]) in the liver and crossed them with mice expressing human apolipoprotein B-100 (apoB-100), generating two lines of Lp[a] mice. One had Lp[a] levels of approximately 700 mg/dl, well above the 30 mg/dl threshold associated with increased risk of atherosclerosis in humans; the other had levels of approximately 35 mg/dl. Most of the LDL in mice with high-level apo[a] expression was covalently bound to apo[a], but most of the LDL in the low-expressing line was free. Using an enzyme-linked sandwich assay with monoclonal antibody EO6, we found high levels of oxidized phospholipids in Lp[a] from high-expressing mice but not in LDL from low-expressing mice or in LDL from human apoB-100 transgenic mice (P <0.00001), even though all mice had similar plasma levels of human apoB-100. The increase in oxidized lipids specific to Lp[a] in high-level apo[a]-expressing mice suggests a mechanism by which increased circulating levels of Lp[a] could contribute to atherogenesis.     

A novel function of lipoprotein [a] as a preferential carrier of oxidized phospholipids in human plasma

Oxidized phospholipids (OxPLs) on apolipoprotein B-100 (apoB-100) particles are strongly associated with lipoprotein [a] (Lp[a]). In this study, we evaluated whether Lp[a] is preferentially the carrier of OxPL in human plasma. The content of OxPL on apoB-100 particles was measured with monoclonal antibody E06, which recognizes the phosphocholine (PC) headgroup of oxidized but not native phospholipids. To assess whether OxPLs were preferentially bound by Lp[a] as opposed to other lipoproteins, immunoprecipitation and ultracentrifugation experiments, in vitro transfer studies, and chemiluminescent ELISAs were performed. Immunoprecipitation of Lp[a] from human plasma with an apolipoprotein [a] (apo[a])-specific antibody demonstrated that more than 85% of E06 reactivity (i.e., OxPL) coimmunoprecipitated with Lp[a]. Ultracentrifugation experiments showed that nearly all OxPLs were found in fractions containing apo[a], as opposed to other apolipoproteins. In vitro transfer studies showed that oxidized LDL preferentially donates OxPLs to Lp[a], as opposed to LDL, in a time- and temperature-dependent manner, even in aqueous buffer. Approximately 50% of E06 immunoreactivity could be extracted from isolated Lp[a] following exposure of plasma to various lipid solvents. These data demonstrate that Lp[a] is the preferential carrier of PC-containing OxPL in human plasma. This unique property of Lp[a] suggests novel insights into its physiological function and mechanisms of atherogenicity.     

A selective bi-site immunoenzymatic procedure for human Lp[a] lipoprotein quantification using monoclonal antibodies against apo[a] and apoB

A selective bi-site ELISA assay procedure for quantification of Lp[a] lipoprotein in human plasma based on linkage of apo[a] to apoB is described. The lipoproteins referred to as apo[a]:B were captured by a mixture of two anti-apo[a] monoclonal antibodies (K07, K09) and were revealed by a mixture of six anti-apoB monoclonal antibodies coupled to peroxidase. Since apo[a] and plasminogen have striking similarities in protein structure, the selective binding of Lp[a]:B in our assay depended upon the marked difference in affinity of the K07 and K09 mixture for Lp[a]:B (Kd = 0.32 x 10(-10) M) versus plasminogen (Kd = 0.47 x 10(-7)M). The high sensitivity (the Lp[a]:B working range 0.06-0.40 micrograms/ml) and the use of anti-apoB as antibody tracer added to the selectivity of the assay. The expression of K07 and K09 epitopes determined by competitive inhibition method and the reactivity of Lp[a]:B particles measured by bi-site ELISA were similar on individual lipoproteins, independent to their plasma levels. The assay is precise, and intra- and interassay coefficients of variation were 4.7% and 9.6%, respectively. It yields quantitative Lp[a]:B values that correlate highly with Lp[a] levels obtained by electroimmunoassay with polyclonal antibody (r = 0.73) or with Lp[a] levels measured by the other bi-site ELISA using only K07 and K09 antibodies (r = 0.96). However, upon analyzing each individual plasma with an arbitrary Lp[a]-cut off of 15 mg/dl, evidence of the qualitative aspect of the lipoprotein was obtained. The group with Lp[a] less than 15 mg/dl had higher frequency of subjects (65%) with the ratio Lp[a]/Lp[a]:B above 1.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chimpanzee lipoprotein(A): Relationship between apolipoprotein(A) isoform size and the density profile of lipoprotein(A) in animals with different heterozygous apo(A) phenotypes

In a previous study [C. Doucet et al., J. Lipid Res 35:263–270, 1994], we have shown that plasma lipoprotein (a) [Lp(a)] levels were significantly elevated in a population of unrelated chimpanzees as compared to those in normolipidemic human subjects. Nonetheless, the inverse correlation between Lp(a) levels and apolipoprotein (a) [apo(a)] isoforms typical of man was maintained in the chimpanzee. In the present study, we describe the density profiles of apo B- and apo A1-containing lipoproteins and of Lp(a) in chimpanzee plasmas heterozygous for apo(a) isoforms after fractionation by single spin ultracentrifugation in an isopycnic gradient. The distribution of apo(a) isoforms in the density gradient was also examined by SDS-agarose gel electrophoresis and immunoblotting using chemiluminescence detection. In all double-band phenotypes examined, the smallest isoform was present along the entire length of the density gradient. The density distribution of the second isoform varied according to the size difference between the respective isoforms. Two isoforms close in size (difference in apparent molecular mass ? 60 kDa) were present together in every gradient subfraction. On the contrary, when the two isoforms displayed distinct molecular mass (maximal difference in apparent molecular mass = 340 kDa), then the largest was principally present in the densest fractions of the gradient (d > 1.1 mg/ml). These observations suggest that Lp(a) particles with small apo(a) isoforms are more susceptible to interact with other lipoproteins than are Lp(a) particles with large isoforms.     

In vivo fate and scavenger receptor recognition of oxidized lipoprotein[a] isoforms in rats.

High levels of Lp[a] in blood form an independent risk factor for atherosclerosis. Oxidative modification of Lp[a] may be involved in the suggested atherogenic action of Lp[a]. After Cu(2+)-mediated oxidative modification of the 440 kDa and 610 kDa apo[a] isoforms of lipoprotein[a] (Ox-Lp[a]), the in vivo fate was investigated in rats. Ox-Lp[a], when injected into rats, was rapidly removed from the blood circulation by the liver, in which the intrahepatic fate is dependent on the degree of oxidation of the isoforms. Upon oxidation to a slightly increased negative charge of Lp[a], the high molecular weight form of Lp[a] is recognized more efficiently by the Kupffer cells than by the endothelial cells. When the liver uptake of Ox-Lp[a] is blocked by preinjection of polyinosinic acid (poly I), the association of Ox-Lp[a] with the rat heart is increased 20-fold. In vitro studies show that the association and degradation of 125I-labeled Ox-Lp[a] with liver endothelial and Kupffer cells was inhibited by oxidized LDL (Ox-LDL), poly I, or Ox-Lp[a] itself by 60-90%, while only a partial competition was found with acetylated-LDL (up to 25%). In conclusion, after oxidative modification of Lp[a], there is recognition of Ox-Lp[a] by specific oxidized-lipoprotein receptors on liver endothelial and Kupffer cells; the relative importance at low degrees of oxidation of Lp[a] is dependent on the molecular weight of the apo[a] isoforms. Under conditions in which liver uptake is not adequate, the deposition of Ox-Lp[a] in the heart may be of potential pathological importance.     

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.     

Characterization of the basis of lipoprotein [a] lysine-binding heterogeneity

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.