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.     

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.     

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.     

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.     

Evaluation of Lp[a] and other independent risk factors for CHD in Asian Indians and their USA counterparts

Conventional risk factors for coronary heart disease (CHD) do not completely account for the observed increase in premature CHD in people from the Indian subcontinent or for Asian Indians who have immigrated to the USA. The objective of this study was to determine the effect of immigration to the USA on plasma levels of lipoprotein [a] (Lp[a]) and other independent risk factors for CHD in Asian Indians. Three subject groups were studied: group 1, 57 subjects living in India and diagnosed with CHD (CHD patients); group 2, 46 subjects living in India and showing no symptoms of CHD (control subjects); group 3, 206 Asian Indians living in the USA. Fasting blood samples were drawn to determine plasma levels of triglyceride (TG), total cholesterol (TC), low density lipoprotein [LDL cholesterol (LDL-Chol)], high density lipoprotein [HDL cholesterol (HDL-Chol)], apolipoprotein B-100 (apoB-100), and Lp[a]. Apolipoprotein [a] (apo[a]) size polymorphism was determined by immunoblotting. Plasma TG, apoB-100, and Lp[a] concentrations were higher in CHD patients than in control and USA groups. CHD patients had higher levels of TC and LDL-Chol and lower HDL-Chol than control subjects. However, the USA population had higher levels of TC, LDL-Chol, and apoB-100 and lower HDL-Chol than control subjects. Plasma Lp[a] levels were inversely correlated with the relative molecular weight of the more abundant of each subject's two apo[a] isoforms (MAI), and CHD patients showed higher frequencies of lower relative molecular weights among MAI. Our observed changes in lipid profiles suggest that immigrating to the USA may place Asian Indians at increased risk for CHD. This study suggests that elevated plasma Lp[a] confers genetic predisposition to CHD in Asian Indians, and nutritional and environmental factors further increase the risk of CHD. This is the first report implicating MAI size as a predictor for development of premature CHD in Asian Indians. Including plasma Lp[a] concentration and apo[a] phenotype in screening procedures may permit early detection and preventive treatment of CHD in this population.     

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)     

Evaluation of a new apolipoprotein(a) isoform-independent assay for serum Lipoprotein(a)

The risk factor, Lipoprotein(a), [(Lp(a)], has been measured in numerous clinical studies by a variety of immunochemical assay methods. It is becoming apparent that for many of these assays antibody specificity towards the apolipoprotein(a) [apo(a)] repetitive component [the kringle 4 - type 2 repeats] and apo(a) size heterogeneity can significantly affect the accuracy of serum Lp(a) measurements. To address this issue, we investigated whether our current in house Lp(a) [Mercodia] assay showed such bias compared to a recently available assay [Apo-Tek], claiming to possess superior capability for isoform-independent measurement of Lp(a). Levels of Lipoprotein(a) by both Apo-Tek and Mercodia assays correlated inversely with apo(a) isoform sizes. No significant differences were observed between assays in ranges of Lp(a) concentration within each isoform group. The Mercodia assay exhibited similar isoform-independent behaviour to that of Apo-Tek for e quantitation of serum Lipoprotein(a). Essentially identical results were obtained by the two methods, suggesting that Mercodia assay's capture monoclonal antibody also (as is the case for Apo-Tek) does not recognize the kringle 4-type 2 repetitive domain of apo(a). Correlation of Lp(a) concentrations in patient specimens between Apo-Tek and Mercodia assays showed good agreement, although an overall higher degree of imprecision and non-linearity was noted for the Apo-Tek procedure. A change-over to the Apo-Tek assay would therefore not improve on our current assessment of risk contribution from Lp(a) for atherosclerotic vascular disease in individuals with measurable levels of circulating Lipoprotein(a).     

Genetic analysis of sexual dimorphism in serum apo AI and HDL-C concentrations in baboons

Sexual dimorphism is evident in many quantitative genetic traits, and there has been much speculation on the evolution of primate sexual dimorphism. Morphological characters have been the main focus of attention, while sexual dimorphism in physiological quantitative traits has been neglected. In either case, the genetic basis of primate sexual dimorphism has received little attention. This study characterizes genotype by sex (GxS) interactions in two physiological traits, serum apolipoprotein AI (apo AI) and high density lipoprotein cholesterol (HDL-C) concentrations, in baboons fed two different diets, a basal diet and a high cholesterol saturated fat (HCSF) diet. A GxS interaction effect on a trait indicates a heritable component of male/female differences in that trait. Using maximum likelihood methods, eight different quantitative genetic models were evaluated. Significant GxS interactions were found for serum apo AI and HDL-C concentrations on the basal diet. GxS interactions were suggested for serum apo AI and HDL-C concentrations on the HCSF diet, but they were not statistically significant. These results reveal that sexual dimorphisms in serum apo AI and HDL-C concentrations in baboons are heritable, with heritabilities that are influenced by diet. © 1992 Wiley-Liss, Inc.     

Polymorphic forms of human apolipoprotein[a]: inheritance and relationship of their molecular weights to plasma levels of lipoprotein[a]

The plasma concentration of human lipoprotein[a], Lp[a], is highly correlated with coronary artery disease. The protein moiety of Lp[a], apoLp[a], consists of two apoproteins, apo[a] and apoB-100, linked by one or more disulfide bonds(s). Apo[a], the protein unique to Lp[a], exists in polymorphic forms that exhibit different apparent molecular weights (Mr). Polyacrylamide gel electrophoresis in sodium dodecyl sulfate followed by immunoblotting was used to separate and visualize these different forms and to determine the polymorphic pattern of apo[a] in the plasma samples of 692 individuals. A total of 11 different polymorph bands ranging in Mr from 419 kD to 838 kD could be resolved, but only 1 or 2 bands were present per individual. The polymorphic band pattern for an individual was assigned to 1 of the 66 different phenotype designations representing the total number of possible single- and double-band combinations of the 11 detectable bands. All 11 of the possible single-band phenotypes but only 32 of the 55 possible double-band phenotypes were represented. There were 412 plasma samples (59.5%) that contained a single band, 274 (39.6%) contained two bands, and only 6 (0.9%) had no detectable apo[a] band. A highly significant inverse correlation was found between the Mr of the band(s) present and the plasma apoLp[a] concentration (r = -0.461; rho = 0.0001). The correlation was better between apoLp[a] and single-band (r = -0.495; rho = 0.0001) than double-band (r = -0.382; rho = 0.0001) phenotypes. Of the 274 individuals exhibiting double-band phenotypes, the lower Mr band was more intense in 141 (51.4%), the two bands were equally intense in 85 (31.0%), while the higher Mr band was more intense in 48 (17.5%). Based upon the hypothesis that apo[a] polymorphism is controlled by different alleles at a single locus, the frequency of the 11 alleles determined from the observe phenotypes (low Mr----high Mr) was: band 1) 419 kD, 0.00875; band 2) 489 kD, 0.00510; band 3) 536 kD, 0.0555; band 4) 553 kD, 0.0758; band 5) 613 kD, 0.135; band 6) 680 kD, 0.0824; band 7) 705 kD, 0.104; band 8) 742 kD, 0.151; band 9) 760 kD, 0.246; band 10) 796 kD, 0.128; band 11) 838 kD, 0.00802. The observed distribution of phenotypes in the population was compared by chi-square analysis to that predicted on the basis of simple Mendelian inheritance, and the hypothesis was rejected (chi 2 = 921.7; rho less than 0.001). Significantly, the singleband phenotypes are over-represented in the population compared to that predicted.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of short term dietary cholesterol and fat on human plasma Lp[a] and LDL levels.

The relationship between plasma levels of Lp[a] and LDL was examined using dietary regimens. In 81 normolipidemic male outpatients, dietary cholesterol was increased by consuming six eggs per day from a mean (SD) level of 311 (162) to 1430 (198) mg per day. Mean (SD) LDL-cholesterol levels increased from 102 (26) mg/dl to 120 (33) mg/dl (P less than 0.001), while mean (SD) Lp[a] levels were 5.5 (6.1) mg/dl on the basal diet and 5.6 (6.4) mg/dl on the cholesterol-rich diet. No significant correlation was observed between increases in either LDL-cholesterol or apolipoprotein B to Lp[a], nor was there any relationship between individual baseline levels of Lp[a] and dietary-induced changes of Lp[a]. Fourteen of the 81 participants were reexamined under strict nutritional control. Four diets with 40% of calories as fat, but differing in the type of fat and the amount of cholesterol, were administered sequentially to all subjects. As expected, mean (SD) LDL-cholesterol and apolipoprotein B levels were highest on the saturated fat, high cholesterol diet (112 (32) mg/dl and 79 (22) mg/dl) and lowest on the polyunsaturated fat, low cholesterol diet (77 (27) mg/dl and 53 (18) mg/dl). In contrast, mean Lp[a] levels did not significantly change among the four diets (range 4.2-4.9 mg/dl). No correlation of Lp[a] responses with changes in plasma lipids, apolipoproteins, or lipoproteins was observed on any diet. These data suggest that determinants of plasma Lp[a] levels are distinctly different from the determinants of plasma LDL levels in normolipidemic males.     

Preparation of a stable fresh frozen primary lipoprotein[a] (Lp[a]) standard

LP[a] is one of the most atherogenic lipoproteins consisting of an LDL-like core particle and a covalently linked glycoprotein of variable size. Due to its structural features, its heterogeneity and instability, there are great difficulties in standardizing quantitative immunochemical Lp[a] assays. One particular problem is the preparation of a pure primary standard, which is sufficiently stable to be used for value assignment of secondary reference material. Here we describe a method to purify Lp[a] to virtual homogeneity. When mixed with glycerol at a ratio of 1:1, the preparation is stable in the deep frozen state for more than 12 months. This latter material gave dose;-response curves in several immunochemical assays that were parallel to fresh or frozen sera, freshly prepared Lp[a], and other proposed reference materials. After determination of the protein content by amino acid analysis, it was possible to assign concentrations in molar and mass units to these preparations considering the theoretical molecular weights of the particular apo[a] isoform. Thus we propose to use this procedure for preparation of a "gold standard" for Lp[a] assays.     

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.     

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.     

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.     

Effect of dietary cis and trans fatty acids on serum lipoprotein[a] levels in humans.

Serum lipoprotein[a] (Lp[a]) is a strong risk factor for coronary heart disease. We therefore examined the effect of dietary fatty acid composition on serum Lp[a] levels in three strictly controlled experiments with healthy normocholesterolemic men and women. In Expt. I, 58 subjects consumed a control diet high in saturated fatty acids for 17 days. For the next 36 days, 6.5% of total energy intake from saturated fatty acids was replaced by monounsaturates plus polyunsaturates (monounsaturated fatty acid diet; n = 29) or by polyunsaturates alone (polyunsaturated fatty acid diet; n = 29). Both diets caused a slight, nonsignificant, increase in median Lp[a] levels, with no difference between diets. In Expt. II, 10% of energy from the cholesterol-raising saturated fatty acids (lauric, myristic, and palmitic acid) was replaced by oleic acid or by trans-monounsaturated fatty acids. Each of the 59 participants received each diet for 3 weeks in random order. The median level of Lp[a] was 26 mg/l on the saturated fatty acid diet; it increased to 32 mg/l (P less than 0.020) on the oleic acid diet and to 45 mg/l (P less than 0.001) on the trans-fatty acid diet. The difference in Lp[a] between the trans-fatty acid and the oleic acid diets was also highly significant (P less than 0.001). Expt. III involved 56 subjects; all received 8% of energy from stearic acid, from linoleic acid, or from trans-monounsaturates, for 3 weeks each. All other nutrients were equal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of the two major apoproteins in human lipoprotein [a]

Human Lp[a] was isolated in preparative amounts from two donors; the native lipoprotein and its constituent apoproteins, apo[a] and apoB, were characterized extensively. Based on differences in apparent molecular weight, four different isoforms of apo[a], a1-a4, were observed between the two donors. The number and relative distribution of these isoforms varied between donors but were constant for each donor. Each apo[a] isoform was shown to be derived from a discrete apo[a]-B100 disulfide-linked complex present before reduction. Complete delipidation of Lp[a] was followed by solubilization, reduction, and carboxamidomethylation of the constituent apoproteins. These apoproteins were then separated by immunoaffinity chromatography using anti-apo[a]- or anti-apoB-Sepharose; their purity and structural integrity were demonstrated by Western blot analysis. ApoB isolated by this procedure was essentially identical to apoB from autologous LDL with respect to molecular weight, secondary structure, amino acid composition, and sialic acid content. However, apo[a] differed from apoB in that it exhibited: a much less alpha-helical, less beta, but much more disordered structure; a lower proportion of aspartate, isoleucine, leucine, phenylalanine, and lysine, but a higher proportion of proline, glycine, and threonine; and a much higher content of sialic acid. These results indicate that apo[a] is not a superglycosylated form of apoB but is distinctly different in its composition and structure.     

Effects of the apolipoprotein(a) size polymorphism on the lipoprotein(a) concentration in 7 ethnic groups

Summary Apolipoprotein(a) [apo(a)] exhibits a genetic size polymorphism explaining about 40% of the variability in lipoprotein(a) [Lp(a)] concentration in Tyroleans. Lp(a) concentrations and apo(a) phenotypes were determined in 7 ethnic groups (Tyrolean, Icelandic, Hungarian, Malay, Chinese, Indian, Black Sudanese) and the effects of the apo(a) size polymorphism on Lp(a) levels were estimated in each group. Average Lp(a) concentrations were highly significantly different among these populations, with the Chinese (7.0mg/dl) having the lowest and the Sudanese (46mg/dl) the highest levels. Apo(a) phenotype and derived apo(a) allele frequencies were also significantly different among the populations. Apo(a) isoform effects on Lp(a) levels were not significantly different among populations. Lp(a) levels were however roughly twice as high in the same phenotypes in the Indians, and several times as high in the Sudanese, compared with Caucasians. The size variation of apo(a) explains from 0.77 (Malays) to only 0.19 (Sudanese) of the total variability in Lp(a) levels. Together these data show (I) that there is considerable heterogeneity of the Lp(a) polymorphism among populations, (II) that differences in apo(a) allele frequencies alone do not explain the differences in Lp(a) levels among populations and (III) that in some populations, e.g. Sudanese Blacks, Lp(a) levels are mainly determined by factors that are different from the apo(a) size polymorphism.     

Oxidative events cause degradation of apoB-100 but not of apo[a] and facilitate enzymatic cleavage of both proteins

Lipoprotein [a] (Lp[a]) contains equimolar amounts of apoB-100 and apolipoprotein [a] (apo[a]). Both proteins are amenable to degradation in vivo by mechanisms yet to be clearly defined. In this study, we examined the in vitro susceptibility of LDL and Lp[a], obtained from the same donor, to oxidation by either Cu(2)+ or the combined Crotalus adamanteus phospholipase A2 and soybean lipoxygenase system, monitoring the course of the reaction by the generation of conjugated dienes and fatty acids. In some experiments, treatment with leukocyte elastase (LE) or matrix metalloproteinase 12 (MMP-12) was administered before and after the oxidative step. In the case of Lp[a] we found that with both oxidizing systems, conditions that caused the breakdown of apoB-100 did not degrade apo[a] although oxidation-mediated changes were detected in the latter by intrinsic tryptophan fluorescence spectroscopy. Similar results were obtained with a reassembled Lp[a] obtained by incubating free apo[a] with LDL. Both apo[a] and apoB-100 were cleaved by LE and MMP-12 but the enzymatic cleavage was more marked when the preoxidized proteins were used as a substrate. Taken together, our in vitro studies indicate that apo[a] but not apoB-100 resists oxidative fragmentation, whereas both proteins are cleaved by enzymes of the serine and metalloproteinase families. We speculate that the fragments of apo[a] observed in vivo may be preferentially generated by proteolytic rather than oxidative events, whereas apoB-100 can be degraded by both mechanisms.     

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)     

Defects of the LDL receptor in WHHL transgenic rabbits lead to a marked accumulation of plasma lipoprotein[a

In this study, we created LDL receptor (LDLr) defective (WHHL) transgenic rabbits expressing human apo[a] to examine whether LDLr mediates the Lp[a] clearance from the plasma. By crossbreeding WHHL rabbits with human apo[a] transgenic rabbits, we obtained two groups of human apo[a] transgenic rabbits with defective LDLr functions: apo[a](1/0) WHHL heterozygous (LDLr(+/-) and apo[a](+/0) WHHL homozygous (LDLr(-/-) rabbits. The lipid and lipoprotein levels of human apo[a] WHHL rabbits were compared to those of human apo[a] transgenic rabbits with normal LDLr functions (LDLr(+/+). The apo[a] production rate was evaluated by analyzing apo[a] mRNA expression in the liver, the major site for apo[a] synthesis in transgenic rabbits. We found that pre-beta lipoproteins were markedly increased accompanied by a 2-fold increase in the plasma Lp[a] in apo[a](+/0)/LDLr(+/-) rabbits and a 4.2-fold increase in apo[a](+/0)/LDLr(-/-) rabbits compared with that in apo[a](+/0) rabbits with normal LDLr function. In apo[a](+/0)/LDLr(-/-) rabbits, there was a marked increase in plasma total cholesterol and triglycerides, as was found in their counterpart non-transgenic WHHL rabbits. Northern blot analysis revealed that hepatic apo[a] expression in WHHL transgenic rabbits was similar to that in LDLr(+/+) transgenic rabbits, suggesting the accumulation of plasma Lp[a] in WHHL transgenic rabbits was not due to increased apo[a] synthesis.In conclusion, absence of a functional LDLr leads to a marked accumulation of plasma Lp[a] in human apo[a] transgenic WHHL rabbits and LDLr may participate in the catabolism of Lp[a] in rabbits.