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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:2165-2175

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:2165-2175.)
© 1995 American Heart Association, Inc.

Articles

Familial Hypobetalipoproteinemia Is Not Associated With Low Levels of Lipoprotein(a)

Maurizio Averna; Santica M. Marcovina; Davide Noto; Thomas G. Cole; Elaine S. Krul; Gustav Schonfeld

From the Division of Atherosclerosis, Nutrition and Lipid Research, Department of Medicine, Washington University School of Medicine (M.A., D.N., T.G.C., E.S.K., G.S.), St Louis, Mo; the Department of Medicine, Northwest Lipid Research Laboratories, University of Washington (S.M.M.), Seattle, Wash; the Istituto di Medicina Interna e Geriatria, Cattedra di Patologia Medica Medicine (M.A., D.N.), Palermo, Italy; and Searle (E.S.K.), St Louis, Mo.

Correspondence to Gustav Schonfeld, MD, William B. Kountz Professor of Medicine, Director, Division of Atherosclerosis, Nutrition and Lipid Research, Washington University School of Medicine, 660 S Euclid Ave, Box 8046, St Louis, MO 63110.


*    Abstract
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Abstract To assess whether very low concentrations of LDL affected lipoprotein(a) [Lp(a)] concentrations and apo(a) associations with lipoproteins, we studied Lp(a) levels and associations in heterozygous subjects with familial hypobeta-lipoproteinemia FHBL) associated with several truncated forms of apoB-100, ranging from apoB-31 to apoB-89. Distributions of apo(a) isotypes were assessed by a combined electrophoresis-immunoblotting procedure that detects 34 isoforms. Lp(a) concentrations were quantified by two ELISAs, one detecting total apo(a) and the other apoB-bound apo(a) in plasma. Associations of apo(a) with plasma lipoproteins were evaluated by gel permeation chromatography (FPLC) and DGUC followed by analyses of elution and gradient fractions by apo(a) ELISA. In addition, associations were examined by nondenaturing electrophoresis or immunoprecipitation of whole plasma and examination of contents by immunoblotting. Finally, interactions between r-apo(a) and LDLs were evaluated in reconstitution experiments. The distributions of apo(a) isotypes did not differ between FHBL-affected and unaffected members of the same kindreds, and concentrations of Lp(a) were similar even when subjects were matched for isotypes both within and across kindreds. In subjects heterozygous for apo(a) isoforms, the smaller isoforms were inversely related to Lp(a) concentrations, the larger isoforms were not. The regression lines between Lp(a) concentrations and the smaller apo(a) isoforms were significant and negative in slope for both FHBL-affected and unaffected subjects, but the slopes of the lines did not differ. In multiple regression analyses, only the sizes of the smaller apo(a) isoforms contributed to the prediction of Lp(a) concentrations. ApoB-size made no difference. In simple apoB-100/apoB-truncation heterozygotes, virtually all apo(a) was complexed with apoB-100–containing particles but not apoB-truncation particles, and r-apo(a) recombined with apoB-100–containing LDLs but not with apoB-89–containing LDLs. Thus, (1) low apoB levels do not affect the plasma concentrations of Lp(a), (2) apo(a) binds apoB-100 to form Lp(a) particles of usual sizes and densities, and (3) apoB truncations even as large as apoB-89 do not form covalent bands with apo(a), although noncovalent associations with apoB-89 may be present in plasma.


Key Words: familial hypobetalipoproteinemia • apo(a) phenotypes • apo(a)-apoB binding • lipoprotein(a) • truncated apolipoprotein B


*    Introduction
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Lp(a) concentrations in plasma are directly associated with risks for coronary and carotid artery atherosclerosis.1 2 3 4 Lp(a) consists of one LDL-like particle and one apo(a) molecule. The apoB-100 of LDL-like particles is coupled to apo(a) primarily, although perhaps not exclusively via a sulfhydryl bond.5 6 The coupling is thought to be a postsecretory event probably occurring within the vascular compartment.7 On the basis of molecular modeling, cysteine availability, and mutagenesis experiments, cysteines at amino acid position 3734 of apoB-100 and position 4057 on apo(a) have been designated as the sites of interaction.8 9 In normolipidemic humans virtually all apo(a) is coupled to LDL-like particles, but because LDL concentrations are ordinarily much higher than apo(a) concentrations, only a minority of LDL-like particles are coupled to apo(a), and Lp(a) forms only a small minority of apoB-100–containing lipoproteins.10 11 This may not be the case in hypobetalipoproteinemia.

Concentrations of Lp(a) are to a large extent determined by the apo(a) gene.12 The gene is highly polymorphic, specifying molecules with a protein mass ranging from {approx}185 to 540 kD. The size polymorphism is due to the variable number of repeats of a kringle structure,13 14 and 34 size-dependent alleles have been identified in human populations by Southern blotting and immunoblotting techniques.15 16 Apo(a) sizes are inversely related to Lp(a) concentrations, but size differences do not account for all of the variation in plasma concentrations. In addition to the varying numbers of kringles, there are also amino acid sequence differences between individual kringles that generate additional apo(a) alleles.17 18 Although genetic influences are very important in the setting of Lp(a) levels in plasma, other factors also contribute. For example, use of estrogens19 or niacin20 affects Lp(a) concentrations. The presence of gene variants that affect plasma apoB concentrations (eg, LDL-receptor gene mutations in familial hypercholesterolemia) may affect Lp(a) levels in some but not all kindreds.21 22 23

Other lipoprotein gene defects that affect plasma apoB concentrations are those specifying FHBL, a condition that in some FHBL kindreds is characterized by the presence in plasma of truncated forms of apoB-100 cosegregating with approximately 5th percentile LDL-cholesterol levels and apoB levels that are approximately 30% of those of unaffected members of the same kindreds.24 The low levels of apoB in plasma are due to decreased production rates of both the apoB-100– and apoB truncation–containing lipoproteins. Some truncation-containing lipoproteins also have increased catabolic rates. Kindreds harboring well-defined FHBL are ideal for testing whether low apoB levels affect Lp(a) levels and apo(a) associations in plasma. Thus, we compared Lp(a) concentrations and phenotypes in the affected and unaffected members of FHBL kindreds and assessed apo(a) associations by several techniques.


*    Methods
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Populations Studied
Forty FHBL simple heterozygotes and 3 compound heterozygotes from kindreds with previously characterized truncations,24 25 26 27 28 29 30 31 52 normolipidemic unaffected members of the same kindreds, and 8 nonrelated subjects were studied (see Table 1Down). The nonrelatives were volunteers from among workers in the laboratory. During a 12- to 14-hour fast blood samples were drawn into EDTA (1 mmol/L), and plasmas were separated by centrifugation and analyzed for lipoprotein lipids by combined ultracentrifugal and enzymatic methods (Wako Pure Chemicals). Total apoB was quantified by immunonephelometry (Behring). Because lipid and Lp(a) levels of blood relatives and spouses of FHBL subjects who married into the kindreds were not significantly different, data for all unaffected relatives were pooled for statistical analyses.


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  		Table 1. Study Subjects

Identification of Truncated Forms of ApoB in Plasma
ApoB was immunoprecipitated from plasma,30 the immunoprecipitated pellets were washed and then dissolved in SDS-PAGE sample buffer, incubated at 100°C for 5 minutes, and applied to 3% to 6% gradient SDS-PAGE gels for electrophoresis. Proteins were transferred to Immobilon-P membranes (Millipore) and immunoblotted with an anti-apoB monoclonal antibody (C1.4).26 Nondenaturing GGE was performed essentially as described elsewhere32 with the exception that the whole plasma or the Lp(a) products reconstituted in vitro were analyzed on gels freshly preparedin the laboratory. Western blots of GGE or SDS-PAGE were performed as previously described using 125I-radiolabeled secondary antibodies.26

Lp(a) Measurements
Two direct binding ELISAs with detecting antibodies of different specificities were developed and performed as reported.33 In both assays the same monoclonal antibody (Mab a-6), specific for apo(a) kringle IV type 2 without cross-reactivity with plasminogen, was linked to the solid-phase to "capture" the apo(a)-containing particles. The apo(a) isoform 17 was used as primary standard, and fresh-frozen serum from the same donor with a high Lp(a) protein concentration was used to calibrate the two ELISA methods. In one assay the detecting antibody was a monoclonal antibody (Mab a-40) specific for apo(a) kringle 4 other than the type 2 repeats. In the second assay, the detecting antibody was a polyclonal antibody specific for the apoB-100 component of Lp(a). The apo(a) detection ELISA can measure both uncomplexed apo(a) and apo(a) associated with apoB, whereas the apoB-detection ELISA can measure only the apo(a)/apoB-Lp(a) complex. The assay measuring the apo(a) component used a monoclonal antibody that does not recognize the multiple repeats of apo(a); therefore, this assay is not affected by the apo(a) size heterogeneity. The two ELISA methods have been extensively evaluated, and nearly identical results have been obtained on a large number of samples regardless of the apo(a) isoforms,33 indicating that very little if any apo(a) is not associated with apoB in plasma. All the samples were analyzed simultaneously with the two assays, and each sample was analyzed in quadruplicate. Results are reported in milligrams per deciliter of Lp(a) protein.

Apo(a) Phenotypes
Apo(a) size isoforms were determined by a high-resolution SDS–agarose gel electrophoretic method followed by immunoblotting.16 With this approach 34 different apo(a) size isoforms can be detected in human plasma. The isoforms are identified by a numeric system in which number 1 is the highest identified molecular weight apo(a) isoform and number 35 is the lowest. Single-band phenotypes are identified by single numbers (eg, 15/) because homozygosity cannot be distinguished from heterozygosity for a null allele or from an allele expressed in low concentrations.

ApoB and Apo(a) Profiles
To assess the distributions of full-length apoB-100 and truncated apoBs and apo(a) among plasma lipoproteins, plasmas were fractionated by gel-permeation chromatography on FPLC30 or by DGUC.34

For gradient ultracentrifugation profiles, 14 mL plasma was adjusted to d=1.040 g/mL and applied to a KBr gradient (density range, 1.210 to 1.006 g/mL) in 40 mL Quickseal tubes (Beckman Instruments). The gradient was centrifuged for 24 hours at 45 000 rpm at 12°C. A blank gradient was used as balance and reference. The gradient was eluted from the top by pumping a solution of d=1.300 g/mL into the bottom of the tube. Fifty 1-mL fractions were collected. Each fraction was analyzed for cholesterol and triglycerides (Wako Pure Chemicals) and Lp(a) by ELISA (Strategic Diagnostics). Equal aliquots (35 µL) of each of the DGUC fractions were removed and dialyzed extensively against 5 mmol/L NH4 HCO3-, pH 8.2, lyophilized, and reconstituted in 35 µL SDS-PAGE sample loading buffer.30 The samples were electrophoresed on 3% to 6% SDS-PAGE gels and immunoblotted using the monoclonal anti-apoB antibody C1.428 that is directed against the NH2-terminal region of apoB and has detected all truncations discovered to date.35 The bands for apoB-100 and the apoB-truncations on the resulting autoradiographs were scanned using a laser densitometer. Areas under the peaks were determined using SigmaScan (Jandel Scientific). The densitometric areas corresponding to either apoB-100 or truncated apoB were summed, and the values representing percentages of the total densitometric area determined for apoB-100 or the truncated apoB in any particular elution fraction were used to generate the distribution curves for each apoB species. The density in each fraction of the reference gradient was measured using a DMA 35 densitometer (PAAR).

For FPLC separation, 1.5 mL plasma was chromatographed at room temperature on two 25-mL Superose 6 columns connected in series.36 The column elution fractions were analyzed enzymatically for cholesterol (Wako Pure Chemicals) and for Lp(a) by ELISA. For apoB, 35-µL aliquots were applied to 3% to 6% gradient SDS-PAGE gels for electrophoresis, immunoblotting, and radiochemical detection of apoB.28 Areas of bands corresponding to apoB-100 or the truncated apoBs on the resulting autoradiograph were quantified by densitometry as described, and apoB contents in each fraction are expressed as percentages of the total summed apoB areas.

Identification of ApoB Subspecies by Immunoprecipitation of Lp(a) From Plasmas of ApoB-Truncation/B-100 Heterozygotes
Lp(a) was immunoprecipitated from heterozygotes' plasmas using a monospecific polyclonal antiserum directed against Lp(a). Sixty-µL aliquots of the proband's plasma were immunoprecipitated with 10 µL each of polyclonal anti-Lp(a) antibody using a standard immunoprecipitation method.27 Aliquots of both pellets and the supernatants were run on a 3% to 6% SDS-PAGE and electrotransferred to Immobilon-P membranes.26 One set of samples was blotted with the anti-apoB monoclonal antibody C1.4 and a replicate set with Mab a-5 directed against kringle 4 of apo(a).

Antibodies Used For Immunoprecipitation and Blotting
A polyclonal goat anti-human apo(a) antibody was obtained from International Enzyme. Mab a-5 was produced and characterized as previously reported.33 Briefly, Mab a-5 is specific for apo(a) without cross-reactivity with plasminogen, has an affinity constant of 2.1x1010 L/mol, is of IgG2b subclass, and is directed to an epitope that is present in apo(a) kringle 4 type 1 and type 2. Mab a-5 was purified from ascitic fluid by absorption to Protein A Sepharose (Affi-Gel Protein A, BioRad) and stored at -80°C until used.

Reconstitution of Apo(a) With ApoB
Reconstitution experiments were carried out essentially as described by Chiesa et al.37 Dr Richard Lawn, Stanford University, Palo Alto, Calif, kindly provided the r-apo(a) that contains 17 kringles. Fifteen micrograms of the lipoprotein under study was incubated at 37°C with 0.3 µg r-apo(a) in 0.9% NaCl in a final volume of 40 µL for 6 hours. At the end of incubation, a 20-µL aliquot was removed and electrophoresed on a 2% to 16% non-denaturing gel as described.32 The gels were immunoblotted and apo(a) detected using the 125I-labeled monoclonal anti-apo(a) antibody Mab a-5.26

Statistical Analysis
For comparisons of Lp(a) concentrations between various groupings, several tests were employed, including the unpaired t test and the Mann-Whitney U test. The Kolgorov-Smirnov test was used to evaluate the normality of Lp(a) concentration distribution in our sample. Parametric tests were used after log transformation of Lp(a) values. Correlations were calculated using both Spearman and Pearson methods. Multiple regression analyses were performed using CRUNCH 4.0 Statistical Software Program (Crunch Software Corp). The specific tests used for any given comparison are provided in the legends to the tables and figures.


*    Results
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 *Results
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Lp(a) Concentrations and Phenotypes
Because the apo(a) genotypes have potent effects on Lp(a) concentrations in plasma, apo(a) phenotypes (reflecting genotypes) were obtained in all subjects (see Table 1Up for subject population). Apo(a) isotypes, based on electrophoretic migration, ranged in size from 1 to 20 for members of kindreds and from 7 to 26 for nonmembers. Numerical designations are inversely related to the sizes of the isoforms. Fifty-six of the 103 individuals had two bands for apo(a) isoforms by immunoblotting. The distributions of both the smaller and larger apo(a) phenotypes overlapped between the various kindreds and between the affected and unaffected subjects of given kindreds. (Fig 1Down, top, for smaller isoforms). Similar results were obtained when the mean numerical values for isoforms were used (not shown). Lp(a) concentrations of members of different kindreds and affected and unaffected members within the same kindreds also overlapped (Fig 1Down, bottom). Examination of the pedigrees (Figs 2Down, 3Down, and 4ADown through 4C) shows exact matches by apo(a) phenotype between FHBL-affected and unaffected siblings. This occurs only once in the apoB-38.9 kindred (III-6 and III-5 in Fig 2Down) in which the two Lp(a) concentrations are 18 and 20 mg/dL, respectively. There are 2 unaffected siblings and 1 affected sibling with the same apo(a) phenotype in the apoB-54.8 kindred (Fig 4ADown), affected V-7, and unaffected V-8 and V-9. The respective Lp(a) concentrations are 0.5, 1.4, and 0.8 mg/dL. In another branch of the apoB-54.8 kindred (Fig 4BDown), there are 3 affected brothers and 1 apo(a) phenotype-matching unaffected sister [II-11, II-17, II-19, and II-22 with Lp(a) levels of l.5, 2.9, 1.4, and 3.2]. Thus, affected individuals may have nearly equal, higher, or lower Lp(a) levels. To increase the numbers of subjects available for comparisons, FHBL-affected and unaffected individuals in given kindreds were matched for one (the smaller sized) apo(a) phenotype. The Lp(a) concentrations of affected and unaffected individuals were not consistently different (Table 2Down). Note that mean values for Lp(a) in the FHBL-affected individuals of the apoB-38.9 and apoB-54.8 kindreds with the 17/x and 10/x isoforms, respectively, were larger than mean Lp(a) values for FHBL-unaffected subjects (Table 2Down). The mean value of FHBL-affected members with the 15/x isoforms was less than the mean for the FHBL-unaffected members. Pooling the subjects into still larger phenotype groups across kindreds yielded compatible results (ie, as expected there were significant differences according to isoform size but not by apoB status, Table 3Down).



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  		Figure 1. Plots show distribution of the smaller apo(a) isoforms (larger in numerical designation) among apoB-truncation kindreds (top). Distribution of plasma Lp(a) concentration among kindreds (bottom). The kindreds are identified on the abscissa by the relative lengths of the truncations expressed as percentages of the apoB-100. Affected indicates for different apoB truncations; unaffected, healthy control subjects (relatives and spouses of the affected subjects, pooled); NM, nonmember group of healthy control subjects who were unrelated to the kindred.



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  		Figure 2. Pedigree of the apoB-38.9 kindred showing the FHBL affected (filled symbols) and unaffected (empty symbols with cross bars) members. Only relevant parts of kindreds are shown. Numbers in parentheses below male and female symbols are apo(a) phenotypes. Numbers next to them are Lp(a) concentrations in mg/dL. Subjects III-5 and III-6 form an unaffected-affected pair, matched for apo(a) phenotype. Circles indicate females; squares, males; empty symbols, no plasma available. All affected subjects are heterozygous for the apoB-38.9 truncation (see Reference 30 for original description).



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  		Figure 3. Pedigree of the apoB-52 kindred. Numbers in parentheses above male and female symbols refer to previously published pedigree (see Reference 29). No FHBL unaffected-affected sibling pairs with identical apo(a) phenotypes are present. See Fig 2Up legend for symbols and abbreviations.



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  		Figure 4. Three branches of the apoB-54.8 kindred are shown in A through C. Numbers above symbols refer to current positions, numbers in parentheses above symbols refer to previously published pedigree (see Reference 27). FHBL unaffected-affected sibling pairs with identical apo(a) phenotypes are present in generations V and II of panels A and B, respectively. The importance of apo(a) phenotypes in determining Lp(a) concentrations is evident in all the pedigrees. See Fig 2Up legend for symbols and abbreviations.


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  		Table 2. Lp(a) Concentrations in Affected and Unaffected Members of FHBL Kindreds with Similar Apo(a) Phenotypes


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  		Table 3. Lp(a) Concentrations and Apo(a) Isoform Sizes

An inverse correlation is known to exist between apo(a) sizes and Lp(a) concentrations. To assess whether the larger or smaller isoform in subjects heterozygous for apo(a) isoforms was the more important determinant of Lp(a) concentrations, a multiple regression analysis was performed. Respective ß and F values for the smaller isoforms were 0.631 and 48.5 (n=57, P<.0001) and R2=.452 for the model. The F value for the larger isoforms was 0.167 (P=.52). The results suggest that the smaller forms (larger size identifying numbers) were the important determinants of Lp(a) concentrations. To assess further the interactions between Lp(a) concentrations, apo(a) phenotypes, and the presence of hypobeta, a multiple regression analysis was preferred using logarithmic Lp(a) concentrations as the dependent variable and the smaller apo(a) isoforms and apoB sizes as predictive variables. With all the available Lp(a) data analyzed, respective ß and F values for apo(a) were 0.642 and 120.0 (n=103, P=.0001) and R2=.475 for the model, suggesting that apo(a) isoforms were predominant in determining Lp(a) concentrations; apoB did not contribute significantly (F=0.324, P=.31). Analogous analyses gave compatible results using only the data of the FHBL heterozygotes or using all the Lp(a) data and the mean values for the two isoforms (rather than the numbers for the short isoforms).

Finally, we assessed whether the well-known inverse correlation between apo(a) phenotypes and Lp(a) concentrations was altered by the apoB truncations using the smaller of the two possible isoforms in any given subject in the regression analyses. The regression equation for unaffected members was Lp(a)=1.02xisoform size+0.36 (r=.44, P<.00001); for affected members, Lp(a)=1.08xisoform size+0.16 (r=.28, P<.0001). The slopes of the two equations were not significantly different (P=.23).

Apo(a) Associations in Plasma
The two Lp(a) ELISAs yielded nearly identical mean values and standard deviations when applied to the plasma samples of simple heterozygotes for apoB truncation/apoB-100. This was also true for samples of unaffected relatives and control subjects. The regression line between the values obtained by the two assays had the following formula: logarithmic apo(a)[B assay]=1.037x[apo(a) assay]-0.061 (r=.983), implying that the apo(a) was almost completely complexed with apoB in most samples. However, samples drawn from the three compound heterozygotes for apoB-40/apoB-89 yielded nonidentical values in the two assays. Respective values for the apo(a)- and apoB-detection ELISAs were 0.5 versus 0.3, 0.4 versus 0.2, and 0.3 versus 0.05 mg/dL, suggesting that some uncomplexed apo(a) may have been present in these selected samples.

On FPLC and DGUC analyses of plasma samples of healthy control subjects, apo(a) was found only in those fractions containing lipoproteins, ie, there was no unassociated apo(a). Nearly all apo(a)s were present in those fractions that also contained apoB-associated lipoproteins, ie, intermediate density lipoprotein and LDL (Fig 5ADown and 5BDown), as predicted from the concordance of the two apo(a) assays. We have reported compatible results in the plasmas of apoB-38.9/apoB-100 simple heterozygotes,30 and compatible results have also been found in the plasma of apoB-89/apoB-100 heterozygotes (Fig 6Down). However, the plasma of an apoB-89/apoB-40 compound heterozygote behaved differently. Although on FPLC most of apo(a) eluted with apoB-89–containing fractions (Fig 7Down, top), on DGUC, in contrast, virtually all of apo(a) eluted in fractions more dense than HDL (Fig 7Down, bottom). In addition, no apo(a) eluted with any fractions that contained only apoB-40 on either FPLC or DGUC.



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  		Figure 5. A, Graphs show FPLC profiles of normal control subjects. Plasma aliquots of 1.5 mL were chromatographed at room temperature on two 25-mL Superose 6 columns connected in series. The eluted fractions were analyzed for cholesterol (dotted lines) and Lp(a) (empty circles). In the healthy control subjects Lp(a) eluted close to intermediate density lipoprotein particles (peak fraction 21). VLDL and LDL peaks are in fractions 10 and 28 through 30, respectively.B, Graphs show DGUC profiles of normal controls. Fourteen mL of plasma was adjusted to d=1.040 g/mL and applied to a KBr gradient (d=1.210 to 1.006 g/mL) in 40-mL Quickseal tubes then centrifuged for 24 hours at 45 000 rpm at 12°C. The gradient was eluted and collected into 50 fractions (see "Methods"). Each fraction was analyzed for density (top), cholesterol (middle, dotted line), and Lp(a) (bottom, empty circles). In the healthy control subjects Lp(a) eluted in a density range intermediate between LDL (fractions 10 through 20) and HDL (fractions 35 through 45).



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  		Figure 6. Graphs show FPLC (top) and DGUC (bottom) profiles of an FHBL heterozygote subject (apoB-100/apoB-89). The FPLC profile shows that apo(a) (open circles) coeluted with apoB-containing particles (apoB-100, solid circles; apoB truncation, solid triangles), and DGUC confirms the presence of Lp(a) eluting in a density range intermediate between LDL and HDL.



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  		Figure 7. Graphs show FPLC (top) and DGUC (bottom) profiles of an FHBL compound heterozygote subject (apoB-89/apoB-40). In this subject the FPLC profile shows that apo(a) (open circles) coeluted with apoB-containing particles (apoB-89, solid circles; apoB-40, solid triangles), whereas DGUC shows that most of the apo(a) eluted in fractions of density >1.210 g/mL.

On GGE-immunoblotting of the whole plasma of the apoB-89/apoB-40 compound heterozygote and of an apoB-100/apoB-100 control subject apo(a) comigrated with apoB-100– (Fig 8Down, lane B) and with apoB-89–containing LDLs (Fig 8Down, lane A), findings compatible with the FPLC (Fig 7Up, top) but not the DGUC analyses (Fig 7Up, bottom).



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  		Figure 8. Blots show nondenaturing GGE (2% to 16%) of whole plasma of the FHBL apoB-89/apoB-40 compound heterozygous subject. Lane A indicates apoB-89/apoB-40 compound heterozygote; lane B, apoB-100/apoB-100 control subject; and lane C, high-molecular-weight markers (Pharmacia). The molecular weight indicators were thyroglobulin (669 kD), ferritin (440 kD), and catalase (232 kD). The gel was electrotransferred to Immobilon-P and immunoblotted using monoclonal antibody Mab a-5 directed toward apo(a) kringle IV type 2 (see "Methods"). The positions of molecular weight markers are drawn onto the film. Bands at the tops of the lanes correspond to Lp(a). The bands at the bottom may represent degradation products.

Next, to assess whether complexes existed between apo(a) and apoB-100 only, or also between apo(a) and truncated apoBs as well, total apo(a) was immunoprecipitated with a monospecific anti–apo(a) antiserum, from plasmas of an apoB-75/apoB-100 heterozygote and the apoB-89/apoB-40 subject. Immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted with either Mab a-5 or with the Mab C1.4 to identify the apo(a) and apoB moieties, respectively, present in the immunoprecipitates. Virtually all of the apoB-75/apoB-100 heterozygote apo(a) was precipitated by the anti-apo(a) antiserum (Fig 9Down, bottom, lane marked A), whereas no apo(a) was detected in the supernatant (Fig 9Down, bottom, lanes marked B). The immunoprecipitate in addition to apo(a) contained only apoB-100 but no apoB-75 (Fig 9Down, top, lane A), whereas apoB-100 and the apoB-75 were both present in the supernatant (Fig 9Down, top, lane B). Immunoprecipitation of the apoB-89/apoB-40 plasma also resulted in complete precipitation of apo(a), but neither apoB-89 nor apoB-40 was seen in the precipitate. ApoB-89 and apoB-40 were found only in the supernatants (Fig 9Down, top, marked B89 and B40).



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  		Figure 9. Blots show immunoprecipitation of Lp(a) particles from plasmas of FHBL heterozygote subjects. Sixty microliters plasma was immunoprecipitated using 10 µL of polyclonal anti-Lp(a) antibody. Aliquots of both pellets (lanes A) and supernatants (lanes B) were loaded onto 3% to 6% SDS polyacrylamide gels for electrophoresis, and then electrotransferred to Immobilon-P and immunoblotted using either monoclonal antibody C1.4 directed toward amino terminal region of apoB (top) or Mab a-5 directed toward apo(a) kringle 4 type 2 (bottom).

Finally, LDLs free of apo(a) were prepared from normal plasma and from the plasma of the apoB-89/apoB-40 compound heterozygote by ultracentrifugation at d=1.019 to 1.05. These LDLs were incubated with r-apo(a), and the incubation mixtures were separated on GGE. The proteins were transferred to membranes and immunoprobed with the radiolabeled 125I–Mab a-5, the anti-apo(a) monoclonal antibody (Fig 10Down). The apoB-100–containing LDL of the control subject readily bound apo(a) (Fig 10Down, lane D), but the apoB-89–containing LDL did not (Fig 10Down, lane C). Control lanes A and E of Fig 10Down contain LDLs without r-apo(a), and lane B contains r-apo(a) without LDL.



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  		Figure 10. Blot shows reconstitution of Lp(a). LDL pools from a healthy control subject (apoB-100/apoB-100) and an FHBL compound heterozygote subject (apoB-89/apoB-40) were obtained by sequential ultracentrifugation and incubated with an r-apo(a) (isoform 17) for 6 hours at 37°C. The samples were subjected to a nondenaturing GGE (2% to 16%) and then electrotransferred to Immobilon-P and immunoblotted using monoclonal antibody Mab a-5 directed toward apo(a) kringle 4 type 2. Lanes A and E indicate apoB-89 LDL and apoB-100 LDL not incubated with r-apo(a); lanes B and D, apoB-89 LDL and apoB-100 LDL incubated with r-apo(a); and lane C, r-apo(a) without LDL. This shows that apoB-100 LDL bound the r-apo(a) to form an Lp(a) particle (lane D), but the apoB-89 LDL did not bind the r-apo(a) (lane B).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Several reports suggest that levels of apoB may affect levels of Lp(a) in plasma. For example, in some but not all kindreds with familial hypercholesterolemia (FH) affected individuals have higher Lp(a) concentrations in their plasmas than do unaffected relatives.21 22 Genetic factors predominate in setting Lp(a) levels in plasma,12 apparently by affecting the production rates of the different apo(a) isotypes,39 while the fractional catabolic rates of the various Lp(a) isotypes are similar.38 39 The low levels of apoB in FHBL due to apoB truncations are also genetically determined with production rates of apoB truncations depending directly on apoB lengths.40 41 42 43 Even the production of apoB-100 in FHBL heterozygotes is reduced to {approx}35% of matched control subjects.43 Thus, both the apoB-100 and apo(a) moieties of the Lp(a) particles are produced at genetically determined but independent rates. However, the postsecretory metabolism of Lp(a) particles is clearly influenced by apo(a) and apoB-100 circulating together as complexes,39 40 in part due to the differing affinities of LDL-apoB and Lp(a) for the LDL receptor.44

Normal plasmas contain high concentrations of apoB-100 relative to concentrations of apo(a), ie, Lp(a)s make up only small subpopulations of the apoB-100–containing lipoproteins. However, in FHBL subjects, lower apoB-100 levels cocirculate with near normal apo(a) levels (Tables 2Up and 3Up). As a result a larger proportion of LDL-like particles are complexed to apo(a) than in healthy control subjects. [Two extreme examples are present: In the plasmas of two simple heterozygotes for apoB-38.9/apoB-100, with Lp(a) levels of 44 and 75 mg/dL and total apoB levels of 38 and 52 mg/dL, the molar concentrations of apo(a) and apoB-100 were such that 92% and 75%, respectively, of their total apoB was associated with apo(a).] Because a larger proportion of LDL-like particles are complexed to apo(a) than in healthy control subjects, we expected FHBL subjects to have Lp(a) concentrations that differed from those of FHBL-unaffected subjects. Initially, we assessed whether there were significant differences between the distributions of apo(a) phenotypes in FHBL-affected and unaffected relatives (Fig 1Up, top) and found no differences, confirming that there were no genetic associations between apo(a) isotypes and apoB truncations; not surprising perhaps because the genes for apo(a) and apoB reside on chromosomes 614 and 2,45 respectively. Plasma Lp(a) levels of FHBL heterozygotes and unaffected relatives were not consistently different whether subjects were matched (1) according to broad categories of apo(a) phenotypes across kindreds (Fig 1Up, bottom, and Table 3Up), (2) according to the smaller phenotypes within kindreds (Fig 1Up, bottom, and Table 2Up), or (3) according to individual sibling pairs in which identical apo(a) isotopes could be safely assumed to represent identical genotypes (alleles) (Figs 2 through 4UpUpUp). In contrast, apo(a) isoforms were strongly related to Lp(a) concentrations (Table 3Up). Furthermore, multivariate regression analysis confirmed that apo(a) isotypes affected Lp(a) concentrations, whereas the presence or absence or sizes of apoB truncations did not. Thus, if steady state apoB concentrations affect Lp(a) concentrations, the effect must be very small. This suggests that the measures designed specifically to lower apoB concentrations are unlikely to affect Lp(a) concentrations. Indeed 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors that significantly lower LDL cholesterol and apoB concentrations have little effect on Lp(a) concentrations.46

We also evaluated associations of apo(a) with apoB truncations in plasma. Previously, we reported that apoB-38.9–containing lipoproteins do not associate with apo(a) probably because the cysteine residue at codon 3734, proposed to be involved in sulfhydryl bond formation,8 is deleted.30 While our experiments were in progress, others reported that human apoB-88, apoB-90, and apoB-94 isolated from hepatoma cell culture media transgenic mouse plasmas or a naturally occurring apoB-86 truncation isolated from an FHBL subject's plasma, respectively, did not associate with r-apo(a) in vitro,47 48 despite the presumed presence of cysteine at apoB-100 codon 3734. The present experiments confirm and expand our own findings30 and those of others47 48 in which plasmas of subjects with the apoB-89, apoB-75, and apoB-40 mutations and several methods to evaluate apo(a)-apoB associations were used.

First, two different quantitative assays for Lp(a) were employed. The "total apo(a)" assay detects all apo(a) whether bound to apoB or not. The other assay detects only that apo(a) complexed to apo(B). Larger values for the total apo(a) assay than for the apoB-bound apo(a) assay would indicate the presence of uncomplexed apo(a) molecules. The two sets of Lp(a) determinations were in fact statistically indistinguishable, suggesting that virtually all apo(a) was complexed to apoB. The presence of three discrepant Lp(a) values for the apoB-89/apoB-4025 compound heterozygotes suggests that some but not all of the apo(a) in plasma was complexed with apoB in these subjects.

Second, lipoproteins were separated by size (FPLC) or density (DGUC) (Figs 5 through 7UpUpUp), and any associations between apo(a) and various lipoprotein classes were examined. In the plasma of the apoB-89/apoB-100 simple heterozygote,26 virtually all apo(a) eluted with (Fig 6Up, top) or floated with (Fig 6Up, bottom) apoB-containing lipoproteins, as in healthy control subjects (Fig 5AUp and 5BUp), confirming the findings of the dual apo(a) ELISAs. In addition, the majority of apo(a) coeluted with particles larger than LDL in size or floated with particles higher in density than LDL just as in healthy control subjects, suggesting that Lp(a) sizes and densities were not affected by the presence of heterozygous FHBL. In contrast, the association between apo(a) and apoB differed in the apoB-40/apoB-89 compound heterozygote (Fig 7Up). Although on FPLC apo(a) coeluted with apoB, on DGUC apo(a) was found in the nonlipoprotein dense fractions. This implies that the complexing between apoB and apo(a) was not due to covalent bonds. Others have demonstrated the existence of noncovalent interactions between apo(a) and apoB-100.49 50 51 Apparently the noncovalent bonds may survive electrophoresis (Fig 8Up, lane A) and gel permeation chromatography (Fig 7Up, top) but not ultracentrifugation in high salt solutions (Fig 7Up, bottom).48 49

Although the elution (FPLC) and floating (DGUC) positions of Lp(a) peaks suggested that apo(a)/apoB-100 LDL-like complexes may have predominated, examination of the profiles showed that neither technique was able to distinguish unequivocally between binding of apo(a) solely to apoB-100 or to apoB-89 or to both (Fig 6Up), since in most cases both apoB-100 and apoB-89 were detectable in apo(a)-containing elution fractions. Therefore, we immunoprecipitated apo(a)-containing fractions from apoB-75/apoB-100 and apoB-89/apoB-40 plasmas and examined the apoB and apo(a) contents of the immunoprecipitates and supernatants. Despite the complete precipitation of apo(a)s (Fig 9Up, bottom, lanes marked A), only apoB-100 but neither apoB-75 nor apoB-89 was found in the immunoprecipitates (Fig 9Up, top, lanes marked A), suggesting that apo(a) was complexed to apoB-100 but not to the truncations. This result was expected for truncations shorter than apoB-82.3, which lack cysteine 3734, and based on our experience with apoB-38.9,30 but at the beginning of these experiments we did expect apoB-89 to bind apo(a) based on the position of the cysteine proposed to be involved in the sulfhydryl bond.8 9

The possibility still remained that apoB-89 was capable of covalent binding to apo(a), but the affinity of binding was lower than for apoB-100 and therefore no covalent binding occurred in the apoB-89/apoB-40 plasma due to the low concentrations of apo(a) present. Accordingly, r-apo(a) was incubated in gross excess37 with the apoB-89–containing LDL isolated from an apoB-89/apoB-40 compound heterozygote subject's plasma and also with an appropriate apo(a)-free control subject's apoB-100–containing LDL. The control apoB-100–containing LDL readily bound r-apo(a) (Fig 10Up, lane D), but the apoB-89 truncation–containing LDL did not (Fig 10Up, lane B), indicating that it was probably incapable of doing so and confirming results that were published while this article was in preparation.47 48 The absence of complexing of apoB-89 and apo(a) could have been due to the absence of cysteine 3734, but this has been ruled out (Groenewegen and Schonfeld, unpublished results, 1995). Other possibilities are that (1) the absence of the COOH terminal portion of apoB-89 could have led to intramolecular sulfhydryl bond formation, which does not occur in the presence of the COOH terminal, making the necessary cysteine unavailable for reaction with apo(a); (2) the conformation of apoB-89 could differ from that of apoB-100, making the necessary bonding sulfhydryl residue inaccessible; or (3) a cysteine distal to the one at 3734 is in fact involved in bond formation with apo(a). ApoB-89–, apoB-87–,52 and apoB-75–containing LDLs also behave unusually in other systems, manifesting enhanced interactions with LDL receptors and more rapid clearance from plasma than normal LDLs41 and suggesting that the COOH-terminal region of apoB-100 may modulate more than one function of LDL.

In summary, our data show (1) that Lp(a) levels are not affected in hypobetalipoproteinemia; (2) that apo(a) is complexed with apoB-100 in the plasmas of apoB-100/apoB-truncation heterozygotes, resulting in Lp(a) particles that resemble Lp(a) particles of normal subjects in size and density; and (3) that truncations as large as apoB-89 do not bind apo(a) normally. The present data add to a growing body of literature that suggests that Lp(a) concentrations are not affected by apoB concentrations and that manipulations of apoB are not necessarily followed by alterations of Lp(a).


*    Selected Abbreviations and Acronyms
 
DGUC = density gradient ultracentrifugation
ELISA = enzyme-linked immunosorbent assay
FHBL = familial hypobetalipoproteinemia
FPLC = fast-performance liquid chromatography
GGE = gradient gel electrophoresis
Lp(a) = lipoprotein(a)
r-apo(a) = recombinant apo(a)
SDS-PAGE = sodium dodecyl sulfate–polyacrylamide gel electrophoresis


*    Acknowledgments
 
This work was supported by NIH grants R01-HL-42460 (to Gustav Schonfeld) and Program Project grant HL-30086 (Santica M. Marcovina). We are grateful to the members of the FHBL kindreds and the nonrelated subjects who permitted us to study them, to Dr Stephen Young of the Gladstone Foundation (San Francisco, Calif) for permitting us to study some of his subjects, to Diana Tessereau, RN, for her good relations with our study subjects and for obtaining the blood samples, to Dr Richard Lawn for providing the r-apo(a), to Dr Mickey LaTour for help with statistical analyses, to Mary Lou Rheinheimer for preparing the manuscript, and to Tom Kitchens, Tish Kettler, and Connie Ferguson for expert technical help.

Received June 14, 1995; accepted October 9, 1995.


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