© 1995 American Heart Association, Inc.
Articles |
Lipid and Lipoprotein Factors Associated With Variation in Lp(a) Density
From the Department of Genetics, Southwest Foundation for Biomedical Research, and the Division of Clinical Epidemiology, Department of Medicine, University of Texas Health Science Center (S.M.H.), San Antonio, Tex.
Correspondence to David L. Rainwater, PhD, Department of Genetics, Southwest Foundation for Biomedical Research, PO Box 28147, San Antonio, TX 78228–0147.
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Abstract |
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Abstract To determine the facets of lipoprotein metabolism associated with variation in lipoprotein(a) [Lp(a)] density, we examined by density gradient ultracentrifugation 246 plasma samples exhibiting single apo(a) isoform band phenotypes. Estimated molecular weights of apo(a) isoforms ranged from 488 to 874 kD, and they accounted for approximately 80% of variation in Lp(a) density. After adjustment for variation in the protein composition, we found in univariate analyses that variation in residual Lp(a) density was associated with 10 different measures of lipoprotein size and concentration. Residual Lp(a) density was positively correlated with measures of apoB-containing lipoproteins and negatively correlated with measures of HDL. HDL size phenotypes were based on nondenaturing gradient gel electrophoresis fractions after staining for esterified cholesterol with Sudan black B and for apoA-I with immunoblotting methods. The HDL size variables in each case had higher correlations with residual Lp(a) density than did the HDL concentration measures. Stepwise regression analyses selected two lipoprotein variables (LDL density and HDL size) that were significantly correlated with residual Lp(a) density, and they accounted for approximately 35% of variation in density. The densities of LDL and Lp(a) were highly correlated, and additional stepwise regression analyses showed that they were similarly correlated with triglyceride concentration and with a measure of HDL size. Thus, with respect to residual Lp(a) density, the results show that small, dense Lp(a) particles are found under conditions leading also to small, dense LDL particles and to small, dense HDLs.
Key Words: Lp(a) • apo(a) • density gradient ultracentrifugation • gradient gel electrophoresis • HDL subclasses
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Introduction |
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Lipoprotein(a) [Lp(a)] is an unusual lipoprotein that in healthy individuals ranges from undetectable (ie, <1 mg/dL) to more than 100 mg/dL.1 High levels of Lp(a) are associated with cardiovascular disease,1 2 although results from some studies are contradictory.3 Lp(a) concentrations are under strong genetic control2 ; Boerwinkle and colleagues4 estimate that more than 90% of Lp(a) variation is attributable to genetic variation at the locus encoding apo(a), the unique apolipoprotein component of Lp(a). Metabolic studies suggest that Lp(a) concentrations are controlled primarily by differences in production rates specified by the apo(a) gene,5 6 and studies of baboon Lp(a) have shown that variation in both transcriptional and posttranslational processes affects serum Lp(a) concentrations.7 8 Because of the overriding effects of biosynthesis, it follows that concentrations of Lp(a) are substantially unaffected by processes governing metabolism of other lipoproteins. Indeed, Lp(a) is generally considered to be refractory to conventional methods of controlling hyperlipidemia.9
However, a number of studies have shown small but significant differences in Lp(a) concentrations, corresponding to administration of certain drugs and hormones9 10 and to metabolic disorders such as renal disease and diabetes.11 Moreover, we have shown baboon Lp(a) to be significantly affected by dietary fat and cholesterol.12 13 These studies point to the likelihood that Lp(a), once secreted by the liver into the vascular compartment, can be modified by some of the same processes that control metabolism of other lipoproteins.
In addition to concentration, Lp(a) composition, including both protein and lipid, can also show variation among individuals. Except for the fact that apo(a) size is known to be genetically determined,1 metabolic factors influencing particle composition are not well understood. Furthermore, the relation of variation in particle composition with cardiovascular disease also is not known. One measure directly related to particle composition is particle density. Previous investigators showed that most variation in Lp(a) density was explained by variation in apo(a) size.14 15 Thus, any residual variation in Lp(a) density [after adjustment for apo(a) size differences] will likely reflect changes in lipid composition. As a first step in understanding metabolic influences on Lp(a), the present study was designed to detect associations of variation in residual density with other plasma lipid and lipoprotein measures. We determined the densities of Lp(a) particles in frozen human plasma samples. After adjusting for the large effects of apo(a) size variation, we discovered that Lp(a) particle densities were associated with variation in both HDLs and apoB-containing lipoproteins. The results suggest that Lp(a) particle compositions are influenced by metabolic pathways that also affect other classes of lipoproteins.
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Methods |
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Study Participants and Samples
Plasma samples were obtained from fasted individuals participating in the San Antonio Family Heart Study, a study of risk factors for cardiovascular disease in a Mexican American population.16 Plasma was isolated by low-speed centrifugation and stored as aliquots in plastic tubing segments17 at -80°C. An Institutional Review Board (University of Texas Health Science Center at San Antonio) approved the procedures, and all subjects gave informed consent. Samples (n=246) for the present study were selected on the basis of exhibiting a single apo(a) isoform band phenotype and having an Lp(a) concentration of at least 2 mg/dL. Given the sensitivities of the Lp(a) assay and apo(a) isoform phenotyping protocols, such samples have sufficient Lp(a) to measure in density gradient fractions, and the measured particle density should represent that of a single apo(a) species. Table 1
shows some of the characteristics of the population
sampled in this study.
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Clinical Measurements
Cholesterol and triglyceride (TG) concentrations were assayed
enzymatically with a Gilford SBA–300 Clinical Chemistry Analyzer with
reagents supplied by Boehringer-Mannheim Diagnostics and Stanbio,
respectively. To measure HDL cholesterol, apoB-containing lipoproteins
were precipitated from freshly collected plasma by use of dextran
sulfate.18 The interassay coefficients of variation for
control products were 1.7% for cholesterol, 5.6% for HDL cholesterol,
and 3.2% for TGs. Non-HDL (ie, apoB-containing) lipid measures were
calculated as the difference between the total and HDL plasma
values.
Apolipoprotein concentrations were measured in the laboratory of Dr Evan A. Stein (Medical Research Laboratories, Cincinnati, Ohio): apoA-I and apoB concentrations were determined by nephelometry19 20 21 ; apoA-II and apoE concentrations were determined by competitive immunoassays22 23 ; and LpA-I (ie, HDL bearing apoA-I but not apoA-II) concentration was determined by electroimmunoassay.24 Lp(a) levels were measured in frozen plasma aliquots with a commercial assay kit [MacraLp(a) ELISA kit, Strategic Diagnostics] with an EL340 Microplate reader (Bio-Tek). The interassay coefficients of variation for quality control samples were 3.5% for apoA-I, 4.4% for apoA-II, 2.9% for apoB, 8.1% for apoE, 6.8% for LpA-I, and 4.6% for Lp(a).
Apo(a) Isoform Phenotype Determinations
Apo(a) isoform phenotypes were determined in plasma samples (1
µL) treated with sodium dodecyl sulfate and ß-mercaptoethanol and
resolved in 3% to 15% acrylamide gradient gels.12 25
After transfer to nitrocellulose, the locations of apo(a) proteins were
detected with rabbit antibodies directed against baboon
apo(a)26 that also bind the human proteins. Molecular
weights of apo(a) bands were estimated by comparison with locations of
four standard bands in an immediately adjacent lane; the molecular
weights of the four bands were estimated to be 776 000, 681 000,
592 000, and 434 000 with standards provided by Dr Joel D. Morissett
(Baylor College of Medicine, Houston, Tex).27 Molecular
weights were based on estimates from an average of 3.37 separate
determinations (SD, 1.42; range, 1 to 8 determinations); the
coefficient of variation for estimates averaged 1.17% (SD, 0.82;
range, 0% to 3.8%).
Density Gradient Ultracentrifugation
Plasma (250 µL) was mixed with 625 µL of d=1.37
g/mL KBr solution and carefully overlaid with KBr solutions of varying
densities (all solutions contained 1 mmol/L EDTA): 1.0 mL of
d=1.21, 3.0 mL of d=1.125, 3.0 mL of
d=1.07, 2.0 mL of d=1.05, and 1.5 mL of
d=1.02. Gradients were subjected to centrifugation (39 000
rpm, 5°C) in an SW41 swinging bucket rotor for 48 hours with an L8
ultracentrifuge (Beckman Instruments). Gradients were displaced from
the tube with Fluorinert FC–40 through an ISCO UA5 monitor and
collected in 0.4-mL fractions. Density in fractions was measured by
refractometry with an Abbe-3L refractometer (Milton Roy). Lp(a)
concentrations in fractions were measured as indicated above; a single
dilution based on the known plasma concentration was used for all
fractions assayed from each gradient. After assay, we identified the
three to six fractions that contained the Lp(a) peak and calculated the
weighted average density for Lp(a) in those fractions. The calculated
density was verified by estimating the density from the absorbance
profile in those cases in which Lp(a) presented a discrete peak. In
most absorbance profiles, there was also a single discrete peak of LDL
whose peak density was measured (n=160). However, for some samples, the
sensitivity of the absorbance detector was not sufficient to identify a
discrete LDL peak.
To test whether freezing altered the estimates of Lp(a) peak densities, we subjected 12 samples to density gradient ultracentrifugation: one aliquot of each sample had never been frozen and one aliquot had been frozen for 4 days before use. We found the average difference between unfrozen and frozen sample pairs to be 0.0003 g/mL (SD, 0.0021; range, -0.0029 to 0.0050), which was not significant (P=.64, paired t test).
Gradient Gel Electrophoresis
Lipoproteins in plasma were subjected to electrophoresis in
nondenaturing 3% to 31% acrylamide gradient gels that were cast in
the laboratory.28 We used immunoblotting
procedures29 30 to detect the HDL distributions of apoA-I.
Proteins in the gel were electrophoretically transferred to
nitrocellulose paper (0.2 µm BA83, Schleicher and Schuell) and
detected by binding with sheep anti-human apoA-I (Boehringer-Mannheim)
followed by a radioiodinated second antibody (donkey anti-sheep IgG,
Chemicon) that was labeled with the chloramine T method.31
Radioactivity was located with autoradiography; HDL distributions were
determined by densitometry (LKB Ultroscan Laser Densitometer with
GSXL software). The distributions of cholesteryl esters
among HDL subclasses were detected with Sudan black B (SBB) staining
and densitometry as described previously.30 32
HDL absorbance profiles were analyzed by fitting gaussian curves33 to the subfractions of HDL.34 The fractional absorbances in HDL1, HDL2b, and HDL2a were summed and divided by the summed fractional absorbances in HDL3a, HDL3b, and HDL3c to calculate a variable related to HDL size distribution. The HDL size variables were calculated from absorbance profiles derived from each stain and in the text are called HDL size (apoA-I) and HDL size (SBB).
Statistical Methods
Multiple regression analyses were done with a statistical
package (STATGRAPHICS, Manugistics). To improve the
assumptions of normality, all lipid, apolipoprotein, and HDL size data
were transformed to their natural logarithm values before analyses.
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Results |
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Density Gradient Ultracentrifugation of Lp(a)
Plasma lipoproteins from samples with a single apo(a) isoform band were resolved in KBr density gradients, and the location of Lp(a) in the gradient was determined with an immunoassay. Fig 1
shows results from one such analysis. As expected, apo(a) and Lp(a)
were colocalized in a density region between LDL and HDL. Because only
samples with a single band were selected for this study, Lp(a) peaks
were generally symmetrical. Lp(a) peak densities were measured in a
total 246 samples, and Fig 2 shows a histogram of Lp(a)
peak densities; the average density was 1.0858 g/mL (range, 1.0640 to
1.1015).
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Relation of Apo(a) Molecular Weight and Lp(a) Density
To assess effects on Lp(a) density, we estimated the size of
apo(a) for each sample (Fig 3). Linear regression
analysis indicated that apo(a) size explained 81% of variation in
Lp(a) density (P<.0001). The y intercept for the
regression line was 1.029 g/mL, which is more buoyant than the average
LDL peak density of 1.045 g/mL (Table 1
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Correlation of Lipid and Lipoprotein Measures With Lp(a)
Density
Using regression analysis, we tested whether lipid and
lipoprotein measures were associated with variation in residual Lp(a)
density, ie, Lp(a) density remaining after correction for the effects
of apo(a) size. Univariate regression analyses showed that five
measures of apoB-containing lipoproteins were all positively correlated
with residual Lp(a) density (Table 2). The significant
measures included LDL density and the concentrations of TG, apoB, apoE,
and non-HDL cholesterol; Lp(a) concentrations were not associated with
variation in Lp(a) density. We also found that five different measures
of HDL particles were negatively correlated with residual Lp(a)
density. The significant measures included assessments of HDL size, as
stained with SBB and anti–apoA-I, and the concentrations of HDL
cholesterol, LpA-I, and apoA-I. Each measure of HDL size had stronger
correlations with variation in residual Lp(a) density than did any of
the four measures of lipid and protein concentrations analyzed.
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We used stepwise regression analysis to select the variables that
significantly explained residual Lp(a) density. The final model
(R2=.3471, n=160) contained two lipoprotein
measures that were correlated with residual Lp(a) density variation:
LDL density (P<.0001) and HDL size (SBB)
(P=.0122). Fig 4 shows the component effects
of the two variables on variation in residual Lp(a) density. In
multiple regression analyses that included both apo(a) size and the
three lipoprotein measures, the regression equation explained 86.3% of
total variation in Lp(a) density.
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Correlates of Lp(a) and LDL Densities
We attempted to determine the common correlates of LDL and
residual Lp(a) densities by conducting stepwise regression analyses in
which we used the same lipoprotein measures as above except that LDL
density was excluded. For residual Lp(a) density, the final model
(R2=.2146, n=206) contained two lipoprotein
measures: HDL size (SBB) (P<.0001) and TG concentration
(P=.0017). Fig 5A and 5B shows the component
effects of the two variables on residual Lp(a) density. With LDL
density as the dependent variable, the final model
(R2=.3653, n=160) contained two lipoprotein
measures that were correlated with LDL density: HDL size (apoA-I)
(P<.0001) and TG concentration (P<.0001). Fig 5C and 5D show the component effects of the two variables on LDL
density. In each model, TG concentration was positively correlated and
the HDL size variable was negatively correlated with density.
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Discussion |
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To determine the lipoprotein factors associated with Lp(a) density variation, we selected 246 samples exhibiting a single apo(a) isoform band. About 45% of participants in the San Antonio Family Heart Study showed a single-banded phenotype. Single-banded apo(a) isoform phenotypes can result from several conditions. The individual could be truly homozygous, which is unlikely given the high index of heterozygosity reported for the apo(a) gene.35 The individual could be heterozygous for two alleles encoding apo(a) proteins of identical or very similar size, thus exhibiting a single-banded phenotype. This possibility is supported by studies demonstrating sequence variation in the apo(a) gene that does not give rise to size variation36 and could pose a problem for interpretation of the present results only if there are allele-specific differences in associations with lipoprotein metabolism. Finally, the individual could be heterozygous with alleles encoding the detected isoform and an undetected or "null" isoform. In studies of baboon Lp(a), we observed that null phenotypes result both from transcriptional and from posttranslational levels of control.8 However, it is possible that the second allele is expressed at very low levels, and in this case sensitivity of detection becomes an issue. Participants in the current study tended to have low Lp(a) concentrations, which would increase the proportion of single-banded phenotypes. For example, a study of Framingham offspring used the same Lp(a) assay as we did. Jenner et al37 reported that the 10th percentile in their population was 1.1 mg/dL for females and 1.0 mg/dL for males. In contrast, the corresponding figures for our population are 0.5 and 0.4 mg/dL, respectively. However, even among those persons with Lp(a) concentrations below 1 mg/dL (n=130), we assigned apo(a) isoform phenotypes to 82 (63%) of the individuals (64 were single-banded and 18 were double-banded phenotypes). For the present study, we selected samples having a minimum Lp(a) concentration of 2 mg/dL. If any sample in fact had an additional very faint apo(a) isoform band, it is likely that the concentration would be so low that the estimated density would still reflect that of particles bearing the dominant band whose size was measured.
For the present analyses, we subjected aliquots of frozen plasma to density gradient ultracentrifugation. Although all samples were subjected to the same freezing protocol before analysis,17 it is possible that the act of freezing could have altered Lp(a) density. However, our earlier study showed that frozen aliquots of baboon serum gave the same estimates of peak densities for Lp(a), LDL, and HDL as were obtained with unfrozen aliquots.26 Furthermore, using human samples, Dormans and colleagues38 observed virtually identical LDL subclass patterns in frozen and unfrozen aliquots of samples subjected to density gradient ultracentrifugation. Finally, data from the present study showed no significant difference in Lp(a) peak densities estimated with frozen and unfrozen aliquots from 12 samples.
We calculated the weighted average Lp(a) density based on Lp(a)
concentrations in three to six fractions. The average Lp(a) peak
density for the 246 individuals ranged from 1.064 to 1.102 g/mL, with a
mean of 1.086 g/mL. This distribution of densities (Fig 2) is very
similar to that observed by Fless and colleagues39 in
studies of 90 subjects. Also similar are results from two other studies
focused on the effects of apo(a) size variation that reported density
ranges for Lp(a) particles bearing different size isoforms to be 1.043
to 1.114 g/mL for 29 subjects14 and 1.057 to 1.091 for 44
subjects.15 Our population tended to have relatively
larger isoforms than reported in other populations; in fact, no isoform
in the present study migrated into the gel farther than apoB. This
may help explain why we have not observed the lower range of particle
densities. It is not clear why our Lp(a) peak densities do not range as
high as in the other study,14 although one possibility is
different gene frequencies in this Mexican American population compared
with other populations studied.
As reported previously,14 the major determinant of variation in Lp(a) peak density is apo(a) protein size variation. Using linear regression analyses, we found that nearly 80% of total variation in Lp(a) density was explained by apo(a) size. The y intercept, ie, the density of an Lp(a) particle with no contribution from apo(a), was calculated to be 1.029 g/mL. The average LDL density for a subset (n=160) of the samples was 1.045 g/mL. On the basis of results from two individuals, Fless and colleagues reported that Lp(a–) particles [the Lp(a) particles remaining after apo(a) was dissociated by chemical reduction] were larger and more buoyant than autologous LDL particles.40 Thus, our data, taken from a much larger population, confirm as a generality that Lp(a–) particles are less dense than autologous LDL.
The primary focus of this study was to determine whether Lp(a) density
variation is associated with any other aspect of lipoprotein
metabolism. Therefore, we tested for correlations with the variation in
residual Lp(a) density after first adjusting for the large effects of
apo(a) protein size. We found, using univariate regression analyses,
that a total of 10 lipoprotein measures were significantly associated
with residual Lp(a) density variation (Table 2). There were two major
classes of variables: measures of HDLs that were negatively correlated
and measures of apoB-containing lipoproteins that were positively
correlated with Lp(a) density.
We used stepwise regression analyses to select a subset of independent measures significantly correlated with variation. The multivariate model included measures of LDL density and HDL size (SBB); nearly 35% of the variation in residual Lp(a) density was related to these variables. The variables selected were among those most strongly correlated in the univariate analyses. When included in this model, neither sex nor age was a significant factor (data not shown).
The different measures of HDLs can be dichotomized into two types,
including measures of concentration and measures of particle size
distribution. HDL size phenotypes were determined with nondenaturing
gradient gel electrophoresis and were constructed as the proportion of
absorbance in HDL1+2 divided by the proportion in
HDL3. The HDL size phenotypes are therefore directly
related to average HDL particle size as indicated by the lipoprotein
distributions of either apoA-I, the major protein species of HDL, or
cholesterol. HDL size phenotypes appeared to predict Lp(a) density
variation better than the concentration measures did; both HDL size
measures had higher correlations with Lp(a) density in the univariate
analyses (Table 2), and each stepwise regression analysis selected
an HDL size variable in preference to the corresponding HDL
concentration measure.
The data suggest that HDL size distributions represent better than HDL concentrations the aspects of HDL metabolism that are associated with Lp(a) density variation. Brinton and colleagues41 recently showed a striking correlation between another HDL size phenotype variable (ie, HDL cholesterol divided by the concentrations of apoA-I plus apoA-II) and apoA-I fractional catabolic rate. They postulated that many metabolic factors such as body fat distribution, sex, insulin resistance, core lipid exchange, and lipase activity affect HDL size phenotype, which in turn controls apoA-I fractional catabolic rate and HDL cholesterol levels. Thus, HDL size phenotype, which may play a central role in HDL metabolism, may affect also the metabolism of Lp(a) in the vascular compartment.
Among apoB lipoprotein measures significantly associated in the
univariate analyses with Lp(a) density variation, LDL density showed
the strongest correlation and was selected in the multivariate model.
Because LDL and residual Lp(a) densities were strongly correlated, we
tested, using stepwise regression analyses, whether similar lipoprotein
size and concentration variables were associated with variation in both
density measures. The multivariate model for variation in residual
Lp(a) density included TG concentration and HDL size (SBB). TG
concentration appeared to substitute for the information provided by
LDL density in the previous model. The multivariate model for variation
in LDL density included TG concentration and HDL size (apoA-I). Given
the high individual correlations with Lp(a) density (Table 2) and the
high degree of intercorrelation (data not shown), we presume that each
HDL size measure provides substantially similar information about HDL
metabolism that is relevant to apoB lipoprotein densities. For example,
substitution of HDL size (SBB) for HDL size (apoA-I) in the model
predicting LDL density variation changed the R2
value from .3653 to .3629. Thus, the densities of LDL and Lp(a) were
significantly correlated with TG concentration and an estimate of HDL
size phenotype.
With the assumption that the inverse relation between size and density applies to variation of residual Lp(a) density, the data demonstrate that small, dense Lp(a) particles are associated both with small, dense LDL and with small, dense HDL particles. Other studies have shown that small, dense LDL particles are associated with elevated TG and decreased HDL concentrations.42 The present data confirm these associations and show they also hold for small, dense Lp(a) particles. In several studies, small, dense LDL particle phenotypes were associated with cardiovascular disease.43 44 45 It is not known, however, whether small, dense Lp(a) particles, which are correlated with small, dense LDL, may be contributing factors in that association with cardiovascular disease.
In summary, we have demonstrated, after adjusting for the large effects of apo(a) size variation, that Lp(a) density variation is correlated with other lipoprotein size and concentration measures. These correlations indicate that lipoprotein metabolic processes of the vascular compartment have significant effects on Lp(a) composition. In particular, small, dense Lp(a) particles occur under conditions that also lead to small, dense LDL particles. Thus, while high concentrations of Lp(a) are associated with cardiovascular disease, it appears that another aspect of Lp(a) phenotype, particle density, forms part of an aggregate lipoprotein phenotype (ie, small, dense lipoproteins) that also may be associated with cardiovascular disease.
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Acknowledgments |
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This work was supported by National Institute of Health grant HL-45522. The excellent assistance of Allen Ford, Pat Moore, Rosa Rosello, Wendy Shelledy, and Jane VandeBerg is gratefully acknowledged.
Received August 31, 1994; accepted December 19, 1994.
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