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Relationship of Apolipoprotein(a) Isoform Size with Clearance and Production of Lipoprotein(a) in a Diverse Cohort

Open AccessPublished:January 24, 2023DOI:https://doi.org/10.1016/j.jlr.2023.100336

      ABSTRACT

      Lipoprotein(a) [Lp(a)] has two main proteins, apolipoprotein B100 (apoB100) and apolipoprotein(a) [apo(a)]. High levels of Lp(a) confer an increased risk for atherosclerotic cardiovascular disease (ASCVD). Most people have two circulating isoforms of apo(a) differing in their molecular mass, determined by the number of Kringle IV Type 2 repeats. Previous studies report a strong inverse relationship between Lp(a) levels and apo(a) isoform sizes. The roles of Lp(a) production and fractional clearance, and how ancestry affects this relationship remains incompletely defined. We therefore examined the relationships of apo(a) size with Lp(a) levels and both apo(a) fractional clearance rates (FCR) and production rates (PR) in 32 individuals not on lipid-lowering treatment. We determined plasma Lp(a) levels and apo(a) isoform sizes and used the relative expression of the two isoforms to calculate a “weighted isoform size” (wIS). Stable isotope studies were performed, using D3-leucine, to determine the apo(a) FCR and PR. As expected, plasma Lp(a) concentrations were inversely correlated with wIS (R2=0.27; p=0.002). The wIS had a modest positive correlation with apo(a) FCR (R2=0.10, p=0.08) and a negative correlation with apo(a) PR (R2=0.11; p=0.06). The relationship between wIS and PR became significant when we controlled for self-reported race and ethnicity (SRRE) (R2=0.24, p=0.03); controlling for SRRE did not affect the relationship between wIS and FCR. Overall, apo(a) wIS plays a role in both apo(a) FCR and PR. Adjustment for SRRE strengthened the correlation between wIS and PR, suggesting an effect of ancestry on the assembly and/or secretion of Lp(a).

      Keywords

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      MATERIALS AND METHODS

      Study Population

      The study subjects had participated in one of three separate stable isotope studies examining the effects on lipoprotein metabolism of (1) an apoB100 antisense, (2) an inhibitor of cholesteryl ester transfer protein, or (3) a monoclonal antibody against PCSK9
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      . The studies were approved by the Columbia University Irving Medical Center (CUIMC) Institutional Review Board. All subjects provided informed consent before enrolling in the studies, which included consent for the use of their study data and samples for future research. Due to liquid chromatography mass spectrometry (LC/MS) assay sensitivity limitations, we only included subjects with Lp(a) concentrations above 10 nmol/L. The present analysis uses the baseline (pre-intervention phase) studies of 32 healthy individuals of varying SRRE. Subjects were not on lipid-lowering agents, nor were they taking over-the-counter supplements. None of the subjects had clinical ASCVD and were considered in good health as assessed by medical history and physical exam. The studies reported in this manuscript abide by the Declaration of Helsinki.

      Study Design

      Complete details of the stable isotope studies on the metabolism of apoB100 and apo(a) have been previously published
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      • Ginsberg H.N.
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      • Hubbard B.
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      • Ginsberg H.N.
      • Millar J.S.
      • Reyes-Soffer G.
      CETP (Cholesteryl Ester Transfer Protein) Inhibition With Anacetrapib Decreases Production of Lipoprotein(a) in Mildly Hypercholesterolemic Subjects.
      . Briefly, isocaloric, low-fat liquid meals (57% carbohydrate, 18% fat, 25% protein) were started 8 hours before stable isotope administration (1:00 AM on Day 1) and provided to subjects every 2 hours for the next 32 hours to maintain steady state metabolic conditions during the kinetic studies. Subjects received a bolus injection of 5,5,5-D3-leucine dissolved in 0.15M NaCl (10 μmol/kg body weight) immediately followed by a constant infusion of D3-leucine dissolved in 0.15M NaCl (10 μmol/kg body weight/hour) for 15 hours. EDTA blood samples were collected at 18 pre-defined times over 24 hours and plasma separated and stored at -80°C. Aliquots of these banked samples were utilized for this study; the samples had not been previously thawed or refrozen.

      Biochemical and Immunological Assays

      Plasma lipids (total cholesterol, triglycerides, and HDL cholesterol) were measured on an Integra400plus (Roche) from samples obtained at baseline. Plasma LDL cholesterol levels were calculated using the Friedewald formula (no subject had a triglyceride level >400 mg/dL). Plasma apoB100 levels were measured by a human enzyme-linked immunosorbent assay (ELISA) kit # 3715-1HP-2, from Mabtech, Inc, Cincinnati, OH.

      Apo(a) Stable Isotope Enrichment Determination

      Apo(a) enrichment with D3-leucine was measured as described by Zhou et al.
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      . In brief, 200 μL of the LDL fraction or equal volumes of LDL (100 μL) and HDL (100 μL) fractions isolated from plasma by ultracentrifugation were desalted. Isolated lipoprotein fractions were then treated with dithiothreitol to open disulfide bonds, alkylated with iodoacetamide, and digested using trypsin. A multiple reaction monitoring method was used to monitor the following precursor-product ion transitions of a peptide specific to apo(a): (LFLEPTQADIALLK): 786.7 > 1069.7 (M0) and 788.2 > 1069.7 (M3). Two μL of the digested samples were analyzed using a nanoAcquity ultra-performance LC system coupled with an ionKey source integrated to a Xevo TQ-S triple quadrupole tandem mass spectrometer (Waters, Milford, MA). The separation was achieved using an iKey Peptide BEH C18 separation device (130 Ǻ, 1.7 μm, 150 μm × 100 mm) maintained at 60°C. The gradient was 90% A (0.1% formic acid in water)/10% B (0.1% formic acid in acetonitrile) ramped linearly to 10% A at 6 min, held for 3 min, and then re-equilibrated to initial conditions (total run time: 12 min; flow rate: 3 μl/min). The multiple reaction transitions were monitored with a collision energy of 24 eV.

      Lp(a) concentration and apo(a) isoform size

      Lp(a) plasma concentration was measured using the isoform-independent sandwich ELISA developed by the Northwest Lipid Metabolism and Diabetes Research Laboratory
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      Use of a reference material proposed by the International Federation of Clinical Chemistry and Laboratory Medicine to evaluate analytical methods for the determination of plasma lipoprotein(a).
      . Apo(a) isoform size measurements were performed by the same laboratory. We started with 250μL of plasma and each sample was diluted in saline to have 100ng of protein in 40 μL, which was combined with an equal volume of reducing buffer and boiled for 10 min. The sample was then loaded onto an agarose gel and run overnight at 123V and 4°C, transferred to a nitrocellulose membrane, immunoblotted, and imaged using the ChemiDoc MP Imaging System to determine the isoforms (separated by size) present in the samples by comparison to in-house standards (combined material containing six apo(a) isoforms: 38, 32, 24, 19, 15, and 12 KIV-2 repeats)
      • Marcovina S.M.
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      Relation between number of apolipoprotein(a) kringle 4 repeats and mobility of isoforms in agarose gel: basis for a standardized isoform nomenclature.
      . The relative expression of each isoform was determined using the Image Lab software, which calculated relative proportions of the two isoforms based on the intensity profile of each lane. The method has an intra-sample variability under 15%.

      Weighted Isoform Size (wIS) Calculation

      Most individuals express two apo(a) isoforms in plasma, and these are inversely correlated with Lp(a) plasma levels, with smaller isoforms generally dominating
      • Kraft H.G.
      • Sandholzer C.
      • Menzel H.J.
      • Utermann G.
      Apolipoprotein (a) alleles determine lipoprotein (a) particle density and concentration in plasma.
      . To ascertain the contribution of isoforms to plasma Lp(a) concentration, we estimated a weighted isoform size (wIS). Each expressed isoform can potentially have a different FCR (equivalently, as used by some investigators, fractional synthetic rate FSR), say, k1 and k2 for the two isoforms. As apo(a) is a slowly turning over protein and we use a primed constant infusion protocol in our studies, the apo(a) enrichment, when expressed as a fraction of the precursor plateau, goes up nearly linearly during the 15-hour infusion period and the rising slope, as a fraction of the plateau enrichment, equals the FCR or FSR. If E1, the enrichment of isoform 1, goes up with slope k1, and E2 goes up with slope k2, it can be seen that the overall enrichment E, which equals m1E1+m2E2, goes up with slope m1k1+m2k2, where m1 and m2 are the relative masses (i.e., mass fractions, m1+m2=1) of the two isoforms, with the total mass denoted by M.
      If there is a linear relationship (with intercept “a” and slope “b”) between isoform-specific FCR, termed k, and the corresponding isoform size, termed S, it means k = a+bS. Applying it to the two isoforms 1 and 2 above, the relationships become k1=a+bS1 and k2=a+bS2. It follows, then, that the combined apo(a) FCR, which is m1k1+m2k2, equals m1(a+b S1)+m2(a+bS2), which simplifies to a+b(m1S1+m2S2). We define m1S1+m2S2 as the weighted isoform size or wIS. Substituting, we see that the combined apo(a) FCR, termed kc, follows the relationship kc=a+b wIS. Thus, when we estimate a single apo(a) FCR, it bears the same linear relationship with wIS as the isoform-specific FCR would bear with the corresponding isoform size. Further, if we look at the total production rate PR, which is the sum of the two isoform-specific PR1 and PR2, where PR1= m1Mk1 and PR2= m2Mk2, it follows that PR= m1Mk1+m2Mk2=M(m1k1+m2k2)=Mkc. That is, the total PR equals the total mass multiplied by the FCR we estimate from the total enrichment data. When we calculate a single apo(a) PR, it bears the same relationship with wIS as the isoform-specific PR would bear with the corresponding isoform size.
      Example: Say the two isoform masses are M1 and M2, so total Lp(a) mass is M=M1+M2. If the two isoform sizes are 20 and 30, with relative expression of 70% and 30%, respectively, the wIS is 0.7*20+0.3*30 = 23. If now, the two isoforms are cleared with rate constants k1=0.4 and k2=0.2, then, by the formulas above, PR1=0.7*M*0.4=0.28M; PR2=0.3*M*0.2=0.06M; kc=0.7*0.4+0.3*0.2=0.34. We see that total PR=PR1+PR2 = 0.34M= kcM.

      Apo(a) Modeling

      The apo(a) enrichment data were modeled as previously described
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      Effects of mipomersen, an apolipoprotein B100 antisense, on lipoprotein (a) metabolism in healthy subjects.
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      Effects of PCSK9 Inhibition With Alirocumab on Lipoprotein Metabolism in Healthy Humans.
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      • Reyes-Soffer G.
      CETP (Cholesteryl Ester Transfer Protein) Inhibition With Anacetrapib Decreases Production of Lipoprotein(a) in Mildly Hypercholesterolemic Subjects.
      . Apo(a) FCR was calculated by fitting the leucine enrichment data in an apo(a)-specific peptide using a single-pool model, with the precursor enrichment set as the VLDL apoB100 D3-leucine enrichment plateau in the same study. The plateau is typically reached during the first 15-hour sampling period and estimated using our model for VLDL apoB100 metabolism
      • Ramakrishnan R.
      • Ramakrishnan J.D.
      Using mass measurements in tracer studies--a systematic approach to efficient modeling.
      ,
      • Ramakrishnan R.
      Studying apolipoprotein turnover with stable isotope tracers: correct analysis is by modeling enrichments.
      . The apo(a) PR in nmol/kg/day was calculated as the product of apo(a) FCR (in pools/day) and the apo(a) concentration (nmol/L) multiplied by the plasma volume (estimated as 0.045 L/kg).

      Statistical Analysis

      All data were analyzed using standard R software functions [summary, lm, estimable, ggplot, etc.] invoked by our cufunctions package

      Holleran S and Ramakrishnan R. cufunctions, a package to facilitate statistical analyses in R. 2021:http://biomath.net/cufunctions.html.

      . Variables found to be normally distributed are summarized by mean and standard deviation, while Lp(a) levels, along with triglycerides, are summarized by median and interquartile range. Pearson correlation coefficients are reported. The relationship of Lp(a) levels with wIS in the three SRRE groups was studied by an analysis of covariance (ANCOVA).

      RESULTS

      The subject demographic data as well as plasma lipid and apoB100 levels are shown in Table 1. We analyzed data from 32 subjects with a mean age of 46.8 years. Seventeen subjects (17) were female, and by SRRE, there were 17 Black (B), 9 Hispanic (H), and 6 White (W) subjects. The mean body mass index (BMI) was 28.9 ± 4.3 kg/m2. Lipid and apoB100 levels were within normal ranges.
      Table 1Population Demographics, Lipid and ApoB100 Levels. Legend: ± – Mean and Standard Deviation; ( ) – Median and Interquartile Range
      Characteristic
      Age, y46.8 ± 12.4
      Range for age26 – 68
      Body Mass Index, kg/m228.9 ± 4.3
      Race (n)FemaleMale
      White24
      Black98
      Hispanic63
      Total Cholesterol, mg/dL173 ± 38.8
      Triglycerides, mg/dL109 (52.5, 143)
      LDL-C, mg/dL103 ± 28.9
      HDL-C, mg/dL51.8 ± 17.2
      Plasma ApoB100, mg/dL81.2 ± 22.1

      Lp(a) levels and Apo(a) kinetics

      The study population had median Lp(a) levels of 54.6 nmol/L (interquartile range 36.8-119.0) (Table 2). Plasma Lp(a) levels did not differ between males and females (data not shown). Participants had a mean wIS of 22.8 ± 4. As expected from published data, Black subjects had a higher median Lp(a) concentration. In our cohort, 9 of the 32 individuals (28%) had only one detectable isoform. Mean ± SD FCR and PR of apo(a) were 0.18 ± 0.08 pools/day and 0.57 ± 0.40 nmol/kg/day, respectively (Table 2). Individual data for the full cohort are provided in Supplemental Table 1.
      Table 2Lp(a) Plasma Levels, Weighted Isoform Size, and Kinetic Parameters of Apo(a)
      Study sample
      Lp(a) (nmol/L)54.6 (36.8, 119.0)
      Black61.2 (43.7, 127.6)
      Hispanic42.0 (21.5, 116.4)
      White49.8 (27.1, 62.3)
      Weighted Isoform Size22.8 ± 4.0
      Apo(a) FCR (pools/day)0.18 ± 0.1
      Apo(a) PR (nmol/kg/day)0.57 ± 0.4
      Legend: ± - Mean and Standard Deviation; ( ) – Median and Interquartile Range
      As observed in larger population data sets, our subjects had an inverse relationship between Lp(a) levels and wIS (R2=0.27, p=0.002) (Figure 1A). Lp(a) levels are impacted by SRRE, hence we examined the relationship between Lp(a) levels and wIS for each SRRE group, Figure 1B. Although adjustment for SRRE strengthened the overall correlation (R2=0.35), SRRE group differences were not statistically significant [Black-Hispanic (p=0.25); Black-White (p=0.11); Hispanic-White (p=0.57)]. The lack of significance may be due to the small number of subjects in each SRRE group. The relationship between individual apo(a) isoforms and the Lp(a) levels associated with each isoform in the combined cohort of all subjects (Blacks, Hispanics, and Whites) was also statistically significant (p<0.0001) (Supplemental Figure 1). Isoform size is a determinant of Lp(a) concentration, and it is known that SRRE plays a role in determining Lp(a) levels at any isoform size, thus we included SRRE in all our data analyses examining the relationships of wIS with the kinetics of apo(a).
      Figure thumbnail gr1
      Figure 1Legend: (A) Negative association of Lp(a) levels with wIS. (B) Negative association of Lp(a) levels with wIS, controlling for SRRE using ANCOVA (Analysis of Covariance). Lp(a) – lipoprotein(a); wIS – weighted isoform size; Self-Reported Race/Ethnicity (SRRE): B – Black, H – Hispanic, W – White.
      The relationship between Lp(a) levels and apo(a) FCR (R2=0.07, p=0.16) was not statistically significant (Supplemental Figure 2). When we examined the relationships of FCR with wIS, we found a positive trend (p=0.08) (Figure 2A). Controlling for SRRE did not affect the relationship of wIS with FCR (Figure 2B).
      Figure thumbnail gr2
      Figure 2Legend: Relationship between apo(a) FCR with wIS before (A) and after controlling for SREE (B) using ANCOVA (Analysis of Covariance). Relationship of apo(a) PR with wIS before (C) and after controlling for SREE (D) using ANCOVA (Analysis of Covariance). Apo(a) – apolipoprotein(a); FCR – Fractional Catabolic Rate; PR – Production Rate; wIS – weighted isoform size; SRRE - Self-Reported Race/Ethnicity; B – Black, H – Hispanic, W – White.
      Additionally, wIS showed a negative trend with apo(a) PR (p=0.06) (Figure 2C), and this relationship became statistically significant when controlling for SRRE (p=0.03) (Figure 2D).
      The results above comprised all 32 subjects, including 9 individuals who expressed only a single isoform (Supplemental Table 1). In this subgroup with single isoforms, we found that wIS and FCR were positively correlated (R2=0.61, p=0.01), but no correlations were found between the single isoforms and PR (R2=0.20, p=0.23) (Supplemental Figure 3A and 3B).

      DISCUSSION

      High plasma Lp(a) levels are associated with an increased risk for ASCVD
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      . Similar to other cohorts
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      Apolipoprotein(a) gene accounts for greater than 90% of the variation in plasma lipoprotein(a) concentrations.
      , the current study finds an inverse association between plasma Lp(a) levels and apo(a) allele size, with smaller isoforms associated with higher Lp(a) levels (Supplemental Figure 1). Some studies have also found smaller apo(a) isoforms to be associated with CAD
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      , although only two of these studies demonstrated that association to be independent of Lp(a) concentration.

      Relationship of Plasma Lp(a) Concentrations with PR and FCR

      Previous reports support a role of production and/or clearance regulating Lp(a) plasma levels; these have been reviewed
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      . Studies by Krempler et al. and Rader et al. found that PR, but not FCR, correlated, with Lp(a) levels
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      . Our study agrees with these findings, as we found no significant relationship between Lp(a) levels and FCR (Supplemental Figure 2). Similarly, in a large study of the effects of a PCSK9 inhibitor on apoB metabolism in individuals without concomitant statin therapy, a treatment-associated decrease in the plasma pool size of Lp(a)-apo(a) was linked with a decrease in the PR of Lp(a)-apo(a), with no effect on FCR in the subjects not on statins. However, in the group taking statins, treatment with the PCSK9 inhibitor resulted in an increase in the FCR of Lp(a)-apo(a) with no treatment-effect on the PR of Lp(a)-apo(a)
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      . These studies and other reports
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      The metabolism of apolipoproteins (a) and B-100 within plasma lipoprotein (a) in human beings.
      support a role of both FCR and PR in the regulation of plasma Lp(a) levels.

      Relationship of apo(a) isoforms with PR and FCR

      As previously stated, an individual’s plasma Lp(a) level, with few exceptions, is highly regulated by the number of KIV-2 repeats present in their apo(a) isoforms, which are determined by their two apolipoprotein(a) gene (LPA) alleles. Early studies in cultured liver cells, using both steady state labeling and pulse chase analyses, showed that the endoplasmic reticulum residence time of secreted apo(a) isoforms is determined by their size, and that this accounted for the inverse relationship between isoform size and level of secretion. The authors concluded that apo(a) post-translational stability is a major determinant of the levels of plasma Lp(a) in baboons
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      . Additional cell work provided support for the important role of the number of KIV-2 repeats in the rate of assembly and secretion of apo(a)
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      . Human studies using externally labeled Lp(a) demonstrated the importance of PR in determining plasma levels of Lp(a) in subjects with varying Lp(a) levels and either similar apo(a) isoforms
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      or varying apo(a) sizes
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      . The latter study showed an inverse correlation between apo(a) size and PR of apo(a). In both of these studies, the FCRs of Lp(a) were not related to the concentration of plasma Lp(a). On the other hand, Jenner et al. reported that isoform size, determined by gel electrophoretic separation, affected both the PR and FCR of apo(a) in studies using endogenous labeling of Lp(a) with stable isotopes
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      . Subjects with smaller isoforms had higher PRs of apo(a), similar to the findings of Rader et al, but they also had lower apo(a) FCRs, the latter similar to our current findings. These previous studies did not determine and take into account participant SRRE.
      Advances in mass spectrometry and methods to isolate Lp(a) have enhanced our ability to interrogate the mechanisms that regulate Lp(a)
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      . In this current study, we used, wIS, an estimated weighted isoform size (see Methods), which captures the contribution of each isoform to the Lp(a) level in the circulation. The use of isoform expression to calculate isoform specific Lp(a) plasma levels has been applied in earlier studies
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      High levels of Lp(a) with a small apo(a) isoform are associated with coronary artery disease in African American and white men.
      . Calculation of the wIS suffers from some limitations listed below, yet it allowed us to assess the effects of a weighted mean of two expressed isoforms on FCR and PR of apo(a). We found that wIS had only modest correlations with both PR and FCR. Our results are consistent with previous reports that found strong relationships between allele size and PR, but also identified trends with FCR
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      PCSK9 inhibition with alirocumab decreases plasma lipoprotein(a) concentration by a dual mechanism of action in statin-treated patients with very high apolipoprotein(a) concentration.
      . As seen in Figure 2A, one individual in our cohort had a very large wIS and excluding this individual from the analysis improved the relationship between wIS and FCR (p<0.02). Relevant to our current findings, a sub-analysis by Chan et al.
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      Lipoprotein(a) Particle Production as a Determinant of Plasma Lipoprotein(a) Concentration Across Varying Apolipoprotein(a) Isoform Sizes and Background Cholesterol-Lowering Therapy.
      of the baseline results obtained from a study of the effects of evolocumab on the kinetic of Lp(a) metabolism, found that levels of Lp(a) were negatively associated with apo(a) size and FCR, and positively associated with PR. Moreover, in subjects with small isoforms (≤22), they found strong correlations between apo(a) concentration and increased apo(a) PR but not with FCR. In subjects with large isoforms (>22), on the other hand, Lp(a) levels were correlated with both kinetic parameters
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      Lipoprotein(a) Particle Production as a Determinant of Plasma Lipoprotein(a) Concentration Across Varying Apolipoprotein(a) Isoform Sizes and Background Cholesterol-Lowering Therapy.
      . The authors found similar associations in the subjects treated with either statin alone, evolocumab alone, or the combination of the two treatments. They demonstrated that Lp(a) lowering with a PCSK9 inhibitor, evolocumab, lowered plasma Lp(a) levels by decreasing apo(a) PR and increasing apo(a) FCR. We found similar results when administering the PCSK9 inhibitor, alirocumab
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      Effects of PCSK9 Inhibition With Alirocumab on Lipoprotein Metabolism in Healthy Humans.
      . Importantly, different non-targeted Lp(a)-lowering treatments decrease Lp(a) by different effects on FCR and PR. Niacin lowered plasma Lp(a) levels in association with decreases in both PR and FCR
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      Effects of Extended-Release Nicotinic Acid on Apolipoprotein (a) Kinetics in Hypertriglyceridemic Patients.
      . Mipomersen, an apoB antisense oligonucleotide (ASO), reduced Lp(a) by increasing FCR, although PR was reduced as well in some individuals
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      Effects of mipomersen, an apolipoprotein B100 antisense, on lipoprotein (a) metabolism in healthy subjects.
      . Anacetrapib, a CETP inhibitor, decreased Lp(a) by decreasing PR
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      CETP (Cholesteryl Ester Transfer Protein) Inhibition With Anacetrapib Decreases Production of Lipoprotein(a) in Mildly Hypercholesterolemic Subjects.
      . The results from those studies support a complex regulatory mechanism of Lp(a) levels. This may be due to the additional proteins and lipids found on and within Lp(a) particles
      • von Zychlinski A.
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      Proteomics of Lipoprotein(a) identifies a protein complement associated with response to wounding.
      . Lastly, the exact location where the covalent linkage of apo(a) to apoB100 assembly occurs (intrahepatic or at the surface of the liver), as well as the site and molecular mechanism of Lp(a) clearance from plasma, are not completely defined
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      Understanding the ins and outs of lipoprotein (a) metabolism.
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      Recent advances in demystifying the metabolism of lipoprotein(a).
      . Recent studies using cell models with a single isoform (17 KIV-2 repeats), found that, in addition to a covalent disulfide bond between apo(a) and apoB100, there are also noncovalent interactions between these two proteins
      • Youssef A.
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      • Koschinsky M.L.
      Apo(a) and ApoB Interact Noncovalently Within Hepatocytes: Implications for Regulation of Lp(a) Levels by Modulation of ApoB Secretion.
      . The latter observations, if true in vivo, could affect measurements of FCR. Additionally, free apo(a) fragments have been found in plasma and urine, but their concentrations are very low and their physiological role, if any, poorly understood
      • Mooser V.
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      Apolipoprotein(a) kringle 4-containing fragments in human urine. Relationship to plasma levels of lipoprotein(a).
      ,
      • Doucet C.
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      Lipoprotein(a) in the nephrotic syndrome: molecular analysis of lipoprotein(a) and apolipoprotein(a) fragments in plasma and urine.
      .
      Due to clear racial differences in the relationship of isoform size and plasma Lp(a) levels
      • Berglund L.
      • Kim K.
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      • Prakash N.
      • Truax K.
      • Anuurad E.
      • Enkhmaa B.
      Lp(a)-Associated Oxidized Phospholipids in Healthy Black and White Participants in Relation to apo(a) Size, Age, and Family Structure.
      ,
      • Rubin J.
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      Apolipoprotein [a] genotype influences isoform dominance pattern differently in African Americans and Caucasians.
      , it is important to control for these when analyzing such data. Since our study population was composed of a diverse cohort, we controlled for any effects of SRRE on the analyzed study outcomes. When we adjusted for SRRE, the relationship between wIS and PR was statistically significant.
      Lp(a) lowering with non-targeted and targeted treatments decreases both isoforms. Of interest, the relative expression of apo(a) isoforms does not change after Lp(a) levels are lowered using antisense (ASO) apo(a) treatment
      • Karwatowska-Prokopczuk E.
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      Prevalence and influence of LPA gene variants and isoform size on the Lp(a)-lowering effect of pelacarsen.
      . The latter result suggests that apo(a) ASO treatment does not preferentially affect one isoform size over the other. Similarly, in data from our lab, we have not observed treatment effects on wIS after various therapies that lower apoB100 and apo(a) (Supplemental Table 2). A recent study using PCSK9 inhibitors showed a positive correlation between apo(a) size and reductions in Lp(a) levels for both small and large isoforms of apo(a)
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      The size of apolipoprotein (a) is an independent determinant of the reduction in lipoprotein (a) induced by PCSK9 inhibitors.
      . Recent studies using a targeted siRNA therapy showed significant Lp(a) lowering, but no isoform size data have been presented
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      .
      Lastly, there are studies examining the roles of single nucleotide polymorphisms (SNPs) in LPA and within the KIV2 region that are linked to high and low Lp(a) levels. The allele frequencies of these SNPs have been found to differ across SRRE groups
      • Enkhmaa B.
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      Lipoprotein (a): impact by ethnicity and environmental and medical conditions.
      ,
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      ,
      • Zekavat S.M.
      • Ruotsalainen S.
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      • Alver M.
      • Bloom J.
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      • Rotter J.I.
      • Rich S.S.
      • Post W.
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      • Correa A.
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      • Wilson J.G.
      • Salomaa V.
      • Kellis M.
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      • Neale B.M.
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      • Group N.T.L.W.
      Deep coverage whole genome sequences and plasma lipoprotein(a) in individuals of European and African ancestries.
      . These differences in SNP presentation could explain why Lp(a) levels differ for similar isoform sizes in different SRRE groups. The effect of these SNPs on the clearance and production of Lp(a) has not been studied.
      Study Limitations: Our results indicate that apo(a) isoforms have a significant, yet modest contribution to the mechanisms regulating apo(a) FCR and PR. Although we did include subjects with different SRRE in this study, our study population was small, with 17 of our 32 subjects identifying as Black, leaving very few subjects in the other groups. Mechanistic studies are costly and labor extensive, hence it will be difficult to perform studies in large populations with adequate sample size for different SRRE groups. Our results, however, highlight the need to recruit diverse cohorts when designing these studies. Berglund et. al. examined the role of isoforms in larger diverse cohorts
      • Berglund L.
      • Kim K.
      • Zhang W.
      • Prakash N.
      • Truax K.
      • Anuurad E.
      • Enkhmaa B.
      Lp(a)-Associated Oxidized Phospholipids in Healthy Black and White Participants in Relation to apo(a) Size, Age, and Family Structure.
      showing associations similar to those found in our cohort, with Blacks having higher Lp(a) level for the same apo(a) isoform size compared to Whites, even though the difference did not reach statistical significance in our study due to the limited subject number. However, the study by Berglund et. al. did not examine metabolic pathways
      • Berglund L.
      • Kim K.
      • Zhang W.
      • Prakash N.
      • Truax K.
      • Anuurad E.
      • Enkhmaa B.
      Lp(a)-Associated Oxidized Phospholipids in Healthy Black and White Participants in Relation to apo(a) Size, Age, and Family Structure.
      .
      There were methodologic limitations: In the current study, we did not isolate individual apo(a) isoforms and calculate their unique FCR and PR; instead, we used the relative expression data from gel electrophoresis to estimate their contributions to the wIS. We examined the kinetics of apo(a) isolated from LDL or LDL+HDL fractions. However, the apo(a) PR and FCR from LDL-only or from LDL plus HDL fractions were not statistically different (Supplemental Figure 4) and the data were, therefore, combined for all analyses. Apo(a) measurements were performed on plasma samples by a validated ELISA
      • Marcovina S.M.
      • Albers J.J.
      • Scanu A.M.
      • Kennedy H.
      • Giaculli F.
      • Berg K.
      • Couderc R.
      • Dati F.
      • Rifai N.
      • Sakurabayashi I.
      • Tate J.R.
      • Steinmetz A.
      Use of a reference material proposed by the International Federation of Clinical Chemistry and Laboratory Medicine to evaluate analytical methods for the determination of plasma lipoprotein(a).
      and not on the mass spectrometry used to obtain enrichments. Various methods have been proposed to measure apo(a) via mass spectrometry
      • Lassman M.E.
      • McLaughlin T.M.
      • Zhou H.
      • Pan Y.
      • Marcovina S.M.
      • Laterza O.
      • Roddy T.P.
      Simultaneous quantitation and size characterization of apolipoprotein(a) by ultra-performance liquid chromatography/mass spectrometry.
      , however we did not have these methods available at the time of the study.

      Data Availability Statement:

      All the data generated during and/or analyzed during the current study are available from the corresponding author and are included in this published article and its supplementary information file.

      Acknowledgements:

      We would like to acknowledge our research volunteers and the nurses and staff of the Irving Institute for Clinical and Translational Science at CUIMC. All apo(a) isoform size and expression data were obtained in the laboratory of Dr. Santica Marcovina at University of Washington.

      Supplementary data

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