Quantifying contribution of Lipoprotein (a) to atherogenic lipoprotein burden: a novel particle-based approach

Background: Elevated lipoprotein (a) [Lp(a)] is an independent risk factor for atherosclerotic cardiovascular disease (ASCVD). As clinical LDL cholesterol [LDL-C] incorporates cholesterol from Lp(a) [Lp(a)-C], there is interest in quantifying the contribution of Lp(a)-C to LDL-C given implications for risk assessment, diagnosis, and treatment. Estimating Lp(a)-C is subject to inaccuracies; measuring Lp(a) particle number [Lp(a)-P] is more accurate. Objective: To capture how Lp(a) contributes to the atherogenic lipoprotein burden, we demonstrate a particle-based approach using readily available measures of Lp(a)-P and apolipoprotein B (apoB). Methods: Using the Very Large Database of Lipids (VLDbL), we compared Lp(a)-P (nmol/L) with all atherogenic particles (non-HDL-P). Non-HDL-P was calculated by converting apoB mass to molar concentration using the preserved molecular weight of apoB100 (512 kg/mol). We calculated the percentage of Lp(a)-P relative to non-HDL-P by Lp(a)-P deciles and stratified across sex, age, triglycerides, LDL-C, and non-HDL-C. Results: 158,260 patients from the VLDbL were included. The fraction Lp(a)-P/non-HDL-P increased with rising Lp(a)-P. Lp(a)-P comprised on average 3% of atherogenic particles among the study population and 15% at the highest Lp(a)-P decile. Findings were similar when stratified by sex. When stratified by age, Lp(a)-P/non-HDL-P was highest among the youngest and oldest patients. Lp(a)-P/non-HDL-P decreased at higher levels of triglycerides and LDL-C owing to larger contributions from VLDL and LDL. Conclusions: We demonstrate a particle-based approach to quantify the contribution of Lp(a) to total atherogenic particle burden using validated and widely available laboratory assays. Future research applying this method could define clinically meaningful thresholds and inform use in risk assessment and management.

Abstract 18416: Discordance Between nonHDLc and Lipoprotein Particle Concentration (apoB and LDLp) in Relation to Future Coronary Events in Women

Background: There remains equipoise as to which plasma lipid/lipoprotein marker most accurately reflects longitudinal risk of coronary heart disease (CHD) events. To compare differences in risk related to nonHDLc (atherogenic particle cholesterol) and LDL particle number (LDLp) or apoB (atherogenic particle number), we examined risk when these markers were discordant. Methods and Results: We divided 27,533 initially-healthy women in the Women’s Health Study (NCT00000479) into concordant/discordant groups based on median nonHDLc (154 mg/dL) and apoB (100 mg/dL) or 1 H NMR-measured LDLp (1,216 nmol/L). Discordance was defined as nonHDLc < median and the alternative measure ≥ median, or vice versa. We compared risks between concordant and discordant groups (using the concordant group as reference) with Cox proportional hazard models adjusted incrementally for: age; and randomization arm, hormone use, postmenopausal status, smoking, and hypertension (“minimally adjusted”); and diabetes, BMI, hsCRP, HDLc, triglycerides, and family history of CHD (“fully adjusted”). Although all 3 biomarkers were strongly correlated - nonHDLc and apoB (Spearman r=0.86, P<0.0001), nonHDLc and LDLp (r=0.77, P<0.0001), and apoB and LDLp (r=0.85, P<0.0001) - discordance between nonHDLc and apoB or LDLp occurred in 13.9% and 20.2% of women, respectively. A total of 1,246 CHD events occurred over median (max) 20.4 (21.7) years of follow-up (514,725 person-years). With nonHDLc < median (Fig. a), CHD risk was underestimated among women with discordant high apoB or LDLp: fully adjusted HR (95% CI) high apoB = 1.33 (1.04, 1.71); high LDLp = 1.27 (1.01, 1.61). Alternately, with nonHDLc ≥ median (Fig. b), CHD risk was overestimated among women with discordant low apoB or LDLp: fully adjusted HR [95% CI] low apoB = 0.74 (0.57, 0.96); low LDLp = 0.93 (0.76, 1.14). Conclusions: For women with discordant levels of nonHDLc with apoB or LDLp, CHD risk may be underestimated or overestimated with nonHDLc.

Abstract 522: Scavenger Receptor-BI’s Cholesterol Transport Functions are Dependent on its Extracellular Tryptophan Residues

High density lipoprotein (HDL) functions as an anti-atherogenic particle, primarily due to its role in reverse cholesterol transport whereby HDL delivers cholesterol to the liver for excretion upon interaction with its receptor, scavenger receptor BI (SR-BI). The extracellular domain of SR-BI is required for its cholesterol transport functions, yet our understanding of the molecular and structural features of this domain remains limited. We designed experiments to test the hypothesis that one or more of the six highly conserved extracellular tryptophan (Trp; W) residues are critical for mediating receptor function. Towards this end, we created a series of Trp-to-Phe mutant receptors of SR-BI, as well as Trp-free SR-BI and assessed the ability of these mutant receptors to mediate cholesterol transport. Wild-type (WT) or mutant SR-BI receptors were transiently expressed in COS7 cells and proper cell surface expression was confirmed by immunoblotting, confocal microscopy and flow cytometry. Next, we showed that Trp-free- and W415F-SR-BI had a significantly decreased ability to bind HDL (12.7% and 31.3% of WT levels, respectively) and promote selective uptake of HDL-cholesteryl esters (35.2% and 70.1% of WT levels, respectively). Interestingly, only Trp-free-, but not W415F-SR-BI, showed an impaired ability to mediate efflux of free cholesterol (FC) (90.8% decrease vs. WT). Furthermore, both W415F- and Trp-free SR-BI were unable to reorganize plasma membrane pools of FC based on lack of sensitivity of FC to exogenous cholesterol oxidase. We then designed an additional set of mutant SR-BI receptors to determine whether restoration of Trp415 alone (or in combination with other Trp residues) could rescue SR-BI function. Restoration of Trp415 into Trp-free-SR-BI partially rescued cholesterol transport functions. Addition of any of the other 5 extracellular Trp residues was also not sufficient to restore WT cholesterol transport function in combination with Trp415. In summary, loss of all Trp residues in SR-BI impairs its cholesterol transport functions, mostly due to the loss of Trp415. Homology modeling of SR-BI based on the crystal structure of LIMP-2, a member of the same protein family, may help identify the importance of this residue in future studies.

Lipoprotein(a) in the Nephrotic Syndrome

Plasma levels of lipoprotein(a) (Lp(a)), an atherogenic particle, are elevated in kidney disease, which suggests a role of this organ in the metabolism of Lp(a). Additional evidence for a role of the kidney in the clearance of Lp(a) is provided by the fact that circulating N-terminal fragments of apolipoprotein(a) (apo(a)) are processed and eliminated by the renal route. To further understand the mechanism underlying such renal excretion, the levels of apo(a) fragments in plasma and urine relative to plasma Lp(a) levels were determined in patients with nephrotic syndrome (n = 15). In plasma, the absolute (24.7 ± 20.4 versus 2.16 ± 2.99 μg/ml, P < 0.0001) as well as the relative amounts of apo(a) fragments (4.6 ± 3.4% versus 2.1 ± 3.3% of total Lp(a), P < 0.0001) were significantly elevated in nephrotic patients compared with a control, normolipidemic population. In addition, urinary apo(a) excretion in patients with nephrotic syndrome was markedly elevated compared with that in control subjects (578 ± 622 versus 27.7 ± 44 ng/ml per mg creatinine, P < 0.001). However, the fractional catabolic rates of apo(a) fragments were similar in both groups (0.68 ± 0.67% and 0.62 ± 0.47% in nephrotic and control subjects, respectively), suggesting that increased plasma concentrations of apo(a) fragments in nephrotic subjects are more dependent on the rate of synthesis rather than on the catabolic rate. Molecular analysis of apo(a) immunoreactive material in urine revealed that the patterns of apo(a) fragments in nephrotic patients were distinct from those of control subjects. Full-length apo(a), large N-terminal apo(a) fragments similar in size to those present in plasma, as well as C-terminal fragments of apo(a) were detected in urine from nephrotic patients but not in urine from controls. All of these apo(a) forms were in addition to smaller N-terminal apo(a) fragments present in normal urine. This study also demonstrated the presence of Lp(a) in urine from nephrotic patients by ultracentrifugal fractionation. These data suggest that in nephrotic syndrome, Lp(a) and large fragments of apo(a) are passively filtered by the kidney through the glomerulus, whereas smaller apo(a) fragments are secreted into the urine.