Search

International audience; Apolipoproteins govern lipoprotein metabolism and are promising biomarkers of metabolic and cardiovascular diseases. Unlike immunoassays, MS enables the quantification and phenotyping of multiple apolipoproteins. Hence, here, we aimed to develop a LC-MS/MS assay that can simultaneously quantitate 18 human apolipoproteins [A-I, A-II, A-IV, A-V, B48, B100, C-I, C-II, C-III, C-IV, D, E, F, H, J, L1, M, and (a)] and determined apoE, apoL1, and apo(a) phenotypes in human plasma and serum samples. The plasma and serum apolipoproteins were trypsin digested through an optimized procedure and peptides were extracted and analyzed by LC-MS/MS. The method was validated according to standard guidelines in samples spiked with known peptide amounts. The LC-MS/MS results were compared with those obtained with other techniques, and reproducibility, dilution effects, and stabilities were also assessed. Peptide markers were successfully selected for targeted apolipoprotein quantification and phenotyping. After optimization, the assay was validated for linearity, lower limits of quantification, accuracy (biases: -14.8% to 12.1%), intra-assay variability [coefficients of variation (CVs): 1.5-14.2%], and inter-assay repeatability (CVs: 4.1-14.3%). Bland-Altman plots indicated no major statistically significant differences between LC-MS/MS and other techniques. The LC-MS/MS results were reproducible over five repeated experiments (CVs: 1.8-13.7%), and we identified marked differences among the plasma and serum samples. The LC-MS/MS assay developed here is rapid, requires only small sampling volumes, and incurs reasonable costs, thus making it amenable for a wide range of studies of apolipoprotein metabolism. We also highlight how this assay can be implemented in laboratories.

AIMS Lipoprotein (a) [Lp(a)] is a lipoprotein species causatively associated with atherosclerosis. Unlike statins, PCSK9 inhibitors (PCSK9i) reduce Lp(a), but this reduction is highly variable. Levels of Lp(a) are chiefly governed by the size of its signature protein, apolipoprotein (a) [apo(a)]. Whether this parameter determines some of the reduction in Lp(a) induced by PCSK9i remains unknown. We aimed to investigate if the Lp(a) lowering efficacy of PCSK9i is modulated by the size of apo(a), which is genetically determined by the variable number of KIV domains present on that protein. METHODS AND RESULTS The levels of Lp(a) and the size of apo(a) were assessed in plasma samples from 268 patients before and after treatment with PCSK9i. Patients were recruited at the Outpatient Lipid Clinic of the Charite Hospital (Berlin) between 2015 and 2020. They were hypercholesterolemic at very high CVD risk with LDL-cholesterol levels above therapeutic targets despite maximally tolerated lipid-lowering therapy. Patients received either Alirocumab (75 or 150 mg) or Evolocumab (140 mg) every 2 weeks. Apo(a), apoB100, and apoE concentrations as well as apoE major isoforms were determined by liquid chromatography high-resolution mass spectrometry. Apo(a) isoforms sizes were determined by Western Blot. PCSK9i sharply reduced LDL-cholesterol (-57%), apoB100 (-47%) and Lp(a) (-36%). There was a positive correlation between the size of apo(a) and the relative reduction in Lp(a) induced by PCSK9i (r = 0.363, p = 0.0001). The strength of this association remained unaltered after adjustment for baseline Lp(a) levels and all other potential confounding factors. In patients with two detectable apo(a) isoforms, there was also a positive correlation between the size of apo(a) and the reduction in Lp(a), separately for the smaller (r = 0.350, p = 0.0001) and larger (r = 0.324, p = 0.0003) isoforms. The relative contribution of the larger isoform to the total concentration of apo(a) was reduced from 29% to 15% (p

Familial hypercholesterolemia (FH) is characterized by elevated low-density lipoprotein-cholesterol and markedly increased cardiovascular risk. In patients with a genetic diagnosis, low-density lipoprotein receptor (LDLR) mutations account for >90% of cases, apolipoprotein B (APOB) mutations for ≈5% of cases, while proprotein convertase subtilisin kexin type 9 (PCSK9) gain of function mutations are rare (T) was found in 5 index patients and cascade screening identified 15 additional carriers. Low-density lipoprotein-cholesterol levels were higher in these 15 carriers compared with the 27 noncarriers (236±73 versus 124±35 mg/dL; P, This work was funded by Regeneron: R727-CL-1555. Grants received from the Fritz Thyssen Foundation, Amsterdam Stollings Foundation, that is, the Jan Wouter ten Cate Prize, Amsterdam University Funds, and the Atheros Foundation to R. Huijgen. Grants received from Academic Medical Center Young Talent Fund, grant received by the Academic Medical Center Foundation, and the Atheros Foundation to M.L. Hartgers. Grants received from Programme de Recherche Hospitalière en Santé ANR-16-RHUS-0007 to K. Chemello and G. Lambert. Grants received from Basque Government (Grupos Consolidados IT1264-19) to A. Benito-Vicente, K.B. Uribe, and C. Martin.

Familial hypercholesterolemia (FH) is one of the most common genetic disorders in humans. It is an extremely atherogenic metabolic disorder characterized by lifelong elevations of circulating LDL-C levels often leading to premature cardiovascular events. In this review, we discuss the clinical phenotypes of heterozygous and homozygous FH, the genetic variants in four genes (LDLR/APOB/PCSK9/LDLRAP1) underpinning the FH phenotype as well as the most recent in vitro experimental approaches used to investigate molecular defects affecting the LDL receptor pathway. In addition, we review perturbations in the metabolism of lipoproteins other than LDL in FH, with a major focus on lipoprotein (a). Finally, we discuss the mode of action and efficacy of many of the currently approved hypocholesterolemic agents used to treat patients with FH, with a special emphasis on the treatment of phenotypically more severe forms of FH., K. C. received a scholarship from the European Union (European Regional Development Fund INTERREG V) and the Région Réunion (Saint-Denis, Réunion, France); J. G.-N. is a recipient of a Ramón y Cajal fellowship from the Spanish Ministerio de Ciencia, Innovación y Universidades; A. G. has received a postdoctoral fellowship from the Fondation pour la Recherche Médicale; C. M. has received research grants from the Basque Government Grupos Consolidados IT-1264-19; GL Research Group is funded by the Agence Nationale de la Recherche (Paris, France) Program Grant CHolesterol Personalized Innovation ANR-16-RHUS-0007 and project grant KRINGLE2 ANR-20-CE14-0009 as well as from La Fondation de France (project grant: 00096274).

Therapeutic antibodies targeting proprotein convertase subtilisin kexin type 9 (PCSK9) (e.g. alirocumab) lower low-density lipoprotein cholesterol (LDL-C) and lipoprotein (a) [Lp(a)] levels in clinical trials. We recently showed that PCSK9 enhances apolipoprotein(a) [apo(a)] secretion from primary human hepatocytes but does not affect Lp(a) cellular uptake. Here, we aimed to determine how PCSK9 neutralization modulates Lp(a) levels in vivo. Six nonhuman primates (NHP) were treated with alirocumab or a control antibody (IgG1) in a crossover protocol. After the lowering of lipids reached steady state, NHP received an intravenous injection of [2H3]-leucine, and blood samples were collected sequentially over 48 h. Enrichment of apolipoproteins in [2H3]-leucine was assessed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Kinetic parameters were calculated using numerical models with the SAAMII software. Compared with IgG1, alirocumab significantly reduced total cholesterol (TC) (−28%), LDL-C (−67%), Lp(a) (−56%), apolipoprotein B100 (apoB100) (−53%), and apo(a) (−53%). Alirocumab significantly increased the fractional catabolic rate of apoB100 (+29%) but not that of apo(a). Conversely, alirocumab sharply and significantly reduced the production rate (PR) of apo(a) (−42%), but not significantly that of apoB100, compared with IgG1, respectively. In line with the observations made in human hepatocytes, the present kinetic study establishes that PCSK9 neutralization with alirocumab efficiently reduces circulating apoB100 and apo(a) levels by distinct mechanisms: apoB primarily by enhancing its catabolism and apo(a) primarily by lowering its production.

International audience; There are 3 isoforms of apolipoprotein E (apo E) in humans (ε2, ε3, and ε4). They differ by single amino acid substitutions that variably affect their affinity for the low-density lipoprotein receptor (LDLR) and for the LDLR-related protein (LRP1), with ε2 having the weakest binding to these receptors (1). The plasma levels of lipoprotein(a) [Lp(a)], a highly atherogenic LDL-like lipoprotein species, are influenced by the polymorphism of apo E, with ε2/ε2 and ε4/ε4 carriers presenting with the lowest and highest Lp(a) plasma concentrations, respectively (1).

Objective— Evolocumab, a PCSK9 (proprotein convertase subtilisin kexin type 9)–neutralizing antibody, lowers low-density lipoprotein cholesterol (LDL-C) in homozygous familial hypercholesterolemic (HoFH) patients with reduced LDLR (low-density lipoprotein receptor) function. However, their individual responses are highly variable, even among carriers of identical LDLR genetic defects. We aimed to elucidate why HoFH patients variably respond to PCSK9 inhibition. Approach and Results— Lymphocytes were isolated from 22 HoFH patients enrolled in the TAUSSIG trial (Trial Assessing Long Term Use of PCSK9 Inhibition in Subjects With Genetic LDL Disorders). Ten patients were true homozygotes (FH1/FH1) and 5 identical compound heterozygotes (FH1/FH2). Lymphocytes were plated with or without mevastatin, recombinant PCSK9 (rPCSK9), or a PCSK9-neutralizing antibody. Cell surface LDLR expression was analyzed by flow cytometry. All HoFH lymphocytes had reduced cell surface LDLR expression compared with non-FH lymphocytes, for each treatment modality. Lymphocytes from FH1/FH2 patients (LDLR defective/negative) displayed the lowest LDLR expression levels followed by lymphocytes from FH1/FH1 patients (defective/defective). Mevastatin increased, whereas rPCSK9 reduced LDLR expression. The PCSK9-neutralizing antibody restored LDLR expression. Lymphocytes displaying higher LDLR expression levels were those isolated from patients presenting with lowest levels of LDL-C and apolipoprotein B, before and after 24 weeks of evolocumab treatment. These negative correlations remained significant in FH1/FH1 patients and appeared more pronounced when patients with apolipoprotein E3/E3 genotypes were analyzed separately. Significant positive correlations were found between the levels of LDLR expression and the percentage reduction in LDL-C on evolocumab treatment. Conclusions— Residual LDLR expression in HoFH is a major determinant of LDL-C levels and seems to drive their individual response to evolocumab.