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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.

International audience; Background and aims: Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) is an endogenous inhibitor of the LDL receptor (LDLR). Mendelian randomization studies suggest that PCSK9 deficiency increases diabetes risk, but the underlying mechanisms remain unknown. The aim of our study was to investigate whether PCSK9 or its inhibition may modulate beta cell function.Methods: We assessed PCSK9 and insulin colocalization in human pancreatic sections by epifluorescent and confocal microscopy. We also investigated the expression and the function of PCSK9 in the human EndoC-8H1 beta cell line, by ELISA and flow cytometry, respectively. PCSK9 was inhibited with Alirocumab or siRNA. LDLR expression and LDL uptake were assessed by flow cytometry.Results: PCSK9 was expressed and secreted from beta cells isolated from human pancreas as well as from EndoC-8H1 cells. PCSK9 secretion was enhanced by statin treatment. Recombinant PCSK9 decreased LDLR abundance at the surface of these cells, an effect abrogated by Alirocumab. Alirocumab as well as PCSK9 silencing increased LDLR expression at the surface of EndoC-8H1 cells. Neither exogenous PCSK9, nor Alirocumab, nor PCSK9 silencing significantly altered glucose-stimulated insulin secretion (GSIS) from these cells. High-low density lipoproteins (LDL) concentrations decreased GSIS, but the addition of PCSK9 or its inhibition did not modulate this phenomenon.Conclusions: While PCSK9 regulates LDLR abundance in beta cells, inhibition of exogenous or endogenous PCSK9 does not appear to significantly impact insulin secretion. This is reassuring for the safety of PCSK9 inhibitors in terms of beta cell function.

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.

Objective: To clarify the association between PCSK9 (proprotein convertase subtilisin/kexin type 9) and Lp(a) (lipoprotein [a]), we studied Lp(a) kinetics in patients with loss-of-function and gain-of-function PCSK9 mutations and in patients in whom extended-release niacin reduced Lp(a) and PCSK9 concentrations. Approach and Results: Six healthy controls, 9 heterozygous patients with familial hypercholesterolemia (5 with low-density lipoprotein receptor [ LDLR ] mutations and 4 with PCSK9 gain-of-function mutations) and 3 patients with heterozygous dominant-negative PCSK9 loss-of-function mutations were included in the preliminary study. Eight patients were enrolled in a second study assessing the effects of 2 g/day extended-release niacin. Apolipoprotein kinetics in VLDL (very-low-density lipoprotein), LDL (low-density lipoprotein), and Lp(a) were studied using stable isotope techniques. Plasma Lp(a) concentrations were increased in PCSK9 -gain-of-function and familial hypercholesterolemia- LDLR groups compared with controls and PCSK9 -loss-of-function groups (14±12 versus 5±4 mg/dL; P =0.04), but no change was observed in Lp(a) fractional catabolic rate. Subjects with PCSK9 -loss-of-function mutations displayed reduced apoE (apolipoprotein E) concentrations associated with a VLDL-apoE absolute production rate reduction. Lp(a) and VLDL-apoE absolute production rates were correlated ( r =0.50; P r =0.96; P r =0.83; P =0.015). In contrast, PCSK9 reduction (−35%; P =0.008) was only correlated with that of VLDL-apoE absolute production rate ( r =0.79; P =0.028). Conclusions: VLDL-apoE production could determine Lp(a) production and/or assembly. As PCSK9 inhibitors reduce plasma apoE and Lp(a) concentrations, apoE could be the link between PCSK9 and Lp(a).

International audience; AimThe benefit of the lipid-lowering drug fenofibrate on cardiovascular outcomes is controversial. Our aim was to find new circulating markers to identify those patients most likely to benefit from fenofibrate prescription.MethodsAnalyses were conducted of plasma samples collected from 102 patients with type 2 diabetes, enrolled in the FIELD trial, before and after fenofibrate treatment (200 mg/day). Non-targeted and targeted lipid analyses and apolipoprotein measurements were made using mass spectrometry methods.ResultsLipidomics revealed a global decrease in ceramide after fenofibrate treatment confirmed by quantitative analysis (−18.2%, P < 0.001). These changes were strongly associated with those found for plasma sphingomyelin (r = 0.80, P < 0.001) and, to a lesser extent, for sphingosine-1-phosphate (r = 0.34, P < 0.001). Ceramide levels decreased in 73.5% of patients. In addition to the expected lipid changes (decreases in triglycerides, total cholesterol and LDL cholesterol, and increase in HDL cholesterol), fenofibrate also lowered plasma apoC-II (−11.1%, P < 0.01), apoC-III (−24.6%; P < 0.001), apoB100 (−27.0%, P < 0.01) and sphingomyelinase (−7.6%, P < 0.001), and increased plasma apoA-II (22.4%, P < 0.001) as well as adiponectin (11.4%, P < 0.001). No significant association was found between ceramide decrease and these modulations except for total cholesterol (r = 0.20, P = 0.047) and HDL protein components. At baseline, only elevated sphingolipid levels were significantly associated with ceramide reduction after fenofibrate treatment.ConclusionFenofibrate lowers plasma ceramide independently of the usual lipid parameters. As ceramide is a strong marker of atherosclerosis, our study underpins the need to further evaluate its contribution to cardiovascular events in fenofibrate-treated patients.AimThe benefit of the lipid-lowering drug fenofibrate on cardiovascular outcomes is controversial. Our aim was to find new circulating markers to identify those patients most likely to benefit from fenofibrate prescription.MethodsAnalyses were conducted of plasma samples collected from 102 patients with type 2 diabetes, enrolled in the FIELD trial, before and after fenofibrate treatment (200 mg/day). Non-targeted and targeted lipid analyses and apolipoprotein measurements were made using mass spectrometry methods.ResultsLipidomics revealed a global decrease in ceramide after fenofibrate treatment confirmed by quantitative analysis (−18.2%, P < 0.001). These changes were strongly associated with those found for plasma sphingomyelin (r = 0.80, P < 0.001) and, to a lesser extent, for sphingosine-1-phosphate (r = 0.34, P < 0.001). Ceramide levels decreased in 73.5% of patients. In addition to the expected lipid changes (decreases in triglycerides, total cholesterol and LDL cholesterol, and increase in HDL cholesterol), fenofibrate also lowered plasma apoC-II (−11.1%, P < 0.01), apoC-III (−24.6%; P < 0.001), apoB100 (−27.0%, P < 0.01) and sphingomyelinase (−7.6%, P < 0.001), and increased plasma apoA-II (22.4%, P < 0.001) as well as adiponectin (11.4%, P < 0.001). No significant association was found between ceramide decrease and these modulations except for total cholesterol (r = 0.20, P = 0.047) and HDL protein components. At baseline, only elevated sphingolipid levels were significantly associated with ceramide reduction after fenofibrate treatment.ConclusionFenofibrate lowers plasma ceramide independently of the usual lipid parameters. As ceramide is a strong marker of atherosclerosis, our study underpins the need to further evaluate its contribution to cardiovascular events in fenofibrate-treated patients.

Objective— ApoM (apolipoprotein M) binds primarily to high-density lipoprotein before to be exchanged with apoB (apolipoprotein B)–containing lipoproteins. Low-density lipoprotein (LDL) receptor–mediated clearance of apoB-containing particles could influence plasma apoM kinetics and decrease its antiatherogenic properties. In humans, we aimed to describe the interaction of apoM kinetics with other components of lipid metabolism to better define its potential benefit on atherosclerosis. Approach and Results— Fourteen male subjects received a primed infusion of 2 H 3 -leucine for 14 hours, and analyses were performed by liquid chromatography–tandem mass spectrometry from the hourly plasma samples. Fractional catabolic rates and production rates within lipoproteins were calculated using compartmental models. ApoM was found not only in high-density lipoprotein (59%) and LDL (4%) but also in a non–lipoprotein-related compartment (37%). The apoM distribution was heterogeneous within LDL and non–lipoprotein-related compartments according to plasma triglycerides ( r =0.86; P r range, 0.55–0.89; P r =0.55; P =0.042). Significant correlations were found between triglycerides and production rates of LDL-apoM ( r =0.73; P Conclusions— In humans, LDL kinetics play a key role in apoM turnover. Plasma triglycerides act on both apoM and sphingosine-1-phosphate distributions between lipoproteins. These results confirmed that apoM could be bound to high-density lipoprotein after secretion and then quickly exchanged with a non–lipoprotein-related compartment and to LDL to be slowly catabolized.

The combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and trypsin proteolysis is an effective tool for accurate quantitation of multiple proteins in a single run. However, expensive samples pre-treatment as immunoenrichment are often required to analyze low abundant proteins. Plasma proprotein convertase subtilisin/kexin type 9 (PCSK9), a circulating regulator of low-density lipoprotein metabolism, was studied as an example of a low abundant plasma protein. We investigated post-proteolysis solid-phase extraction (SPE) as an alternative strategy to improve its detection. After optimization of pretreatment, including denaturation, reduction, alkylation, tryptic digestion and selective SPE concentration, 91 +/- 7% of PCSK9 was recovered from human plasma samples and coefficients of variation were less than 13.2% with a lower limit of quantification of 37.5 ng/ml. This LC-MS/MS method was compared with standard enzyme-linked immunosorbent assay in 30 human plasma samples with a broad range of PCSK9 concentrations. Both methods were significantly correlated (r =0.936, p < 0.001) with less than 7% of the values out of the 95% confidence interval and similar concentrations were measured using either LC-MS/MS or ELISA methods (514.2 +/- 217.2 vs. 504.2 +/- 231.0 ng/ml, respectively p = NS). This method involving SPE is an effective measurement tool for low abundant plasma protein analysis that could be easily included in multiplexed assays.

Objective— To determine the mechanisms by which extended-release nicotinic acid reduces circulating lipoprotein (a) concentrations in hypertriglyceridemic patients. Approach and Results— Eight nondiabetic, obese male subjects (aged 48±12 years; body mass index, 31.2±1.8 kg/m 2 ) with hypertriglyceridemia (triglycerides, 226±78 mg/dL) were enrolled in an 8 week, double blind, placebo-controlled cross-over study. At the end of each treatment phase, fasted subjects received a 10 µmol/L per kg bolus injection of [5,5,5- 2 H 3 ]- l -Leucine immediately followed by constant infusion of [5,5,5- 2 H 3 ]- l -Leucine (10 µmol L −1 kg −1 h −1 ) for 14 hours, and blood samples were collected. A liquid chromatography–tandem mass spectrometry method was used to study apolipoprotein (a) (Apo(a)) kinetics. The fractional catabolic rate of Apo(a) was calculated with a single compartmental model using the apolipoprotein B100 (ApoB100) containing very low density lipoprotein tracer enrichment as a precursor pool. Extended-release nicotinic acid decreased plasma triglycerides (−46%; P =0.023), raised high-density lipoprotein cholesterol (+20%; P =0.008), and decreased Apo(a) plasma concentrations (−20%; P =0.008). Extended-release nicotinic acid also decreased ApoB100 (22%; P =0.008) and proprotein convertase subtilisin/kexin type 9 (PCSK9, −29%; P =0.008) plasma concentrations. Apo(a) fractional catabolic rate and production rates were decreased by 37% (0.58±0.28 versus 0.36±0.19 pool/d; P =0.008) and 50% (1.4±0.8 versus 0.7±0.4 nmol/kg per day; P =0.008), respectively. Conclusions— Extended-release nicotinic acid treatment decreased Apo(a) plasma concentrations by 20%, production rates by 50%, and catabolism by 37%. ApoB100 and PCSK9 concentrations were also decreased by treatment, but no correlation was found with Apo(a) kinetic parameters.