Kinetics of plasma apolipoprotein E isoforms by LC-MS/MS: a pilot study
2018; Elsevier BV; Volume: 59; Issue: 5 Linguagem: Inglês
10.1194/jlr.p083576
ISSN1539-7262
AutoresValentin Blanchard, Stéphane Ramin‐Mangata, Stéphanie Billon‐Crossouard, Audrey Aguesse, Manon Durand, Kévin Chemello, Brice Nativel, Laurent Flet, Maud Chétiveaux, David Jacobi, Jean‐Marie Bard, Khadija Ouguerram, Gilles Lambert, Michel Krempf, Mikaël Croyal,
Tópico(s)Diabetes Management and Research
ResumoHuman apoE exhibits three major isoforms (apoE2, apoE3, and apoE4) corresponding to polymorphism in the APOE gene. Total plasma apoE concentrations are closely related to these isoforms, but the underlying mechanisms are unknown. We aimed to describe the kinetics of apoE individual isoforms to explore the mechanisms for variable total apoE plasma concentrations. We used LC-MS/MS to discriminate between isoforms by identifying specific peptide sequences in subjects (three E2/E3, three E3/E3, and three E3/E4 phenotypes) who received a primed constant infusion of 2H3-leucine for 14 h. apoE concentrations and leucine enrichments were measured hourly in plasma. Concentrations of apoE2 were higher than apoE3, and concentrations of apoE4 were lower than apoE3. There was no difference between apoE3 and apoE4 catabolic rates and between apoE2 and apoE3 production rates (PRs), but apoE2 catabolic rates and apoE4 PRs were lower. The mechanisms leading to the difference in total plasma apoE concentrations are therefore related to contrasted kinetics of the isoforms. Production or catabolic rates are differently affected according to the specific isoforms. On these grounds, studies on the regulation of the involved biochemical pathways and the impact of pathological environments are now warranted. Human apoE exhibits three major isoforms (apoE2, apoE3, and apoE4) corresponding to polymorphism in the APOE gene. Total plasma apoE concentrations are closely related to these isoforms, but the underlying mechanisms are unknown. We aimed to describe the kinetics of apoE individual isoforms to explore the mechanisms for variable total apoE plasma concentrations. We used LC-MS/MS to discriminate between isoforms by identifying specific peptide sequences in subjects (three E2/E3, three E3/E3, and three E3/E4 phenotypes) who received a primed constant infusion of 2H3-leucine for 14 h. apoE concentrations and leucine enrichments were measured hourly in plasma. Concentrations of apoE2 were higher than apoE3, and concentrations of apoE4 were lower than apoE3. There was no difference between apoE3 and apoE4 catabolic rates and between apoE2 and apoE3 production rates (PRs), but apoE2 catabolic rates and apoE4 PRs were lower. The mechanisms leading to the difference in total plasma apoE concentrations are therefore related to contrasted kinetics of the isoforms. Production or catabolic rates are differently affected according to the specific isoforms. On these grounds, studies on the regulation of the involved biochemical pathways and the impact of pathological environments are now warranted. apoE plays a key role in lipoprotein metabolism (especially triglyceride-rich lipoproteins) and as a ligand for the LDL receptor (LDLR), the LDLR-related protein (LRP), and heparan sulfate proteoglycans (HSPGs) (1.Mahley R.W. Rall Jr, S.C. Apolipoprotein E: far more than a lipid transport protein.Annu. Rev. Genomics Hum. Genet. 2000; 1: 507-537Crossref PubMed Scopus (1334) Google Scholar, 3.Dominiczak M.H. Caslake M.J. Apolipoproteins: metabolic role and clinical biochemistry applications.Ann. Clin. Biochem. 2011; 48: 498-515Crossref PubMed Scopus (128) Google Scholar). Human apoE is a 299 amino acid protein mostly expressed by the liver and the brain. The APOE gene is localized on chromosome 19 and exhibits three common alleles (ε2, ε3, and ε4) coding for three isoforms (apoE2, apoE3, and apoE4) that differ by single cysteine (C)-arginine (R) substitutions. apoE3 (C112, R158) is the most common isoform in 50–90% of the population. apoE2 (C112, C158) and apoE4 (R112, R158) are less frequent and found in 1–15% and 5–35% of the population, respectively (1.Mahley R.W. Rall Jr, S.C. Apolipoprotein E: far more than a lipid transport protein.Annu. Rev. Genomics Hum. Genet. 2000; 1: 507-537Crossref PubMed Scopus (1334) Google Scholar, 2.Martínez-Morillo E. Nielsen H.M. Batruch I. Drabovich A.P. Begcevic I. Lopez M.F. Minthon L. Bu G. Mattsson N. Portelius E. et al.Assessment of peptide chemical modifications on the development of an accurate and precise multiplex selected reaction monitoring assay for apolipoprotein e isoforms.J. Proteome Res. 2014; 13: 1077-1087Crossref PubMed Scopus (43) Google Scholar). Six phenotypes are found in humans: three homozygotes (E4/E4, E3/E3, and E2/E2) and three heterozygotes (E3/E4, E2/E3, and E2/E4) (2.Martínez-Morillo E. Nielsen H.M. Batruch I. Drabovich A.P. Begcevic I. Lopez M.F. Minthon L. Bu G. Mattsson N. Portelius E. et al.Assessment of peptide chemical modifications on the development of an accurate and precise multiplex selected reaction monitoring assay for apolipoprotein e isoforms.J. Proteome Res. 2014; 13: 1077-1087Crossref PubMed Scopus (43) Google Scholar). The LDLR, LRP, and HSPG binding functions of apoE2 are reduced compared with apoE3. This may lead, in the homozygote E2/E2 state, to type III combined hyperlipoproteinemia and increased CVD risk. In contrast, apoE4 and apoE3 show similar affinities for those receptors. apoE4 has been associated with increased CVD risk and also appears to be a strong genetic determinant for Alzheimer's disease (1.Mahley R.W. Rall Jr, S.C. Apolipoprotein E: far more than a lipid transport protein.Annu. Rev. Genomics Hum. Genet. 2000; 1: 507-537Crossref PubMed Scopus (1334) Google Scholar, 3.Dominiczak M.H. Caslake M.J. Apolipoproteins: metabolic role and clinical biochemistry applications.Ann. Clin. Biochem. 2011; 48: 498-515Crossref PubMed Scopus (128) Google Scholar, 4.Mahley R.W. Huang Y. Rall Jr, S.C. Pathogenesis of type III hyperlipoproteinemia (dysbetalipoproteinemia): questions, quandaries and paradoxes.J. Lipid Res. 1999; 40: 1933-1949Abstract Full Text Full Text PDF PubMed Google Scholar, 5.Mahley R.W. Apolipoprotein E: from cardiovascular disease to neurodegenerative disorders.J. Mol. Med. (Berl.). 2016; 94: 739-746Crossref PubMed Scopus (258) Google Scholar). apoE plasma concentrations are closely related to APOE genotypes. Carriers of at least one ε2 or one ε4 allele respectively present with higher and lower plasma apoE levels than ε3/ε3 homozygotes (6.Moriarty P.M. Varvel S.A. Gordts P.L. McConnell J.P. Tsimikas S. Lipoprotein (a) mass levels increase significantly according to APOE genotype.Arterioscler. Thromb. Vasc. Biol. 2017; 37: 580-588Crossref PubMed Scopus (57) Google Scholar, 8.Rasmussen K.L. Plasma levels of apolipoprotein E, APOE genotype and risk of dementia and ischemic heart disease: a review.Atherosclerosis. 2016; 255: 145-155Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The mechanisms underlying these differences are still unknown. Lipoprotein turnover can be assessed in vivo by measuring the incorporation of an injected tracer, usually 2H3-leucine, in apolipoproteins over time, allowing the determination of lipoprotein kinetic parameters such as their production rates (PRs) and fractional catabolic rates (FCRs) (9.Barrett P.H. Chan D.C. Watts G.F. Design and analysis of lipoprotein tracer kinetics studies in humans.J. Lipid Res. 2006; 47: 1607-1619Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). This approach has been improved by new analytical techniques involving enzymatic proteolysis and LC-MS/MS (10.Croyal M. Fall F. Ferchaud-Roucher V. Chétiveaux M. Zaïr Y. Ouguerram K. Krempf M. Nobécourt E. Multiplexed peptide analysis for kinetic measurements of major human apolipoproteins by LC/MS/MS.J. Lipid Res. 2016; 57: 509-515Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 11.Pan Y. Zhou H. Mahsut A. Rohm R.J. Berejnaia O. Price O. Chen Y. Castro-Perez J. Lassman M.E. McLaren D. et al.Static and turnover kinetic measurement of protein biomarkers involved in triglyceride metabolism including apoB48 and apoA5 by LC/MS/MS.J. Lipid Res. 2014; 55: 1179-1187Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). LC-MS/MS is a powerful tool to simultaneously quantify several plasma proteins even at low concentrations (12.Tavori H. Christian D. Minnier J. Plubell D. Shapiro M.D. Yeang C. Giunzioni I. Croyal M. Duell P.B. Lambert G. et al.PCSK9 association with lipoprotein(a).Circ. Res. 2016; 119: 29-35Crossref PubMed Scopus (82) Google Scholar, 13.Ceglarek U. Dittrich J. Becker S. Baumann F. Kortz L. Thiery J. Quantification of seven apolipoproteins in human plasma by proteotypic peptides using fast LC-MS/MS.Proteomics Clin. Appl. 2013; 7: 794-801Crossref PubMed Scopus (32) Google Scholar). This technique also allows the determination of protein polymorphisms (2.Martínez-Morillo E. Nielsen H.M. Batruch I. Drabovich A.P. Begcevic I. Lopez M.F. Minthon L. Bu G. Mattsson N. Portelius E. et al.Assessment of peptide chemical modifications on the development of an accurate and precise multiplex selected reaction monitoring assay for apolipoprotein e isoforms.J. Proteome Res. 2014; 13: 1077-1087Crossref PubMed Scopus (43) Google Scholar, 14.Croyal M. Ouguerram K. Passard M. Ferchaud-Roucher V. Chétiveaux M. Billon-Crossouard S. de Gouville A.C. Lambert G. Krempf M. Nobécourt E. Effects of extended-release nicotinic acid on apolipoprotein (a) kinetics in hypertriglyceridemic patients.Arterioscler. Thromb. Vasc. Biol. 2015; 35: 2042-2047Crossref PubMed Scopus (40) Google Scholar). An LC-MS/MS method was recently developed to quantify apoE isoforms in both human plasma and cerebrospinal fluid (2.Martínez-Morillo E. Nielsen H.M. Batruch I. Drabovich A.P. Begcevic I. Lopez M.F. Minthon L. Bu G. Mattsson N. Portelius E. et al.Assessment of peptide chemical modifications on the development of an accurate and precise multiplex selected reaction monitoring assay for apolipoprotein e isoforms.J. Proteome Res. 2014; 13: 1077-1087Crossref PubMed Scopus (43) Google Scholar). In this study, plasma apoE2 was more abundant than apoE3, and apoE3 more abundant than apoE4 in patients with ε2/ε3 and ε3/ε4 genotypes, respectively (7.Martínez-Morillo E. Hansson O. Atagi Y. Bu G. Minthon L. Diamandis E.P. Nielsen H.M. Total apolipoprotein E levels and specific isoform composition in cerebrospinal fluid and plasma from Alzheimer's disease patients and controls.Acta Neuropathol. 2014; 127: 633-643Crossref PubMed Scopus (100) Google Scholar). To investigate why apoE isoform concentrations differ in vivo, we measured the isotopic enrichment of apoE isoforms in whole plasma and in the lipoproteins to determine their kinetics in a series of ε2/ε3, ε3/ε3, and ε3/ε4 patients who received a primed constant infusion of 2H3-leucine. UPLC/MS-grade acetonitrile, water, methanol, and 99% formic acid were purchased from Biosolve (Valkenswaard, The Netherlands). The 2H3-leucine was obtained from Cambridge Isotope Laboratories Inc. (Andover, MA). Ammonium bicarbonate was obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France). Synthetic labeled and unlabeled peptides were purchased from Thermo Scientific Biopolymers (Darmstadt, Germany). Stock solutions of synthetic peptides were prepared at 1 mmol/l in 50% acetonitrile containing 0.1% formic acid and stored at −20°C until use. LC-MS/MS analyses were performed on a Xevo® TQD mass spectrometer with an ESI interface and an Acquity H-Class® UPLC™ device (Waters Corporation, Milford, MA). Data acquisition and analyses were performed with MassLynx® and TargetLynx® software, respectively (version 4.1; Waters Corporation). Nine overweight male subjects (three ε2/ε3, three ε3/ε3, and three ε3/ε4; 49 ± 11 years old; BMI of 29 ± 3 kg/m2) with hypertriglyceridemia (plasma triglycerides: 248 ± 70 mg/dl) were included in this study. They did not receive any treatment. After an overnight fast, each subject received an intravenous bolus of 10 μmol/kg 2H3-leucine immediately followed by a constant intravenous infusion at 10 μmol/kg/h for 14 h. Blood samples were collected hourly in EDTA tubes (Venoject, Paris, France), and the plasma was separated by centrifugation at 4°C for 30 min and stored at −80°C until use. The Ethics Committee of Nantes University Hospital approved the clinical protocols, and written informed consent was obtained from each subject (trial numbers: NCT01216956 and V00002CA101). Cholesterol and triglyceride concentrations were measured using enzymatic kits from Boehringer Mannheim GmbH (Mannheim, Germany) according to the supplier's instructions. Proprotein convertase subtilisin kexin type 9 (PCSK9) concentrations were measured in plasma by enzyme-linked immunosorbent assay according to the supplier's instructions (R&D Systems, Lille, France). Plasma lipoprotein fractions, including VLDL, IDL, LDL, and HDL, were separated by fast protein LC (FPLC) or by sequential ultracentrifugation methods (15.Chétiveaux M. Nazih H. Ferchaud-Roucher V. Lambert G. Zaïr Y. Masson M. Ouguerram K. Bouhours D. Krempf M. The differential apoA-I enrichment of prebeta1 and alphaHDL is detectable by gel filtration separation.J. Lipid Res. 2002; 43: 1986-1993Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 16.Ouguerram K. Chetiveaux M. Zair Y. Costet P. Abifadel M. Varret M. Boileau C. Magot T. Krempf M. Apolipoprotein B100 metabolism in autosomal-dominant hypercholesterolemia related to mutations in PCSK9.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1448-1453Crossref PubMed Scopus (164) Google Scholar). Total cholesterol and triglyceride contents were measured in each FPLC fraction. FPLC fractions corresponding to the same lipoprotein class were pooled. Lipoprotein fractions (2 ml for FPLC, 800 μl for ultracentrifugation) were desalted and concentrated with 3 ml of 50 mmol/l ammonium bicarbonate buffer (pH 8) using a 5 kDa molecular mass cut-off filter for apolipoprotein enrichment measurements. Apolipoproteins (apoA-I, apoB100, apoC-II, apoC-III, and apoE) were analyzed in plasma, lipoprotein fractions, and concentrated lipoprotein fractions using a validated multiplexed assay involving trypsin proteolysis and the subsequent analysis of proteotypic peptides by LC-MS/MS (10.Croyal M. Fall F. Ferchaud-Roucher V. Chétiveaux M. Zaïr Y. Ouguerram K. Krempf M. Nobécourt E. Multiplexed peptide analysis for kinetic measurements of major human apolipoproteins by LC/MS/MS.J. Lipid Res. 2016; 57: 509-515Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The method was updated for the quantification of apoE isoforms as described previously (2.Martínez-Morillo E. Nielsen H.M. Batruch I. Drabovich A.P. Begcevic I. Lopez M.F. Minthon L. Bu G. Mattsson N. Portelius E. et al.Assessment of peptide chemical modifications on the development of an accurate and precise multiplex selected reaction monitoring assay for apolipoprotein e isoforms.J. Proteome Res. 2014; 13: 1077-1087Crossref PubMed Scopus (43) Google Scholar). A pool solution of unlabeled synthetic peptides (M0; Table 1) was constituted and serially diluted in water to obtain seven standard solutions ranging from 0.5 to 50 μmol/l (apoA-I), from 0.25 to 25 μmol/l (apoB100, apoC-II, and apoC-III), and from 0.1 to 10 μmol/l (apoE and isoforms). Plasma, lipoprotein, and standard samples (60 μl) were reduced [addition of 120 μl of ammonium bicarbonate (50 mmol/l) containing 7 mg/ml of RapidGest detergent (Waters Corporation) and incubation for 10 min at 80°C; then addition of dithiothreitol (100 mmol/l, 20 μl) and incubation for 20 min at 60°C], alkylated [addition of iodoacetamide (200 mmol/l, 20 μl) and incubation for 20 min at room temperature in the dark], and trypsin digested overnight [5 mg/ml in HCl (1 mmol/l, 30 μl) at 37°C] using the ready-to-use solutions of the ProteinWorks™ eXpress kit (Waters Corporation) according to the manufacturer's instructions. Labeled proteotypic peptides (Table 1) were used as internal standards (ISs) and a mixed solution of standards was added to the digestion buffer to a final concentration of 0.5 μmol/l. After digestion, samples were cleaned using 30 mg Oasis HLB 1 cc cartridges (Waters Corporation). Cartridges were conditioned, equilibrated, loaded, washed, and eluted with methanol (1 ml), water (1 ml), samples (∼250 μl), 5x% methanol (1 ml), and 80% methanol (500 μl), respectively. Eluates were dried under a nitrogen stream, reconstituted with 100 μl of 5% acetonitrile containing 0.1% formic acid, and 10 μl were injected into the LC-MS/MS system.TABLE 1Analytical parameters used for each proteotypic peptideProteinNamePeptideFragmentCone/Collision (V)MRM (m/z)apoA-IM0ATEHLSTLSEKy102+25/15406.2 → 573.2ISATEHLSTLSE-[13C615N4]Ry102+25/15408.9 → 577.2apoB100M0ATGVLYDYVNKy6+34/23622.4 → 915.6M3ATGVLYDYVNKy6+34/23623.9 → 915.6ISATGVLYDYVN-[13C615N2]Ky6+34/23626.4 → 923.6apoC-IIM0TAAQNLYEKy7+35/20519.7 → 865.7ISTAAQNLYE-[13C615N2]Ky7+35/20523.7 → 873.7apoC-IIIM0GWVTDGFSSLKy8+40/35598.2 → 854.1ISGWVTDGFSSL-[13C615N2]Ky8+40/35602.2 → 862.1apoEM0LGPLVEQGRy5+25/30484.8 → 588.3M3LGPLVEQGRy5+25/30486.3 → 588.3ISLGPLVEQG-[13C615N4]Ry5+25/30489.8 → 598.3apoE2M0[C]LAVYQAGARy8+40/22555.2 (527.7) → 835.7M3[C]LAVYQAGARy8+40/22556.7 (529.3) → 835.7IS[C]LAVYQAGA-[13C615N4]Ry8+40/22560.2 → 845.7apoE4M0LGAD[M]EDVRy8+35/20503.6 (511.6) → 892.6 (908.6)M3LGADMEDVRy8+35/20505.1 (513.1) → 892.6 (908.6)ISLGADMEDV-[13C615N4]Ry8+35/20508.6 → 902.6apoE2/E3M0LGADMEDV[C]GRy6+35/20612.0 → 735.6M3LGADMEDV[C]GRy6+35/20613.5 → 735.6ISLGADMEDV[C]G-[13C615N4]Ry6+35/20612.0 → 735.6apoE3/E4M0LAVYQAGARy7+40/22475.0 → 764.7M3LAVYQAGARy7+40/22476.5 → 764.7ISLAVYQAGA-[13C615N4]Ry7+40/22480.0 → 774.7Parentheses indicate secondary transitions that may occur. [C] indicates carbamydomethyl-cysteine (+57) and [M] indicates oxidized methionine. Underlined L indicates the putative incorporation site(s) of 2H3-leucine. M0, unlabeled peptide; M3, 2H3-leucine labeled peptide; MRM, multiple reaction monitoring. Open table in a new tab Parentheses indicate secondary transitions that may occur. [C] indicates carbamydomethyl-cysteine (+57) and [M] indicates oxidized methionine. Underlined L indicates the putative incorporation site(s) of 2H3-leucine. M0, unlabeled peptide; M3, 2H3-leucine labeled peptide; MRM, multiple reaction monitoring. Apolipoprotein analyses were carried out by LC-MS/MS. Proteotypic peptides were separated over 9 min on an Acquity® BEH C18 column (2.1 × 100 mm, 1.7 μm; Waters Corporation) held at 60°C with a linear gradient of mobile phase B (100% acetonitrile) in mobile phase A (5% acetonitrile), each containing 0.1% formic acid, at a flow rate of 600 μl/min. Mobile phase B was linearly increased from 1% to 50% for 5 min, kept constant for 1 min, returned to the initial condition over 1 min, and kept constant for 2 min before the next injection. Proteotypic peptides were then detected by the mass spectrometer with the ESI interface operating in the positive ion mode (capillary voltage, 3 kV; desolvatation gas (N2) flow and temperature, 900 l/h and 400°C; source temperature, 150°C). The multiple reaction monitoring mode was applied for MS/MS detection as detailed in Table 1. apoE genotypes were confirmed by LC-MS/MS in plasma samples according to the presence of different combinations of peptides (2.Martínez-Morillo E. Nielsen H.M. Batruch I. Drabovich A.P. Begcevic I. Lopez M.F. Minthon L. Bu G. Mattsson N. Portelius E. et al.Assessment of peptide chemical modifications on the development of an accurate and precise multiplex selected reaction monitoring assay for apolipoprotein e isoforms.J. Proteome Res. 2014; 13: 1077-1087Crossref PubMed Scopus (43) Google Scholar) and illustrated in Fig. 1: E2/E2 phenotype (LGADMEDVCGR, CLAVYQAGAR), E2/E3 phenotype (LGADMEDVCGR, CLAVYQAGAR, LAVYQAGAR), E2/E4 phenotype (LGADMEDVCGR, CLAVYQAGAR, LGADMEDVR, LAVYQAGAR), E3/E3 phenotype (LGADMEDVCGR, LAVYQAGAR), E3/E4 phenotype (LGADMEDVCGR, LAVYQAGAR, LGADMEDVR), and E4/E4 phenotype (LGADMEDVR, LAVYQAGAR). Chromatographic peak area ratios between unlabeled peptides (M0) and their respective ISs constituted the detector responses. Standard solutions were used to plot calibration curves for peptide quantification. The linearity was expressed by the mean r2, which was greater than 0.985 for all peptides (linear regression, 1/x weighting, origin excluded). Each sample was assayed three times and the coefficients of variation did not exceed 11.3% for all peptides in all samples. Apolipoprotein concentrations were expressed in micromoles per liter, assuming 1 mol of peptide equivalent to 1 mol of protein. Concentrations were then converted to their standard unit (milligrams per deciliter) assuming a molecular mass of 28,079, 512,858, 8,204, 8,765, and 34,237 g/mol for apoA-I, apoB100, apoC-II, apoC-III, and apoE, respectively (www.uniprot.org). For the quantification of apoE isoforms, specific CLAVYQAGAR and LGADMEDVR peptides were used for apoE2 and apoE4, respectively. Unlike apoE2 and apoE4, the apoE3 isoform does not display any specific peptide. apoE3 concentration was therefore calculated by subtracting the concentrations measured for apoE2 (E2/E3 phenotype) or apoE4 (E3/E4 phenotype) from the total apoE (LGPLVEQGR) concentration. The common peptides of apoE2/E3 (LGADMEDVCGR) and apoE3/E4 (LAVYQAGAR) were used to confirm these apoE3 concentrations with acceptance criteria set at a maximum of 10% of variation between both approaches (2.Martínez-Morillo E. Nielsen H.M. Batruch I. Drabovich A.P. Begcevic I. Lopez M.F. Minthon L. Bu G. Mattsson N. Portelius E. et al.Assessment of peptide chemical modifications on the development of an accurate and precise multiplex selected reaction monitoring assay for apolipoprotein e isoforms.J. Proteome Res. 2014; 13: 1077-1087Crossref PubMed Scopus (43) Google Scholar). Chemical modifications that might occur within some peptides were taken into account (secondary MRM transitions shown within parentheses, Table 1) and integrated to determine the exact concentrations of each apoE isoform (2.Martínez-Morillo E. Nielsen H.M. Batruch I. Drabovich A.P. Begcevic I. Lopez M.F. Minthon L. Bu G. Mattsson N. Portelius E. et al.Assessment of peptide chemical modifications on the development of an accurate and precise multiplex selected reaction monitoring assay for apolipoprotein e isoforms.J. Proteome Res. 2014; 13: 1077-1087Crossref PubMed Scopus (43) Google Scholar). 2H3-leucine enrichments were assessed in apoE isoforms in plasma and concentrated lipoprotein fractions, as previously validated (10.Croyal M. Fall F. Ferchaud-Roucher V. Chétiveaux M. Zaïr Y. Ouguerram K. Krempf M. Nobécourt E. Multiplexed peptide analysis for kinetic measurements of major human apolipoproteins by LC/MS/MS.J. Lipid Res. 2016; 57: 509-515Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 14.Croyal M. Ouguerram K. Passard M. Ferchaud-Roucher V. Chétiveaux M. Billon-Crossouard S. de Gouville A.C. Lambert G. Krempf M. Nobécourt E. Effects of extended-release nicotinic acid on apolipoprotein (a) kinetics in hypertriglyceridemic patients.Arterioscler. Thromb. Vasc. Biol. 2015; 35: 2042-2047Crossref PubMed Scopus (40) Google Scholar). Enrichments were calculated as described previously from unlabeled (M0) and 2H3-leucine-labeled (M3) peptides (10.Croyal M. Fall F. Ferchaud-Roucher V. Chétiveaux M. Zaïr Y. Ouguerram K. Krempf M. Nobécourt E. Multiplexed peptide analysis for kinetic measurements of major human apolipoproteins by LC/MS/MS.J. Lipid Res. 2016; 57: 509-515Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 17.Zhou H. Castro-Perez J. Lassman M.E. Thomas T. Li W. McLaughlin T. Dan X. Jumes P. Wagner J.A. Gutstein D.E. et al.Measurement of apo(a) kinetics in human subjects using a microfluidic device with tandem mass spectrometry.Rapid Commuxn. Mass Spectrom. 2013; 27: 1294-1302Crossref PubMed Scopus (28) Google Scholar). Briefly, the isotope ratio (IR), corresponding to the M3/M0 percent ratio (%), was divided by the number of leucine residues in the peptide sequence. After baseline subtraction, the IR was converted to enrichment as follows: enrichment = (IR × 100) ÷ (100 + IR). Both apoE2 and apoE4 kinetics were investigated from their respective signature peptides (CLAVYQAGAR and LGADMEDVR, respectively). To minimize variability, two peptides located in the same areas were used for apoE3 kinetics, as illustrated in Fig. 1 (LAVYQAGAR for E2/E3 phenotype, LGADMEDVCGR for E3/E4 phenotype, and the average of both LAVYQAGAR and LGADMEDVCGR for the E3/E3 phenotype). Apolipoprotein enrichment measurements were performed on three replicates for all kinetic time points: coefficients of variation did not exceed 12.6%. Total apoE kinetics were investigated in plasma and concentrated lipoprotein fractions by the use of the common LGPLVEQGR peptide, as previously described and validated (10.Croyal M. Fall F. Ferchaud-Roucher V. Chétiveaux M. Zaïr Y. Ouguerram K. Krempf M. Nobécourt E. Multiplexed peptide analysis for kinetic measurements of major human apolipoproteins by LC/MS/MS.J. Lipid Res. 2016; 57: 509-515Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Apolipoprotein enrichment measurements were performed on three replicates for all kinetic time points: coefficients of variation did not exceed 7.1%. The 2H3-leucine enrichments were investigated in VLDL apoB100 (10.Croyal M. Fall F. Ferchaud-Roucher V. Chétiveaux M. Zaïr Y. Ouguerram K. Krempf M. Nobécourt E. Multiplexed peptide analysis for kinetic measurements of major human apolipoproteins by LC/MS/MS.J. Lipid Res. 2016; 57: 509-515Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Enrichment measurements were performed on three replicates for all kinetic time points and coefficients of variation did not exceed 5.1%. Kinetic analysis was achieved using the Simulation, Analysis, and Modeling II software (SAAM II; Epsilon Group, Charlottesville, VA). The labeling of apoE nearly reached the asymptotic maximal enrichment (precursor pool), which suggested a relatively rapid turnover over the time course of the study (17.Zhou H. Castro-Perez J. Lassman M.E. Thomas T. Li W. McLaughlin T. Dan X. Jumes P. Wagner J.A. Gutstein D.E. et al.Measurement of apo(a) kinetics in human subjects using a microfluidic device with tandem mass spectrometry.Rapid Commuxn. Mass Spectrom. 2013; 27: 1294-1302Crossref PubMed Scopus (28) Google Scholar). Fractional synthetic rates (FSRs) were estimated using the following mono-exponential equation: protein labelingtime = protein labelingsteady-state × [1 − e−FSR×(time-delay)] (17.Zhou H. Castro-Perez J. Lassman M.E. Thomas T. Li W. McLaughlin T. Dan X. Jumes P. Wagner J.A. Gutstein D.E. et al.Measurement of apo(a) kinetics in human subjects using a microfluidic device with tandem mass spectrometry.Rapid Commuxn. Mass Spectrom. 2013; 27: 1294-1302Crossref PubMed Scopus (28) Google Scholar). The protein labeling at steady state (precursor pool) was assumed to be close to that of a surrogate protein with fast turnover (i.e., VLDL apoB100, supplemental Fig. S1), and the delay parameter was set adjustable (0.01–1.00 h) for calculation. As expected (fasting state), apolipoprotein pool sizes were considered constant, as no significant variation was observed in apoE concentrations during the time course of the kinetic study. PRs were calculated as the product of the FSR and the average apoE concentration by assuming a plasma volume of 4.5% of body weight. At steady-state, the FCR is equal to the FSR. Total apoE FCR was deduced from those of specific isoforms with the following equation: FCRTotal = [(Q1 × k1) + (Q2 × k2)] ÷ (Q1 + Q2). Pool sizes of isoforms 1 and 2 are expressed by Q1 and Q2, respectively, and the FCRs of isoforms 1 and 2 are expressed by k1 and k2, respectively. Kinetic parameters of total apoE were also investigated by the use of the common LGPLVEQGR peptide (10.Croyal M. Fall F. Ferchaud-Roucher V. Chétiveaux M. Zaïr Y. Ouguerram K. Krempf M. Nobécourt E. Multiplexed peptide analysis for kinetic measurements of major human apolipoproteins by LC/MS/MS.J. Lipid Res. 2016; 57: 509-515Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Kinetic parameters obtained from both approaches (i.e., sum of isoforms vs. total apoE) were then compared. Concentrations of apoE isoforms were measured in concentrated samples (60 μl) taking into account the concentration factor. Total apoE, apoA-I (HDL), and apoB100 (VLDL/IDL/LDL) contents were measured simultaneously. Results are expressed as mean ± standard deviation. The nonparametric Spearman correlation test was carried out with GraphPad Prism software (version 6.0; GraphPad Software Inc., La Jolla, CA) and results were considered statistically significant at P < 0.05. apoE genotypes were confirmed by LC-MS/MS in the nine subjects according to the presence or the absence of proteotypic peptides (Fig. 2). Although we used a limited number of patients, precluding adequate statistical analyses, the lipid/lipoprotein/apolipoprotein levels (Table 2) between E2/E3, E3/E3, and E3/E4 groups were similar, including the parameters PCSK9, apoB100, LDL cholesterol, triglyceride, apoC-II, and apoC-III. In contrast, apoE plasma concentrations appeared quite different between groups: 7.2 ± 1.1 mg/dl for E2/E3, 3.8 ± 0.7 mg/dl for E3/E3, and 3.1 ± 0.4 mg/dl for E3/E4. As shown in Fig. 3, E3/E3 patients displayed nearly twice as much apoE3 (3.8 ± 0.7 mg/dl) as E2/E3 (2.1 ± 0.8 mg/dl) and E3/E4 (2.1 ± 0.5 mg/dl) patients. In E2/E3 individuals, the apoE2 plasma concentration was higher than apoE3 (5.1 ± 0.3 mg/dl vs. 2.1 ± 0.8 mg/dl, respectively). In E3/E4 individuals, apoE4 plasma concentrations were lower than apoE3 (1.0 ± 0.1 mg/dl vs. 2.1 ± 0.5 mg/dl, respectively).TABLE 2Clinical and biochemical characteristics of patientsParametersE2/E3E3/E3E3/E4Number333Age (years)51 ± 545 ± 1544 ± 9BMI (kg/m2)31 ± 327 ± 328 ± 4TC (mg/dl)193 ± 38209 ± 28231 ± 9TG (mg/dl)207 ± 43290 ± 80228 ± 47HDL-C (mg/dl)41 ± 541 ± 1240 ± 5LDL-C (mg/dl)114 ± 28116 ± 28150 ± 8apoA-I (mg/dl)128 ± 27139 ± 28138 ± 14apo
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