Kinetics of plasma apolipoprotein E isoforms by LC-MS/MS: a pilot study

Abstract

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.

Trial registration: ClinicalTrials.gov NCT01216956.

Keywords: lipoprotein/kinetics; lipoprotein/metabolism; liquid chromatography; peptide; stable isotope tracers; tandem mass spectrometry.

Conflict of interest statement

The authors declare no competing financial interest.

Copyright © 2018 Blanchard et al.

Figures

Fig. 1.

Selection of signature peptides from apoE sequences for isoform analysis. Phenotype identification and apoE isoform concentrations were assessed using different combinations of peptides. Both apoE2 and apoE4 carry a single specific peptide (CLAVYQAGAR and LGADMEDVR, respectively) unlike apoE3. For enrichment measurements in heterozygous patients, LAVYQAGAR and LGADMEDVCGR were used for apoE3 kinetics in E2/E3 and E3/E4 phenotypes, respectively. In homozygous E3/E3 patients, enrichments of LAVYQAGAR and LGADMEDVCGR were averaged. Blue indicates cysteine-arginine interchanges between isoforms.

Fig. 2.

Identification of apoE phenotypes by selective combination of proteotypic peptides. LC-MS/MS chromatograms obtained in plasma from representative subjects.

Fig. 3.

Kinetics of plasma apoE isoforms. Mean plasma concentrations and mean changes in 2H3-leucine incorporation over the course of the tracer infusion in subjects with E2/E3 phenotype (A), E3/E3 phenotype (B), and E3/E4 phenotype (C). Values are presented as mean ± standard deviation (n = 3). Total apoE concentrations are shown as indicative in heterozygote patients.

Fig. 4.

Kinetic parameters of plasma apoE isoforms. FCRs (A) and PRs (B) estimated with a mono-exponential equation in subjects with E2/E3 phenotype, E3/E3 phenotype, and E3/E4 phenotype.

Fig. 5.

Validation of plasma apoE kinetic parameters. Total FCRs and PRs were calculated for each subject from kinetic data of apoE isoforms, and then compared (Spearman correlation test) with those obtained directly from the LGPLVEQGR peptide used for total apoE detection. Gray, black, and white circles indicate E2/E3, E3/E3, and E3/E4 phenotypes, respectively.

Fig. 6.

Distribution of apoE isoforms within lipoproteins in hypertriglyceridemic patients. ApoE isoforms were assayed in apoB100-containing lipoproteins (i.e., VLDL + IDL + LDL) (A) and in apoA-I-containing lipoproteins (i.e., HDL) (B) obtained by ultracentrifugation. C: Summary of apoE isoform distribution within lipoproteins. Concentrations were normalized to the total content of apoE recovered in each lipoprotein subclass. Values are expressed as mean ± standard deviation.