Current dyslipidemia management in patients with atherosclerotic cardiovascular disease (ASCVD) is based on traditional serum lipids. Yet, there is some indication from basic research that serum apolipoproteins A-I, (a), B, C-I, C-II, C-III, and E may give better pathophysiological insight into the root causes of dyslipidemia. To facilitate the future adoption of clinical serum apolipoprotein (apo) profiling for precision medicine, strategies for accurate testing should be developed in advance. Recent discoveries in basic science and translational medicine set the stage for the IFCC Working Group on Apolipoproteins by Mass Spectrometry. Main drivers were the convergence of unmet clinical needs in cardiovascular disease (CVD) patients with enabling technology and metrology. First, the residual cardiovascular risk after accounting for established risk factors demonstrates that the current lipid panel is too limited to capture the full complexity of lipid metabolism in patients. Second, there is a need for accurate test results in highly polymorphic and atherogenic apolipoproteins such as apo(a). Third, sufficient robustness of mass spectrometry technology allows reproducible protein quantification at the molecular level. Fourth, several calibration hierarchies in the revised ISO 17511:2020 guideline facilitate metrological traceability of test results, the highest achievable standard being traceability to SI. This article outlines the conceptual approach aimed at achieving a novel, multiplexed Reference Measurement System (RMS) for seven apolipoproteins based on isotope dilution mass spectrometry and peptide-based calibration. This RMS should enable standardization of existing and emerging apolipoprotein assays to SI, within allowable limits of measurement uncertainty, through a sustainable network of Reference Laboratories.

Background Medical results generated by European CE Marking for In Vitro Diagnostic or in-house tests should be traceable to higher order reference measurement systems (RMS), such as International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)-endorsed reference measurement procedures (RMPs) and reference materials. Currently, serum apolipoprotein (a) [apo(a)] is recognized as a novel risk factor for cardiovascular risk assessment and patient management. The former RMS for serum apo(a) is no longer available; consequently, an International System of Units (SI)-traceable, ideally multiplexed, and sustainable RMS for apo(a) is needed. Methods A mass spectrometry (MS)-based candidate RMP (cRMP) for apo(a) was developed using quantitative bottom-up proteomics targeting 3 proteotypic peptides. The method was provisionally validated according to ISO 15193 using a single human serum based calibrator traceable to the former WHO-IFCC RMS. Results The quantitation of serum apo(a) was by design independent of its size polymorphism, was linear from 3.8 to 456 nmol/L, and had a lower limit of quantitation for apo(a) of 3.8 nmol/L using peptide LFLEPTQADIALLK. Interpeptide agreement showed Pearson Rs of 0.987 and 0.984 for peptides GISSTVTGR and TPENYPNAGLTR, and method comparison indicated good correspondence (slopes 0.977, 1.033, and 1.085 for LFLEPTQADIALLK, GISSTVTGR, and TPENYPNAGLTR). Average within-laboratory imprecision of the cRMP was 8.9%, 11.9%, and 12.8% for the 3 peptides. Conclusions A robust, antibody-independent, MS-based cRMP was developed as higher order RMP and an essential part of the apo(a) traceability chain and future RMS. The cRMP fulfils predefined analytical performance specifications, making it a promising RMP candidate in an SI-traceable MS-based RMS for apo(a).

Apolipoprotein E (apoE) occurs on the majority of plasma lipoproteins and plays a major role in the lipid metabolism in the periphery and in the central nervous system. ApoE is a polymorphic protein with three common isoforms, apoE2, apoE3 and apoE4, derived from respective alleles ε2, ε3 and ε4. The aim of this study was to develop a sample pretreatment protocol combined with rapid mass spectrometry (MS)-based assay for simultaneous apolipoprotein profiling and apoE phenotype identification. This assay was validated in 481 samples from patients with stable atherosclerotic cardiovascular disease (ASCVD) and applied to study association with mild cognitive impairment (MCI) in the LIFE Adult study, including overall 690 study subjects. Simultaneous quantification of 8–12 major apolipoproteins including apoA-I, apoB-100 and apoE could be performed within 6.5 min. Phenotyping determined with the developed MS assay had good agreement with the genotyping by real-time fluorescence PCR (97.5%). ApoE2 isoform was associated with the highest total apoE concentration compared to apoE3 and apoE4 (p < 0.001). In the subgroup of diabetic atherosclerotic cardiovascular disease (ASCVD) patients, apoE2 isoform was related to higher apoC-I levels (apoE2 vs. apoE3, p < 0.05), while in the subgroup of ASCVD patients under statin therapy apoE2 was related to lower apoB-100 levels (apoE2 vs. apoE3/apoE4, p < 0.05). A significant difference in apoE concentration observed between mild cognitive impairment (MCI) subjects and controls was confirmed for each apoE phenotype. In conclusion, this study provides evidence for the successful implementation of an MS-based apoE phenotyping assay, which can be used to assess phenotype effects on plasma lipid and apolipoprotein levels.

Plant sterols (PSs) cannot be synthesized in mammals and are exclusively diet-derived. PSs cross the blood-brain barrier and may have anti-neuroinflammatory effects. Obesity is linked to lower intestinal uptake and blood levels of PSs, but its effects in terms of neuroinflammation—if any—remain unknown. We investigated the effect of high-fat diet-induced obesity on PSs in the brain and the effects of the PSs campesterol and β-sitosterol on in vitro microglia activation. Sterols (cholesterol, precursors, PSs) and polyunsaturated fatty acid-derived lipid mediators were measured in the food, blood, liver and brain of C57BL/6J mice. Under a PSs-poor high-fat diet, PSs levels decreased in the blood, liver and brain (>50%). This effect was reversible after 2 weeks upon changing back to a chow diet. Inflammatory thromboxane B2 and prostaglandin D2 were inversely correlated to campesterol and β-sitosterol levels in all brain regions. PSs content was determined post mortem in human cortex samples as well. In vitro, PSs accumulate in lipid rafts isolated from SIM-A9 microglia cell membranes. In summary, PSs levels in the blood, liver and brain were associated directly with PSs food content and inversely with BMI. PSs dampen pro-inflammatory lipid mediators in the brain. The identification of PSs in the human cortex in comparable concentration ranges implies the relevance of our findings for humans.