Metrologically Traceable Quantification of 3 Apolipoprotein E Isoforms in Cerebrospinal Fluid.

BACKGROUND: APOE genotype is associated with Alzheimer disease. Thus, the concentration of apolipoprotein E (apoE) isoforms in cerebrospinal fluid (CSF) could be altered in dementia. However, conflicting results have been obtained in different studies. Carefully validated and standardized assays could improve the interpretation of research findings, allow their replication in other laboratories, and generalize their application. METHODS: To evaluate this hypothesis, we aimed to develop, validate, and standardize a new measurement procedure using LC-MS/MS. Purified recombinant apoE protein standards (E2, E3, E4) were thoroughly characterized and used to assign the concentration of a matrix-matched calibration material that contained each apoE isoform, which ensured the metrological traceability of results. RESULTS: The assay of each isoform in human CSF was precise (≤11%CV) and of moderate throughput (approximately 80 samples per day). It demonstrated good linearity and parallelism for lumbar CSF, ventricular CSF, and bovine CSF. The use of an SI-traceable matrix-matched calibrator enabled precise and accurate measurements. There was no association observed between total apoE concentration and the number of Ɛ4 alleles in a cohort of 322 participants. However, the concentration of each isoform was significantly different in heterozygotes, with E4 > E3 > E2. Isoform concentrations were associated with cognitive and motor symptoms but contributed negligibly to a predictive model of cognitive impairment that included established CSF biomarkers. CONCLUSIONS: Our method simultaneously measures each apoE isoform in human CSF with excellent precision and accuracy. A secondary matrix-matched material has been developed and is available to other laboratories to improve interlaboratory agreement.

Development of an LC-MS/MS Proposed Candidate Reference Method for the Standardization of Analytical Methods to Measure Lipoprotein(a).

BACKGROUND: Use of lipoprotein(a) concentrations for identification of individuals at high risk of cardiovascular diseases is hampered by the size polymorphism of apolipoprotein(a), which strongly impacts immunochemical methods, resulting in discordant values. The availability of a reference method with accurate values expressed in SI units is essential for implementing a strategy for assay standardization. METHOD: A targeted LC-MS/MS method for the quantification of apolipoprotein(a) was developed based on selected proteotypic peptides quantified by isotope dilution. To achieve accurate measurements, a reference material constituted of a human recombinant apolipoprotein(a) was used for calibration. Its concentration was assigned using an amino acid analysis reference method directly traceable to SI units through an unbroken traceability chain. Digestion time-course, repeatability, intermediate precision, parallelism, and comparability to the designated gold standard method for lipoprotein(a) quantification, a monoclonal antibody-based ELISA, were assessed. RESULTS: A digestion protocol providing comparable kinetics of digestion was established, robust quantification peptides were selected, and their stability was ascertained. Method intermediate imprecision was below 10% and linearity was validated in the 20-400 nmol/L range. Parallelism of responses and equivalency between the recombinant and endogenous apo(a) were established. Deming regression analysis comparing the results obtained by the LC-MS/MS method and those obtained by the gold standard ELISA yielded y = 0.98*ELISA +3.18 (n = 64). CONCLUSIONS: Our method for the absolute quantification of lipoprotein(a) in plasma has the required attributes to be proposed as a candidate reference method with the potential to be used for the standardization of lipoprotein(a) assays.