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.

A Targeted Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Quantification of Peptides from the Carboxyl-terminal Region of Type III Procollagen, Biomarkers of Collagen Turnover.

BACKGROUND: The development of analytical approaches to help reduce the risk of growth hormone (GH) doping is important to fair competition and the health of athletes. However, the reliable detection of GH use remains challenging. The identification of novel biomarkers of GH administration could lead to a better understanding of the physiological response to GH, more sensitive detection of the illicit use of GH in sport, and better management of patients treated for GH disorders. METHODS: We developed a targeted liquid chromatography-tandem mass spectrometry method to simultaneously quantify the carboxyl-terminal propeptide of type III procollagen (P-III-CP) and type III collagen degradation products in human serum. Following proteolysis, we instituted a simple acid precipitation step to reduce digested sample complexity before peptide immunoenrichment, which improved the recovery of one target peptide from serum. We evaluated the concentration of each biomarker at different age ranges and after GH administration in healthy participants. RESULTS: The assay was linear over an estimated concentration range of 0.3 to1.0 nM and 0.1 to 0.4 nM for each surrogate peptide of P-III-CP and collagen fragments, respectively. Intra-day and inter-day coefficients of variation were ≤15%. Biomarker concentrations appeared to vary with age and to reflect age-specific collagen turnover. Moreover, their concentrations changed after GH administration. CONCLUSIONS: Our method quantifies the proteins belonging to the family of P-III-CP and type III collagen degradation products in human serum, which could be used to detect GH administration in athletes and better understand diseases involving GH therapy or altered type III collagen turnover.