Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, United States.
Department of Epidemiology and Population Health, Stanford University, Stanford, CA, United States.
Clin Chem. 2023 Jul 5;69(7):734-745. doi: 10.1093/clinchem/hvad056.
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.
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.
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.
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.
载脂蛋白 E（APOE）基因型与阿尔茨海默病有关。因此，脑脊液（CSF）中载脂蛋白 E （apoE）同工型的浓度可能在痴呆症中发生改变。然而，不同的研究得出了相互矛盾的结果。经过仔细验证和标准化的检测方法可以改善研究结果的解释，允许在其他实验室中进行复制，并推广其应用。
为了验证这一假设，我们旨在开发、验证和标准化一种使用 LC-MS/MS 的新测量程序。经过彻底表征的纯化重组 apoE 蛋白标准品（E2、E3、E4）被用于分配含有每种 apoE 同工型的基质匹配校准材料的浓度，这确保了结果的计量溯源性。
该方法对人 CSF 中每种同工型的检测具有良好的精密度（≤11%CV）和中等的通量（每天约 80 个样本）。它在腰椎 CSF、脑室 CSF 和牛 CSF 中表现出良好的线性和平行性。使用 SI 可溯源的基质匹配校准品可实现精确和准确的测量。在 322 名参与者的队列中，总 apoE 浓度与 Ɛ4 等位基因的数量之间没有关联。然而，在杂合子中，每种同工型的浓度存在显著差异，E4>E3>E2。同工型浓度与认知和运动症状相关，但对包括已建立的 CSF 生物标志物在内的认知障碍预测模型的贡献可以忽略不计。
我们的方法可以同时以优异的精密度和准确性测量人 CSF 中的每种 apoE 同工型。已经开发了一种辅助基质匹配材料，并可供其他实验室使用，以提高实验室间的一致性。
