Division of Metabolism, Endocrinology, and Nutrition, Northwest Lipid Metabolism and Diabetes Research Laboratories, University of Washington, Seattle, WA, USA.
Division of Metabolism, Endocrinology, and Nutrition, University of Washington, Seattle, WA, USA.
Clin Chem. 2021 Mar 1;67(3):490-499. doi: 10.1093/clinchem/hvaa324.
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.
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.
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).
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.
载脂蛋白（a）浓度大小的多态性严重影响免疫化学方法，导致结果不一致，从而阻碍了脂蛋白（a）用于识别心血管疾病高危个体。因此，需要有一种具有准确 SI 单位值的参考方法，以实施分析标准化策略。
基于通过同位素稀释定量的特征肽，建立了用于定量载脂蛋白（a）的靶向 LC-MS/MS 方法。为了实现准确测量，使用由人重组载脂蛋白（a）组成的参考物质进行校准。其浓度使用通过不间断的可追溯链直接可溯源到 SI 单位的氨基酸分析参考方法来赋值。评估了参考材料的消化时程、重复性、中间精密度、平行性以及与脂蛋白（a）定量的指定金标准方法（基于单克隆抗体的 ELISA）的可比性。
建立了提供可比消化动力学的消化方案，选择了稳健的定量肽，并确定了其稳定性。方法中间精密度低于 10%，并且验证了线性范围在 20-400nmol/L 之间。建立了响应的平行性和内源性 apo(a)之间的等效性。通过 LC-MS/MS 方法和金标准 ELISA 获得的结果之间的 Deming 回归分析得到 y=0.98*ELISA+3.18（n=64）。
我们的血浆载脂蛋白（a）绝对定量方法具有被提议作为候选参考方法的所需属性，有可能用于载脂蛋白（a）分析的标准化。
