目的:评估Wilson病很复杂,既没有铜蓝蛋白,血清或尿铜,是可靠的。两个新指数,准确的非铜蓝蛋白铜(ANCC)和相对ANCC被开发并应用于71名患者的队列,作为威尔逊病注册研究的一部分。
方法:使用强阴离子交换色谱与三重四极杆电感耦合等离子体质谱法,开发了元素铜蛋白形态用于全铜蓝蛋白定量。使用梯度洗脱分离血清蛋白,并使用氧反应模式和Cu-EDTA作为校准标准在m/z63(63Cu+)和48(32S16O+)下测量。通过从总血清铜减去铜蓝蛋白结合铜来计算ANCC,RelANCC是作为ANCC存在的总铜的百分比。
结果:使用两种认证的参考材料建立了全铜蓝蛋白测量的准确性,平均回收率为94.2%。患者样品中含铜物质的总和与总铜浓度之间的回归分析是可接受的(斜率=0.964,截距=0,r=0.987)和差异图,铜的平均差为0.38μmol/L。血清铜浓度为0.48和3.20μmol/L时,全铜蓝蛋白的日内精密度分别为5.2和5.6%CV,浓度为0.80和5.99μmol/L时的中间精密度分别为6.4和6.4%CV。分别。全铜蓝蛋白的检测限(LOD)和定量下限(LLOQ)分别为0.08和0.27μmol/L,分别。
结论:ANCC和相对ANCC是威尔逊病(WD)重要的新的诊断和监测生物标志物指标。
OBJECTIVE: Assessment of Wilson disease is complicated, with neither
ceruloplasmin, nor serum or urine copper, being reliable. Two new indices, accurate non-
ceruloplasmin copper (ANCC) and relative ANCC were developed and applied to a cohort of 71 patients, as part of a Wilson Disease Registry Study.
METHODS: Elemental copper-protein speciation was developed for holo-
ceruloplasmin quantitation using strong anion exchange chromatography coupled to triple quadrupole inductively coupled plasma mass spectrometry. The serum proteins were separated using gradient elution and measured at m/z 63 (63Cu+) and 48 (32S16O+) using oxygen reaction mode and Cu-EDTA as calibration standard. The ANCC was calculated by subtraction of the
ceruloplasmin bound copper from the total serum copper and the RelANCC was the percentage of total copper present as the ANCC.
RESULTS: The accuracy of the holo-ceruloplasmin measurement was established using two certified reference materials, giving a mean recovery of 94.2 %. Regression analysis between the sum of the copper containing species and total copper concentration in the patient samples was acceptable (slope=0.964, intercept=0, r=0.987) and a difference plot, gave a mean difference for copper of 0.38 μmol/L. Intra-day precision for holo-
ceruloplasmin at serum copper concentrations of 0.48 and 3.20 μmol/L were 5.2 and 5.6 % CV and the intermediate precision at concentrations of 0.80 and 5.99 μmol/L were 6.4 and 6.4 % CV, respectively. The limit of detection (LOD) and lower limit of quantification (LLOQ) for holo-ceruloplasmin were 0.08 and 0.27 μmol/L as copper, respectively.
CONCLUSIONS: ANCC and Relative ANCC are important new diagnostic and monitoring biomarker indices for Wilson disease (WD).