Institute of Clinical Chemistry and Laboratory Medicine, Medical Center of the Johannes Gutenberg University, Mainz, Germany.
Clin Chem Lab Med. 2010 Feb;48(2):189-98. doi: 10.1515/CCLM.2010.048.
Fabry disease is an X-chromosomally inherited lysosomal storage disorder leading to accumulation of glycosphingolipids, mainly globotriaosylceramide (ceramide-trihexoside, Gb3). Concentrations of Gb3 in plasma and urine have been used to diagnose Fabry disease and to monitor enzyme replacement therapy with recombinant alpha-galactosidase.
Gb3 was purified from plasma or urine by combined liquid extraction/protein precipitation and solid-phase extraction, and was detected by flow-injection analysis electrospray mass spectrometry (MS) using multi-reaction-monitoring. Calibration was performed via standard addition using C17-Gb3 as internal standard. The most abundant isoforms were monitored for calculation of total Gb3.
A MS-based assay for quantification of Gb3 in plasma and urine was established and validated. Intra- and inter-assay coefficient of variation (CV) of the method were <or=12%. However, at low concentrations the CV was 16%. The linear range covers roughly two orders of magnitude, down to 0.54 mg/L in plasma and 0.07 mg/L in urine. Careful adjustment of tuning parameters was necessary to obtain identical isoform intensities and quantitative results on different mass spectrometers. Gb3 concentrations in healthy controls were <4 mg/L in EDTA-plasma and <10 microg/mmol creatinine in urine. Significantly increased Gb3 concentrations were found in plasma and urine from male and female patients with Fabry disease.
An improved MS protocol for Gb3 quantification has been developed, validated, and shown to be suitable for diagnosis and monitoring of Fabry patients.
法布里病是一种 X 连锁溶酶体贮积病，导致糖鞘脂的积累，主要是神经酰胺三己糖苷（Gb3）。Gb3 在血浆和尿液中的浓度已被用于诊断法布里病，并监测用重组α-半乳糖苷酶进行的酶替代治疗。
通过组合液提取/蛋白沉淀和固相萃取，从血浆或尿液中纯化 Gb3，并通过使用多反应监测的流动注射分析电喷雾质谱（MS）进行检测。通过使用 C17-Gb3 作为内标进行标准添加进行校准。监测最丰富的异构体以计算总 Gb3。
建立并验证了一种用于定量检测血浆和尿液中 Gb3 的基于 MS 的测定法。该方法的日内和日间变异系数（CV）<或=12%。然而，在低浓度时，CV 为 16%。线性范围大致涵盖两个数量级，在血浆中低至 0.54mg/L，在尿液中低至 0.07mg/L。需要仔细调整调谐参数，才能在不同的质谱仪上获得相同的异构体强度和定量结果。健康对照者的 EDTA 血浆中 Gb3 浓度<4mg/L，尿液中 Gb3 浓度<10μg/mmol 肌酐。在法布里病男性和女性患者的血浆和尿液中发现 Gb3 浓度显著升高。
已开发、验证了一种改进的 Gb3 定量 MS 方案，该方案适用于法布里病的诊断和监测。
