Division of Medical Genetics, Department of Laboratory Medicine, CIUSSS de l'Estrie-CHUS, Sherbrooke, Quebec, Canada.
Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada.
Curr Protoc. 2024 Jun;4(6):e1087. doi: 10.1002/cpz1.1087.
Fabry disease (FD) is a lysosomal storage disorder caused by variants in the GLA gene encoding α-galactosidase A, an enzyme required for catabolism of globotriaosylceramide (Gb). Accumulation of Gb in patients' cells, tissues, and biological fluids causes clinical manifestations including ventricular hypertrophy, renal insufficiency, and strokes. This protocol describes a methodology to analyze urinary Gb and creatinine. Samples are diluted with an internal standard solution containing Gb(C17:0) and creatinine-D, centrifuged, and directly analyzed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) using an 8.7-min method. Eight Gb isoforms [C16:0, C18:0, C20:0, C22:1, C22:0, C24:1, C24:0, and (C24:0)OH] are analyzed and the total is normalized to creatinine. Confirmation ions are monitored to detect potential interferences. The Gb limit of quantification is 0.023 µg/ml. Its interday coefficients of variation (3 concentrations measured) are ≤15.4%. This method minimizes matrix effects (≤6.5%) and prevents adsorption or precipitation of Gb. Urine samples are stable (bias <15%) for 2 days at 21°C, 7 days at 4°C, and 4 freeze/thaw cycles, whereas prepared samples are stable for 5 days at 21°C, and 14 days at 4°C. The Gb/creatinine age-related upper reference limits (mean + 2 standard deviations) are 29 mg/mol creatinine (<7 years) and 14 mg/mol creatinine (≥7 years). This simple, robust protocol has been fully validated (ISO 15189) and provides a valuable tool for diagnosis and monitoring of FD patients. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Analysis of urinary globotriaosylceramide (Gb) and creatinine by UHPLC-MS/MS Support Protocol 1: Preparation of the urinary quality controls Support Protocol 2: Preparation of the urine matrix used for the Gb calibration curve Support Protocol 3: Preparation of the Gb calibrators Support Protocol 4: Preparation of the working solution containing the internal standards Support Protocol 5: Preparation of the creatinine calibrators Support Protocol 6: Preparation of the UHPLC solutions and mobile phases.
法布里病（FD）是一种溶酶体贮积症，由编码α-半乳糖苷酶 A 的 GLA 基因突变引起，该酶是糖鞘脂（Gb）分解代谢所必需的。患者细胞、组织和生物液中 Gb 的积累导致临床症状，包括心室肥厚、肾功能不全和中风。本方案描述了一种分析尿液 Gb 和肌酐的方法。将样品用含有 Gb(C17:0)和肌酐-D 的内标溶液稀释，离心，然后通过超高效液相色谱-串联质谱联用仪（UHPLC-MS/MS）在 8.7 分钟的方法直接分析。分析了 8 种 Gb 异构体[C16:0、C18:0、C20:0、C22:1、C22:0、C24:1、C24:0 和(C24:0)OH]，并将总量归一化为肌酐。监测确认离子以检测潜在干扰。Gb 的定量下限为 0.023μg/ml。其日内变异系数（3 个浓度测量值）≤15.4%。该方法最大限度地减少了基质效应（≤6.5%），并防止了 Gb 的吸附或沉淀。尿液样品在 21°C 下稳定（偏差<15%）2 天，在 4°C 下稳定 7 天，4 次冻融循环；而制备的样品在 21°C 下稳定 5 天，在 4°C 下稳定 14 天。与年龄相关的 Gb/肌酐上参考限（均值+2 个标准差）为 29mg/mol 肌酐（<7 岁）和 14mg/mol 肌酐（≥7 岁）。本简单、稳健的方案已全面验证（ISO 15189），为 FD 患者的诊断和监测提供了有价值的工具。©2024 作者。Wiley Periodicals LLC 出版的《当代协议》。基础方案：通过 UHPLC-MS/MS 分析尿液糖鞘脂（Gb）和肌酐支持方案 1：尿液质控品的制备支持方案 2：用于 Gb 校准曲线的尿液基质的制备支持方案 3：Gb 校准品的制备支持方案 4：含内标工作溶液的制备支持方案 5：肌酐校准品的制备支持方案 6：UHPLC 溶液和流动相的制备
