Clinical Biochemistry Department, Vall d'Hebron University Hospital, Barcelona 08035, Spain.
Clinical Biochemistry, Drug Delivery & Therapy (CB-DDT) Research Group, Vall d'Hebron Research Institute (VHIR), Vall d'Hebron Barcelona Hospital Campus, Barcelona 08035, Spain.
Anal Chem. 2024 Aug 6;96(31):12875-12882. doi: 10.1021/acs.analchem.4c02654. Epub 2024 Jul 24.
Mutations in GBA1, encoding glucocerebrosidase beta 1 (GCase), are the most common genetic risk factor for Parkinson's disease (PD). GCase dysfunction leads to an accumulation of glucosylceramide (GluCer) substrates in different organs and fluids. Despite the challenges in quantifying GluCer isoforms in biological samples, their potential clinical interest as PD biomarkers justifies the development of robust assays. An extensively evaluated high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for quantifying 14 GluCer and galactosylceramide (GalCer) isoforms in human cerebrospinal fluid (CSF) samples is presented. Sample pretreatment, HPLC, and MS/MS parameters were optimized. Evaluation was performed according to the recommendations of the Clinical and Laboratory Standards Institute and European Medicines Agency guidelines. Four 7-point calibration curves were generated, with a linearity interval from 2.5 to 200 nM ( ≥ 0.995). The limit of quantification was set at 5 nM. Between-run precision and accuracy were up to 12.5 and 9%, respectively. After method validation, we measured the levels of GluCer and GalCer isoforms in CSF human samples, including 6 healthy controls (HC), 22 idiopathic GBA1 wild-type PD (iPD) patients, and 5 GBA1-associated PD (PD-GBA) patients. GluCer/GalCer median ratios were found to be higher in the CSF of PD-GBA patients, particularly in severe GBA1 mutations, than those in iPD and HC. The observed trends in GluCer/GalCer ratios among groups provide novel information for the comprehensive analysis of sphingolipids as potential biomarkers of PD.
GBA1 基因突变，编码β-葡萄糖脑苷脂酶 1（GCase），是帕金森病（PD）最常见的遗传风险因素。GCase 功能障碍导致不同器官和液体中葡萄糖脑苷脂（GluCer）底物的积累。尽管在生物样本中定量 GluCer 异构体存在挑战，但它们作为 PD 生物标志物的潜在临床意义证明了开发稳健测定法的合理性。本文介绍了一种经过广泛评估的高效液相色谱-串联质谱（HPLC-MS/MS）方法，用于定量人脑脊液（CSF）样本中的 14 种 GluCer 和半乳糖脑苷脂（GalCer）异构体。优化了样品预处理、HPLC 和 MS/MS 参数。根据临床和实验室标准协会和欧洲药品管理局指南的建议进行了评估。生成了 4 个 7 点校准曲线，线性区间为 2.5 至 200 nM（≥0.995）。定量限设定为 5 nM。批间精密度和准确度分别高达 12.5%和 9%。方法验证后，我们测量了 CSF 人类样本中 GluCer 和 GalCer 异构体的水平，包括 6 名健康对照（HC）、22 名特发性 GBA1 野生型 PD（iPD）患者和 5 名 GBA1 相关 PD（PD-GBA）患者。与 iPD 和 HC 相比，PD-GBA 患者的 CSF 中 GluCer/GalCer 中位数比值更高，尤其是在严重的 GBA1 突变中。各组之间 GluCer/GalCer 比值的观察趋势为全面分析鞘脂作为 PD 的潜在生物标志物提供了新信息。
