Fabry disease is an X-linked lysosomal storage disorder with marked variability in the phenotype and genotype. Glycosphingolipids such as globotriaosylceramide (Gb3) isoforms/analogs, globotriaosylsphingosine (lyso-Gb3) and analogs, and galabiosylceramide (Ga2) isoforms/analogs may accumulate in biological fluids and different organs. The aims of this study were to: 1) develop/validate a novel UHPLC-MS/MS method for relative quantitation of Gb3 in leukocytes (unfractionated white blood cells), B lymphocytes and monocytes; 2) evaluate these biomarkers in a cohort of Fabry patients and healthy controls; and 3) assess correlations between these biomarkers, treatment and genotype. Whole blood, plasma and urine samples from 21 Fabry patients and 20 healthy controls were analyzed. Samples were purified by liquid-liquid extraction and analyzed by UHPLC-MS/MS in positive electrospray ionization. Methylated Gb3 isoforms were detected, showing that a methylation process occurs at the cellular level. Our results show that there were no significant differences in the distribution of the different Gb3 isoforms/analogs in blood cells between Fabry patients and healthy controls. In leukocyte, Gb3[(d18:1)(C14:0)], Gb3[(d18:1)(C16:0)], Gb3 [(d18:1)(C16:0)]Me, Gb3 [(d18:1)(C16:1)], Gb3 [(d18:1)(C18:0)], Gb3 [(d18:1)(C18:1)], Gb3 [(d18:1)(C20:1)], Gb3 [(d18:1)(C24:2)], Gb3 [(d18:1)(C26:1)] and total Gb3 allowed good discrimination between male Fabry patients and male controls, patients having higher biomarker levels than controls. Regarding B lymphocytes and monocytes, the same tendency was observed without reaching statistical significance. A positive concordance between mutation types and biomarker levels in white blood cells was established. Our results might provide a deeper mechanistic comprehension of the underlying biochemical processes of Gb3 biomarkers in white blood cells of Fabry patients.

中文翻译：

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使用超高效液相色谱/串联质谱法分析法布里患者未分级白细胞、B 淋巴细胞和单核细胞中的球三糖基神经酰胺 (Gb 3 ) 同种型/类似物

法布里病是一种 X 连锁溶酶体贮积症，表型和基因型存在显着变异。鞘糖脂，例如球三糖神经酰胺 (Gb3) 异构体/类似物、球三糖神经鞘氨醇 (lyso-Gb3) 和类似物，以及半乳糖苷神经酰胺 (Ga2) 异构体/类似物可能会在生物体液和不同器官中积聚。本研究的目的是： 1) 开发/验证一种新的 UHPLC-MS/MS 方法，用于对白细胞（未分离的白细胞）、B 淋巴细胞和单核细胞中的 Gb3 进行相对定量；2) 在一组 Fabry 患者和健康对照中评估这些生物标志物；3) 评估这些生物标志物、治疗和基因型之间的相关性。分析了 21 名法布里患者和 20 名健康对照的全血、血浆和尿液样本。样品通过液-液萃取纯化，并通过 UHPLC-MS/MS 在正电喷雾电离中进行分析。检测到甲基化的 Gb3 异构体，表明甲基化过程发生在细胞水平。我们的结果表明，法布里患者和健康对照之间血细胞中不同 Gb3 同种型/类似物的分布没有显着差异。在白细胞中，Gb3[(d18:1)(C14:0)]、Gb3[(d18:1)(C16:0)]、Gb3[(d18:1)(C16:0)]Me、Gb3[(d18 :1)(C16:1)], Gb3 [(d18:1)(C18:0)], Gb3 [(d18:1)(C18:1)], Gb3 [(d18:1)(C20:1) ]、Gb3 [(d18:1)(C24:2)]、Gb3 [(d18:1)(C26:1)] 和总 Gb3 可以很好地区分男性法布里患者和男性对照，患者的生物标志物水平高于对照. 关于 B 淋巴细胞和单核细胞，观察到相同的趋势，但没有达到统计显着性。建立了白细胞中突变类型和生物标志物水平之间的正一致性。我们的结果可能会提供对 Fabry 患者白细胞中 Gb3 生物标志物的潜在生化过程的更深层次的机械理解。