Division of Medical Genetics, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke , 3001 12th Avenue North, Sherbrooke, Quebec, Canada J1H 5N4.
Anal Chem. 2017 Dec 19;89(24):13382-13390. doi: 10.1021/acs.analchem.7b03609. Epub 2017 Nov 27.
Fabry disease is an X-linked lysosomal storage disorder caused by α-galactosidase A (α-GAL A) deficiency. This enzyme contributes to the cellular recycling of glycosphingolipids such as galabiosylceramide (Ga), globotriaosylceramide (Gb), and globotriaosylsphingosine (lyso-Gb) by hydrolyzing the terminal α-galactosyl moiety. Urine and plasma α-GAL A substrates are currently analyzed as biomarkers for the detection, monitoring, and follow-up of Fabry disease patients. The sensitivity of the analysis of Ga is decreased by the co-analysis of its structural isomer, lactosylceramide (LacCer), which is not an α-GAL A substrate. A normal-phase ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) methodology, allowing the baseline separation of 12 Ga isoforms/analogues from their lactosylceramide counterparts, was developed and validated in urine. The method was multiplexed with the analysis of 12 Gb isoforms/analogues having the same fatty acid moieties as those of Ga for comparison, and with creatinine for sample normalization. Urine samples were studied from 34 untreated and 33 Fabry males treated by enzyme replacement therapy (ERT) and 54 untreated and 19 ERT-treated Fabry females, along with 34 male and 25 female healthy controls. The chromatographic separation of Ga from LacCer increased the sensitivity of analysis, especially in women. One untreated Fabry female and two treated Fabry females presented abnormal levels of Ga but normal levels of Gb, supporting the importance of analyzing Ga in addition to Gb. Our results show that urine LacCer levels from females were significantly higher than those from males. Moreover, LacCer levels were not affected by Fabry disease for both males and females.
法布里病是一种 X 连锁溶酶体贮积症，由α-半乳糖苷酶 A（α-GAL A）缺乏引起。这种酶通过水解末端α-半乳糖基部分，有助于糖鞘脂如半乳糖基神经酰胺（Ga）、Globotriaosylceramide（Gb）和Globotriaosylsphingosine（lyso-Gb）的细胞再循环。尿液和血浆α-GAL A 底物目前被分析为法布里病患者的检测、监测和随访的生物标志物。Ga 的分析灵敏度因与其结构异构体乳糖基神经酰胺（LacCer）的共同分析而降低，LacCer 不是α-GAL A 的底物。开发并验证了一种正相超高效液相色谱-串联质谱（UPLC-MS/MS）方法，该方法允许基线分离 12 种 Ga 异构体/类似物与其乳糖基神经酰胺对应物，该方法在尿液中进行了验证。该方法与 12 种 Gb 异构体/类似物的分析进行了复用，这些类似物具有与 Ga 相同的脂肪酸部分，用于比较，并与肌酐一起用于样品归一化。研究了 34 名未经治疗和 33 名接受酶替代治疗（ERT）的法布里男性以及 54 名未经治疗和 19 名接受 ERT 治疗的法布里女性以及 34 名男性和 25 名女性健康对照者的尿液样本。Ga 与 LacCer 的色谱分离提高了分析的灵敏度，尤其是在女性中。一名未经治疗的法布里女性和两名接受治疗的法布里女性 Ga 水平异常，但 Gb 水平正常，这支持了除 Gb 之外还分析 Ga 的重要性。我们的结果表明，女性尿液 LacCer 水平明显高于男性。此外，法布里病对男女两性的 LacCer 水平均无影响。
