Beschreibung:
Abstract Background Gangliosides are a family of complex lipids that are molecules composed of glycosphingolipid with one or more sialic acid linked on the sugar moieties. Gangliosides are recognized to play an important role in many biological processes and the alterations in ganglioside profiles have been observed in some neurodegenerative diseases, neuropathies and lysosomal disorders. Gangliosidoses are a group of inherited lysosomal enzyme deficiencies characterized by an accumulation of incompletely degraded gangliosides in the lysosome. While the diagnosis of gangliosidoses can be accomplished by measurement of enzymatic activities and/or molecular genetic testing, biomarker analysis could facilitate monitoring affected patients, including those enrolled in clinical trials of new treatment approaches (ie., substrate reduction or gene therapies). We developed a rapid and sensitive LC-MS/MS assay that allows simultaneous quantification of multiple gangliosides and other glycosphingolipids and tested it in serum and plasma of patients with a diagnosis of GM2-gangliosidosis. Methods Gangliosides and other glycosphingolipids were extracted from 20 µL serum or plasma using chloroform:methanol (1:2, v/v). A multiplexed LC-MS/MS assay was developed that targeted several ganglioside species (GM3, GM2, GM1, GD3, GD2, GD1, GT3, GT2, GT1 and GQ1) along with several glycosphingolipid species (GlcCer, LacCer, Gb3, Gb4 and GA2). This assay was performed on a triple quadrupole mass spectrometer with scheduled MRM in both negative and positive ion modes after internal standards (GM3 18:0-d5, GM1 17:0, GA2 17:0 and Gb3 18:0-d3) were spiked into clinical samples. Results The multiplexed targeted assay applied to serum and plasma from controls (47 and 49 samples, respectively) and three GM2-gangliosidosis patients for identification and quantification of the targeted gangliosides and other glycosphingolipids. The abundance of GM2 and GM1 gangliosides inserum and plasma of patients with GM2-gangliosidosis was significantly higher than in the control group (P < 0.0001) whereas GM3 levels were significantly lower (P < 0.005). Other gangliosides including GD3, GD2, GD1 and GT1 and glycosphingolipids such as GlcCer, LacCer and Gb3 were not significantly different between these patients and controls. Conclusion A multiplexed targeted assay for glycosphingolipids including gangliosides was developed. This assay will be further validated for potential use in the diagnosis and monitoring of patients with additional gangliosidoses.