Mass Spectrometric Quantification of Plasma Glycosphingolipids in Human GM3 Ganglioside Deficiency

Background: Among Amish communities of North America, biallelic mutations of (c.694C > T) eliminate synthesis of GM3 and its derivative downstream a- and b-series gangliosides. Systemic ganglioside deficiency is associated with infantile onset psychomotor retardation, slow brain growth, intractable epilepsy, deafness, and cortical visual impairment. We developed a robust quantitative assay to simultaneously characterize glycan and ceramide moieties of plasma glycosphingolipids (GSLs) among c.694C > T homozygotes (n = 8), their heterozygous siblings (n = 24), and wild type control (n = 19) individuals.

Methods: Following extraction and saponification of total plasma lipids, GSLs were purified on a tC18 cartridge column, permethylated, and subjected to nanospray ionization mass spectrometry utilizing neutral loss scanning and data-dependent acquisition. Plasma GSLs were quantified against appropriate synthetic standards.

Results: Our method demonstrated linearity from 5 to 250 μl of plasma. Recovery of synthetic GSLs spiked into plasma was 99-104% with no matrix interference. Quantitative plasma GSL profiles discriminated among genotypes: GM3 and GD3 were undetectable in c.694C > T homozygotes, who had markedly elevated lactosylceramide (19.17 ± 4.20 nmol/ml) relative to heterozygous siblings (9.62 ± 2.46 nmol/ml) and wild type controls (6.55 ± 2.16 nmol/ml). Children with systemic ganglioside deficiency had a distinctive shift in ceramide composition toward higher mass species.

Conclusions: Our quantitative glycolipidomics method discriminates among c.694C > T genotypes, can reveal subtle structural heterogeneity, and represents a useful new strategy to diagnose and monitor GSL disorders in humans.