Urinary neutral glycosphingolipid analysis of patients with Fabry's disease; rapid isocratic elution from high-performance liquid chromatography as per-o-benzoyl derivatives.

Neutral glycosphingolipids from urinary sediments of six patients with Fabry's disease and 11 members of the family of one propositus were analyzed using high-performance liquid chromatography. Per-o-benzoyl derivatives of GlcCer, LacCer, GbOse3Cer and GbOse4Cer were clearly resolved by a solvent mixture of hexane/dioxane/isopropanol (75:25:1, v/v) on a normal-phase silica column. Using our isocratic solvent system, the analysis was completed within 15 min. The smallest amount of glycolipid that could be detected by HPLC was 50 pmol and a linear response was shown at 230 nm up to 400 pmol. The calculated peak area of GbOse4Cer was higher than those of GlcCer, LacCer and GbOse3Cer. The molar ratios of GbOse3Cer to monohexosyl ceramide (CMH) in the urinary sediments were: Fabry hemizygotes, 36.33 +/- 25.54 (n = 6); heterozygotes, 0.94 +/- 0.50 (n = 4); and controls, 0.11 +/- 0.06 (n = 5). The molar ratio CDH/CMH was also higher in patients (8.42 +/- 6.23) than in controls (0.77 +/- 0.23). The female H was a rare example of a carrier with typical clinical manifestations. From the urinary sediment analysis, females E, G and J were suspected to be Fabry heterozygotes, although no clinical signs were observed at the time of examination.