Pulsed amperometric detection of carbohydrates in lysosomal storage disease fibroblasts: a new screening technique for carbohydrate storage diseases.

A first step in determining the metabolic defect in patients with an unknown storage disease is to identify the stored material. In the case of fibroblasts storing carbohydrates, this can be accomplished by trifluoroacetic acid (TFA) hydrolysis producing monosaccharides which are separated by anion-exchange chromatography and quantitated by pulsed amperometric detection. This technique separates neutral, amino, and acidic monosaccharides in a single run with a detection limit of 50 pmol. The method, applied to hydrolyzed 100,000 g supernatants of ten normal fibroblast sonicates, revealed a mean +/- S.D. content of the following monosaccharides (in nmol/mg of protein): fucose, 7 +/- 3; galactosamine, 4 +/- 2; glucosamine, 20 +/- 3; galactose, 11 +/- 3; mannose, 27 +/- 6; glucuronic acid, 56 +/- 28; iduronic acid, 17 +/- 11. Six mucopolysaccharidosis fibroblast strains (types I, II, IIIB, IVA, VI and VII) contained 2 to 8 times the normal glucuronic acid levels, and types I and II exhibited 10- to 30-fold normal levels of iduronic acid and 40-fold increases in galactosamine. All the mucopolysaccharidoses could be distinguished from normal based upon an increased concentration of some monosaccharide. Fibroblasts from patients with mannosidosis and fucosidosis contained 7-fold normal amounts of mannose and 11-fold normal amounts of fucose, respectively. The quantitation of monosaccharides in fibroblasts after TFA hydrolysis can identify cells that store excess amounts of a glycosaminoglycan, glycoprotein, oligosaccharide or, presumably, a glycolipid. This may comprise the first step toward identifying novel lysosomal storage disorders and point the way toward new glycoconjugate degradative pathways.