SYNTHESIS AND USE OF NOVEL FLUORESCENT GLYCOSPHINGOLIPIDS FOR ESTIMATING BETA-GLUCOSIDASE ACTIVITY IN-VITRO IN THE ABSENCE OF DETERGENTS AND SUBTYPING GAUCHER DISEASE VARIANTS FOLLOWING ADMINISTRATION INTO INTACT-CELLS

Two novel fluorescent glycolipids, LRO-glucosylceramide (LRO-GC) and L RO-trihexosylceramide (LRO-THC) were synthesized and utilized for esti mating activities of the lysosomal, acid beta-glucosidase in cell extr acts and intact skin fibroblasts, derived from normal individuals and patients with Gaucher disease subtypes. The uniqueness of the glycolip ids is the fact that a fluorescent probe (lissamine rhodamine) is link ed in a sulfonylamide linkage to the sphingosyl residue of the sphingo lipid. Thus, the product of enzymatic hydrolysis, lissamine rhodamine sulfonylamido sphingosine (LRO-ceramide) cannot be further hydrolyzed and remains a metabolic end product. A unique property of LRO-GC as a substrate for the lysosomal, acid beta-glucosidase in vitro was the ob servation that enzymatic hydrolysis occurs in the absence of detergent s and that hydrolytic rates are, in fact, reduced in the presence of T riton X-100 and/or sodium taurocholate. Also, both glycolipids penetra ted the membrane of intact fibroblasts in the absence of serum and wer e hydrolyzed in lysosomes of the intact cells. The rates of intracellu lar hydrolysis decreased with the severity of the Gaucher disease subt ypes. Using LRO-THC as substrate, the intracellular ratio of LRO-ceram ide to LRO-glucosylceramide was an indicator for the specific GD-subty pe.