ACCURATE DIFFERENTIATION OF NEURONOPATHIC AND NONNEURONOPATHIC FORMS OF NIEMANN-PICK DISEASE BY EVALUATION OF THE EFFECTIVE RESIDUAL LYSOSOMAL SPHINGOMYELINASE ACTIVITY IN INTACT-CELLS

Niemann-Pick disease types A and B are two clinical forms of an inheri ted lysosomal storage disorder characterized by accumulation of sphing omyelin due to deficient activity of the lysosomal enzyme, acid sphing omyelinase. Patients with both types have hepatosplenomegaly, but only those with type A have nervous system involvement leading to death in early infancy. The residual activities of lysosomal sphingomyelinase in types A and B have never been well characterized because of limitat ions in both in vitro enzymatic assays and loading tests on intact cel ls. To evaluate the effective level of sphingomyelinase activity, inta ct, living cultured Epstein-Barr virus-transformed lymphoid cells were incubated with a radiolabeled sphingomyelin that was first associated to human low-density lipoproteins. This lipoprotein-associated sphing omyelin was targeted to lysosomes, thereby permitting selective hydrol ysis by the lysosomal sphingomyelinase. Short-term pulse-chase experim ents allowed the determination of the initial rates of degradation; in normal cells, the half-time of sphingomyelin degradation averaged 4.5 h. Whereas cells from the severe neuronopathic type A form of Niemann -Pick disease exhibited about 0.15% residual sphingomyelinase activity , cells from patients with the visceral type B form exhibited about 4% , i.e., 27 times more. Cells from heterozygous Niemann-Pick subjects s howed about 70% residual activity. These results provide the first app roach to measuring the effective activity of a lysosomal enzyme and re present an accurate method for the differential diagnosis of Niemann-P ick disease types A and B. They also support the hypothesis of relatio nships among the effective in situ residual enzyme activity, the amoun t of stored substrate, and the severity of the ensuing lysosomal stora ge disorder.