Sphingomyelin Storage in Lymphoid Cell Lines from Patients with Niemann-Pick Disease Types A, B and C: Influence of Culture Conditions

Abstract

Epstein-Barr-virus-transformed lymphoid cell lines from normal individuals and from patients with Niemann-Pick disease types A, B or C were subjected to various culture conditions in order to study the source of the characteristic lysosomal storage of sphingomyelin (SPM) observed in the tissues of Niemann-Pick patients. RPMI 1640 containing L-glutamine and antibiotics was used as culture medium and was supplemented with 2% Ultroser HY (a serum substitute devoid of lipoproteins and lipids) or with one of the following lipid sources: fetal calf serum (10%), human LDL (100 to 400 µg protein/ml) or human HDL (400 µg protein/ml). By determining the phosphorus content in the SPM fraction of cell lipid extracts, storage of SPM was demonstrated under all tested culture conditions in cells deficient in acid sphingomyelinase, i.e., lymphoid cell lines from Niemann-Pick disease types A and B. In contrast, the lymphoid cell line from a Niemann-Pick type C patient (not deficient in sphingomyelinase), like normal cells, exhibited no storage of SPM. Even after growing the cells for more than 30 days in a medium devoid of SPM, the Niemann-Pick types A and B lymphoid cell lines showed considerable accumulation of SPM (about 2.0–2.3 times more SPM than in the control lymphoid cell lines). SPM concentration was higher when cells were grown in a medium supplemented with lipids, particularly human LDL or HDL. These results are consistent with the hypothesis that there are both exogenous and endogenous factors in the lysosomal storage of SPM observed in lymphoid cell lines from patients with Niemann-Pick disease types A and B.