Abnormal neutral lipase activity in Acid-Lipase-Deficient cultured human fibroblasts

Acid and neutral lipase activities of homogenized or sonicated cultured fibroblasts were examined using [2-³H]glycerol triolein, glycerol tri[1-¹⁴Coleate or cholesterol [1-¹⁴C)oleate as substrates. In normal fibroblasts, optimal conditions for acid lipase activity were pH 4.5-5.0, 0.15-0.2 mM triacylglycerol, and 0.25% Triton X-100. Fatty acid release was linear to 2 h and between 0.2-2.0 mg fibroblast protein/ml. For the neutral lipase, activity was optimal at pH 6.0-7.0, >1.5 mM triacylglycerol or cholesterol oleate (suspended in 8 mg albumin/ml), and 160 pg phosphaddyl- serine/ml. The reaction was linear to 60-120 min, and up to 1.0 mg protein/ml. In contrast to the situation at neutral pH, very little [³H]glycerol was released under acid conditions, suggesting little monoglyceride lipase activity at acid pH. Acid lipase activity in fibroblasts from Wolman’s disease (WD) or cholesterol ester storage disease (CESD) patients was <1% of activity in normal fibroblasts. Neutral lipase activity (as % of control values) was <15%, <15%, and undetectable when measured as fatty acid release or diglyceride or monoglyceride appearance, respectively, and was less than 25% of controls when cholesterol oleate was the substrate. Neutral lipase activity in mixtures of control and WD fibroblast homogenates was similar to that predicted from individual activities. The triacylglycerol content of the control and mutant cells was within normal limits, and cholesterol content was only slightly elevated, indicating that endogenous dilution of substrate was not the reason for the low activity in mutant cells. Hydrolysis of labeled endogenous triacylglycerol was reduced by >75-80% in the WD cells, indicating that low levels of activity measured with the exogenous substrate were not due solely to lack of accessibility to the neutral lipase. These results suggest a close relationship between the acid and neutral activities of the normal cell in that mutations which affect one apparently affect the activity of the other.