Pyrenemethyl laurate (PMLes), a fluorogenic substrate for determining in vitro 
lipase activity [Nègre, Salvayre, Dagan and Gatt (1989) Biochim. Biophys. Acta 
1006, 84-88], has been administered to cultured lymphoblastoid cells from normal 
subjects and from a patient affected with Wolman disease, which is characterized 
by a deficiency of lysosomal acid lipase. The intracellular degradation of PMLes 
was dependent on the mode of administration of the substrate into the cells, and 
occurred by two separate pathways involving lysosomal and extra-lysosomal 
hydrolases. PMLes incorporated into LDL was taken up by normal lymphoblastoid 
cells through the apolipoprotein-B/E-receptor-mediated pathway and degraded in 
the lysosomal compartment, as suggested by the degradation block in Wolman 
cells. In contrast, when PMLes dissolved in 2% dimethyl sulphoxide was added 
directly to the culture medium, its hydrolysis was similar in lymphoblastoid 
cells from controls and from patients affected with Wolman disease, neutral 
lipid storage disease or familial hypercholesterolaemia. This suggested that the 
administered PMLes was degraded by a non-lysosomal enzyme which is not deficient 
in Wolman cells. This enzyme also differs from the neutral lipase system which 
is deficient in lymphoblastoid cells from patients with neutral lipid storage 
disease. When pyrenemethanol was administered directly to the cell culture, it 
was only poorly acylated and was rapidly released into the culture medium. These 
results and the fluorescence properties of PMLes ('monomeric' emission in a 
hydrophobic environment and 'excimeric' emission in a hydrophilic environment) 
and pyrenemethanol ('monomeric' emission in a hydrophilic environment) allowed 
us to design a 'direct reading' procedure by monitoring (without any lipid 
extraction) the fluorescence of intact living cells and that of the culture 
medium during pulse-chase experiments. This method allowed the direct evaluation 
of the time course of in situ degradation of PMLes. In pulse-chase experiments 
with LDL-PMLes, the fluorescence of normal cells decreased relatively rapidly 
with time whereas the fluorescence of the culture medium increased 
concomitantly. With Wolman cells, the cellular fluorescence decreased only very 
slightly, whereas that of the culture medium remained at the basal level; this 
demonstrates the catabolic block in intact living cells from patients with 
Wolman disease. In vitro degradation of PMLes indicated the existence of two 
PMLes-degrading enzymes in lymphoblastoid cell homogenates: one is the acid 
lipase which is involved in PMLes degradation in the lysosomal compartment (and 
is deficient in Wolman cells), while the second is a cytoplasmic enzyme (not 
deficient in Wolman cells).