Fluorescent
pyrene-methyl lauryl ester (
PMLes) was synthesized and used for the determination of cellular
lipase activities in lymphoblasts and fibroblasts from normal subjects and from patients affected with Wolman's or
cholesteryl ester storage diseases (both exhibiting a deficiency of the
lysosomal acid lipase). The hydrolysis of
PMLes by
acid lipase could be followed directly in a spectrofluorometer; this was possible because of the very high fluorescence emission of
pyrene-
methanol at 378 nm (monomeric form) in aqueous medium, whereas the substrate has practically no monomeric emission at 378 nm but emits only at 475 nm (excimeric form) in the experimental conditions used: this property permitted us to use
PMLes as a
fluorogenic substrate. In an alternative procedure, the enzymatic reaction could be determined after partition of the reaction mixture in a biphasic system of
heptane and aqueous
ethanol; the residual undegraded substrate partitioned into the upper
heptane phase and the fluorescence of the product (i.e.
pyrene-
methanol) was read in the lower aqueous-ethanolic phase, at 378 nm.
PMLes was hydrolyzed in extracts of normal lymphoblasts and fibroblasts by at least two lipases, one acidic
lipase (pH 4.0) and a second more neutral
enzyme (pH 6.5). The acidic
lipase activity was practically absent in lymphoblasts and fibroblasts from Wolman's or
cholesteryl ester storage diseases. This demonstrates that the fluorescent
PMLes is hydrolyzed by the
lysosomal acid lipase and can be used as a very sensitive
fluorogenic substrate which permits direct recording of product formation and is suitable for the enzymatic diagnosis of either of these diseases.