Cholesteryl Ester Storage Disease
Diagnosis of LAL deficiency is possible using peripheral lymphocytes or cultured fibroblast cells, where activity of the enzyme is significantly decreased. There is no role for assay of serum enzymes (ie, serum acid phosphatase). Activity can been measured by measuring the lipase activity with C-triolein, C-cholesteryl oleate or 4-methyl-umbelliferyl-oleate. By studying the catabolic turnover of radiolabelled cholesteryl oleate in intact cells, lower in situ residual activity has been shown in cells with a C→T substitution at position 233, which induces a premature stop codon, than in cells having the His274 → Tyr substitution.C-cholesteryl oleate and 4-methy-umbelliferyl-oleate have been used for prenatal diagnosis in cultured chorionic villus cells.
Alternatively, again using live cells a fluorimetric technique where pyrinemethyl laurate (PMLes) is administered to cultured lymphoblastoid cells has been developed. PMLes hydrolysis by acid lipase can be followed directly in a spectrofluorometer 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 an alternative procedure, the enzymatic reaction can 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 (ie, pyrene-methanol) is read in the lower aqueous-ethanolic phase, at 378 nm. PMLes is hydrolysed 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 is practically absent in lymphoblasts and fibroblasts from WD or CESD. The two separate pathways of intracellular degradation of PMLes are mediated by lysosomal and extra-lysosomal hydrolases. PMLes incorporated into LDL is taken up by normal lymphoblastoid cells through the apolipoprotein-B/E-receptor-mediated pathway and degraded in the lysosomal compartment, resulting in a degradation block in Wolman cells. If PMLes dissolved in 2% dimethyl sulfoxide is added directly to the culture medium, its hydrolysis in lymphoblastoid cells from controls and from patients affected with WD, neutral lipid storage disease or familial hypercholesterolaemia is similar, indicating a pathway dependent on non-lysosomal enzyme, which is not deficient in Wolman cells.
A recent advance in the biochemical assessment of LAL has been the development of a dried blood spot assay: Total acid lipase activity is measured using a 4-methyl-umbelliferyl palmitate substrate. Activity is then also measured with Lalistat 2 (a selective inhibitor of LAL) and the LAL activity is the difference between the two results.
Biochemical Testing and Diagnosis
Diagnosis of LAL deficiency is possible using peripheral lymphocytes or cultured fibroblast cells, where activity of the enzyme is significantly decreased. There is no role for assay of serum enzymes (ie, serum acid phosphatase). Activity can been measured by measuring the lipase activity with C-triolein, C-cholesteryl oleate or 4-methyl-umbelliferyl-oleate. By studying the catabolic turnover of radiolabelled cholesteryl oleate in intact cells, lower in situ residual activity has been shown in cells with a C→T substitution at position 233, which induces a premature stop codon, than in cells having the His274 → Tyr substitution.C-cholesteryl oleate and 4-methy-umbelliferyl-oleate have been used for prenatal diagnosis in cultured chorionic villus cells.
Alternatively, again using live cells a fluorimetric technique where pyrinemethyl laurate (PMLes) is administered to cultured lymphoblastoid cells has been developed. PMLes hydrolysis by acid lipase can be followed directly in a spectrofluorometer 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 an alternative procedure, the enzymatic reaction can 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 (ie, pyrene-methanol) is read in the lower aqueous-ethanolic phase, at 378 nm. PMLes is hydrolysed 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 is practically absent in lymphoblasts and fibroblasts from WD or CESD. The two separate pathways of intracellular degradation of PMLes are mediated by lysosomal and extra-lysosomal hydrolases. PMLes incorporated into LDL is taken up by normal lymphoblastoid cells through the apolipoprotein-B/E-receptor-mediated pathway and degraded in the lysosomal compartment, resulting in a degradation block in Wolman cells. If PMLes dissolved in 2% dimethyl sulfoxide is added directly to the culture medium, its hydrolysis in lymphoblastoid cells from controls and from patients affected with WD, neutral lipid storage disease or familial hypercholesterolaemia is similar, indicating a pathway dependent on non-lysosomal enzyme, which is not deficient in Wolman cells.
A recent advance in the biochemical assessment of LAL has been the development of a dried blood spot assay: Total acid lipase activity is measured using a 4-methyl-umbelliferyl palmitate substrate. Activity is then also measured with Lalistat 2 (a selective inhibitor of LAL) and the LAL activity is the difference between the two results.