Assays / Genetic Enzymes / Cholesterol Esterification

Principle Of Method:
Cultured cells are grown in medium containing lipoprotein deficient serum to deplete their cholesterol stores. They are then incubated in medium containing low density lipoprotein (LDL), usually supplied as fasting human serum, and [³H]-oleic acid in order to quantify their ability to esterify exogenously derived cholesterol. The cholesterol esters formed are quantitated by thin-layer chromatography and scintillation counting after harvesting and extraction of the cells.

Uses And Limitations Of The Method:
The method is used to diagnose Niemann-Pick disease type C (NPC) using cultured fibroblasts. It is applied in conjunction with the filipin staining of stored cholesterol in these cells. In NPC esterification of exogenous cholesterol is usually markedly decreased, although in some patients, described as having a variant biochemical phenotype, the deficiency is only partial. Cells from other patients in which there is a block somewhere in the LDL pathway also have reduced rates of cholesterol ester synthesis. These include cases of hypercholesterolaemia, cholesterol ester storage disease, Wolman disease and I-cell disease.

Prenatal diagnosis of NPC is possible in families where the biochemical phenotype is of the classic type but is not reliable in variant families. The SAS laboratory does not offer prenatal diagnosis routinely but will offer it for known classical families that have been thoroughly studied. Filipin staining for cholesterol storage is used in conjunction with the assay. Cultured chorionic villi are used for prenatal diagnosis; amniotic cells are not considered reliable. Heterozygote detection is reported to be possible for about 50% of carriers but is not offered for random screening. Some NPC patients have a partial deficiency of sphingomyelinase in their cultured fibroblasts, but not white cells, and this assay is always routinely carried out in suspected cases.

Specimen Requirements:
Fibroblasts cultured from a skin biopsy are needed. Biopsy material should be collected aseptically into a sterile bottle containing tissue culture medium (available from the laboratory), and sent at room temperature to arrive at the laboratory within 24 hours. Biopsies for tissue culture should not be frozen. Fibroblast cultures established in other laboratories should be sent in plastic 25 cm² flasks filled with medium.

THE LABORATORY RECOMMENDS USE OF A COURIER SERVICE OR ROYAL MAIL SPECIAL DELIVERY FOR SENDING ALL SPECIMENS TO THE LABORATORY.

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