Niemann-Pick disease type C.

Niemann-Pick type C (NPC) is an autosomal recessive lysosomal storage disease. Fibroblasts from individuals with Niemann-Pick type C exhibit defective intracellular cholesterol transport. Linkage analysis has led to the recent cloning of the NPC1 gene on human chromosome 18, which is the major disease locus. Analysis of NPC1 reveals homologies with key regulators of cholesterol homeostasis and a Drosophila morphogen receptor.

Compartmental isolation of cholesterol participating in the cytoplasmic cholesteryl ester cycle in Chinese hamster ovary 25-RA cells.

Using the Chinese hamster ovary cell line, 25-RA, we have demonstrated that lipoprotein-derived cholesterol and endogenously synthesized cholesterol are selectively differentiated with respect to their cellular locations. These cells lack sterol-mediated regulation, spontaneously storing large amounts of esterified cholesterol, which turns over with a half-time of 7.5 h. When [3H]cholesterol was provided to the cells in serum to trace cellular cholesterol, the specific activities of cellular free and esterified cholesterol (6238 +/- 273 and 5128 +/- 277 cpm/ microg, respectively) failed to equilibrate, indicating that bulk cellular free cholesterol is isolated from that participating in the cholesteryl ester cycle. Using [3H]acetate to trace the fate of endogenously synthesized cholesterol, a failure of equilibration was also observed (specific activities of free and esterified cholesterol = 280 +/- 37 and 458 +/- 8 cpm/ microg, respectively). The lower specific activity of the precursor indicates that endogenously synthesized cholesterol is preferentially esterified. When cells radiolabeled with [3H]acetate were post-incubated in the absence of radiolabel, the specific activity of the esterified cholesterol pool remained significantly higher than that of the free cholesterol, suggesting that cholesterol derived from hydrolysis of esterified cholesterol is preferentially re-esterified.

Cholesterol quantitation by GLC: artifactual formation of short-chain steryl esters.

A simple and rapid method for the quantitation of total cholesterol in lipid extracts using gas-liquid chromatography is presented here as a modification of an earlier saponification procedure (Ishikawa, T. T., J. MacGee, J. A. Morrison, and C. J. Glueck. 1974. Quantitative analysis of cholesterol in 5 to 20 microliters of plasma. J. Lipid Res. 15: 286-291). Using the original method, as well as a slightly modified version, we found a systematic loss of cholesterol measured as total cholesterol that was attributable to the formation of a byproduct during the procedure. Depending on the nature of the solvent mixture used for extraction after saponification, different byproducts were produced that had longer retention times than cholesterol. The byproducts were identified as cholesteryl butyrate (produced when methyl butyrate was included in the solvent mix) and cholesteryl propionate (with ethyl propionate in the solvent mix) by comparison to authentic standards using gas chromatography-mass spectroscopy. Using mixtures of cholesterol standards, we compared several solvents in lieu of the solvent mixture used in the original extraction procedure to identify those that eliminate the formation of the byproducts. Our optimized microsaponification procedure uses a single solvent, tetrachloroethylene, to extract lipids after the saponification reaction, and improves the accuracy of the cholesterol determination.