desmosterol and zymosterol

We developed a simple and robust liquid chromatographic/mass spectrometric method (LC-MS) for the quantitative analysis of 10 sterols from the late part of cholesterol synthesis (zymosterol, dehydrolathosterol, 7-dehydrodesmosterol, desmosterol, zymostenol, lathosterol, FFMAS, TMAS, lanosterol, and dihydrolanosterol) from cultured human hepatocytes in a single chromatographic run using a pentafluorophenyl (PFP) stationary phase. The method also avails on a minimized sample preparation procedure in order to obtain a relatively high sample throughput. The method was validated on 10 sterol standards that were detected in a single chromatographic LC-MS run without derivatization. Our developed method can be used in research or clinical applications for disease-related detection of accumulated cholesterol intermediates. Disorders in the late part of cholesterol synthesis lead to severe malformation in human patients. The developed method enables a simple, sensitive, and fast quantification of sterols, without the need of extended knowledge of the LC-MS technique, and represents a new analytical tool in the rising field of cholesterolomics.

Topics: Cholecalciferol; Cholesterol; Chromatography, Liquid; Desmosterol; Fluorobenzenes; Gene Deletion; Hep G2 Cells; Hepatocytes; Humans; Lanosterol; Mass Spectrometry; Phenols; Reproducibility of Results; Sterols

The disorders in cholesterol biosynthesis pathway and various diseases manifest in the accumulation of cholesterol precursors in the human tissues and cellular membranes. In this paper the effect of desmosterol--one of cholesterol precursors--on model lipid membranes was studied. The investigations were performed for binary SM/desmo and POPC/desmo and ternary SM/POPC/desmo monolayers. Moreover, the experiments based on the gradual substitution of cholesterol by desmosterol in SM/POPC/chol=1:1:1 system were done. The obtained results allowed one to conclude that desmosterol is of lower domains promoting and stabilizing properties and packs less tightly with the lipids in monolayers. Moreover, desmosterol probably could replace cholesterol in model membranes, but only at its low proportion in the system (2%), however, at a higher degree of cholesterol substitution a significant decrease of the monolayer stability and packing and alterations in the film morphology were detected. The results collected in this work together with those from previous experiments allowed one to analyze the effect of a double bond in the sterol side chain as well as its position in the ring system on membrane activity of the molecule and to verify Bloch hypothesis.

Topics: Cholesterol; Desmosterol; Lipid Bilayers; Membranes, Artificial; Molecular Structure; Phosphatidylcholines; Sphingomyelins; Structure-Activity Relationship

A major biologic role of the ubiquitous mitochondrial P450 enzyme CYP27A1 is the generation of ligands such as 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid, which regulate the expression of nuclear receptors that govern many aspects of cholesterol homeostasis. We now report that sterol intermediates in cholesterol synthesis, beginning with the initial post-cyclization sterol, lanosterol, continuing with zymosterol, and ending with desmosterol are also substrates for the enzyme. Using the human enzyme expressed in Escherichia coli, we characterized the retention times and major mass fragments of these novel metabolites. Although sequestration of the enzyme in the inner mitochondrial membrane and normal subcellular organization probably greatly restrict the proportion of these and other intermediates in cholesterol synthesis that undergo side chain oxidation, disruption of compartmentalization can bypass cholesterol as the end product and give rise to potent ligands that further modify gene expression.

Topics: Catalysis; Cholestanetriol 26-Monooxygenase; Cholesterol; Dehydrocholesterols; Desmosterol; Enzyme Activation; Gene Expression Regulation, Enzymologic; Humans; Lanosterol; Recombinant Proteins; Steroid Hydroxylases; Sterols; Substrate Specificity

The biochemical basis for the cholesterol-dependent growth phenotype of the NS-1 myeloma cell line has been investigated. In one series of experiments, the growth response of NS-1 cells to several of the intermediates of cholesterol biosynthesis was studied in serum-free medium. The cholesterol precursors, squalene and lanosterol, were totally ineffective in promoting NS-1 cell growth. In contrast, cholesterol precursors downstream from lanosterol, i.e., desmosterol and 7-dehydrocholesterol, completely replaced cholesterol in supporting NS-1 cell growth. In a second series of experiments, NS-1 cells and NS-1-503 cells (a cholesterol growth-independent variant of NS-1 cells) were labelled with [2-14C]acetate and the distributions of radioactivity between cholesterol and its precursors were determined by thin-layer chromatography using two different solvent systems. The major labelled sterol product (greater than 80%) in NS-1 cells after a 24-h exposure to [2-14C]acetate was lanosterol. In contrast, the major labelled sterol product (greater than 95%) in NS-1-503 cells after a 24-h exposure to [2-14C]acetate was cholesterol. Taken together, these results indicate that NS-1 cells are defective in cholesterol biosynthesis and identify the site of lesion as the demethylation of lanosterol to C-29 sterol intermediates.

Topics: Acetates; Animals; Cell Division; Cell Line; Cholesterol; Culture Media; Dehydrocholesterols; Desmosterol; Lanosterol; Mice; Multiple Myeloma; Phenotype; Squalene; Sterols

Several effects of hydroxylated sterols on cell cultures are known. Most of these can be explained by an inhibition of the cholesterol synthesis at the level of the 3-hydroxy-3-methylglutaryl CoA reductase. When studying cholesterol metabolism in rat adrenal cells, an inhibitory action of some sterols on the ACTH-stimulated corticosterone production was observed. The effects of one sterol, 22S-OH-cholesterol, were investigated further. The sterol had no effect on the ACTH-stimulated cyclic AMP production, suggesting an intact receptor-adenylate cyclase complex and cellular membrane. In the presence of ACTH and 22-OH-cholesterol particularly the free cholesterol concentration was elevated; 22S-OH-cholesterol therefore probably exerts its inhibitory effect at a step located after hydrolysis of the cholesterol esters. 22S-OH-cholesterol had no effect on the conversion of exogenous pregnenolone into corticosterone. These results make it probable, that the inhibitory effect of 22S-OH-cholesterol on the ACTH-stimulated corticosterone production is situated at the level of the cholesterol side-chain cleavage.

Topics: Adrenal Glands; Adrenocorticotropic Hormone; Animals; Cholestadienols; Cholesterol; Corticosterone; Cyclic AMP; Desmosterol; Hydroxycholesterols; Ketocholesterols; Male; Pregnenolone; Rats; Sterols