Chromatographic behavior of oxygenated derivatives of cholesterol.

Oxygenated derivatives of cholesterol have important functions in many biochemical processes. These oxysterols are difficult to study because of their low physiological concentrations, the facile formation of cholesterol autoxidation artifacts, and lack of information on their chromatographic behavior. Focusing on metabolites and autoxidation products of cholesterol, we have documented the chromatographic mobilities of 35 oxysterols under a variety of conditions: eight solvent systems for thin-layer chromatography on silica gel, several mobile phases for reversed-phase high-performance liquid chromatography (HPLC), and two types of stationary phase for capillary gas chromatography (GC) using trimethylsilyl derivatives.

Alternative pathways of sterol synthesis in yeast. Use of C(27) sterol tracers to study aberrant double-bond migrations and evaluate their relative importance.

Yeast produce traces of aberrant sterols by minor alternative pathways, which can become significant when normal metabolism is blocked by inhibitors or mutations. We studied sterols generated in the absence of the delta(8)-delta(7) isomerase (Erg2p) or delta(5) desaturase (Erg3p) by incubating three mutant strains of Saccharomyces cerevisiae with 5 alpha-cholest-8-en-3beta-ol, 8-dehydrocholesterol (delta(5,8) sterol), or isodehydrocholesterol (delta(6,8) sterol), together with the corresponding 3 alpha-3H isotopomer. Nine different incubations gave altogether 16 sterol metabolites, including seven delta(22E) sterols formed by action of the yeast C-22 desaturase (Erg5p).

A colorimetric assay for 7-dehydrocholesterol with potential application to screening for Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS; MIM 270400) is a genetic disorder characterized by hypocholesterolemia and elevated 7-dehydrocholesterol (7DHC) levels resulting from mutations affecting 7-dehydrocholesterol reductase. We describe a colorimetric assay for 7DHC with potential application to large-scale screening for SLOS. Reaction of 7DHC and its esters with the Liebermann-Burchard reagent resulted in a brief initial absorbance at 510 nm (pink color) followed by an absorbance at 620 nm (blue color) after 2 min, while cholesterol samples were essentially colorless.

Sterols in blood of normal and Smith-Lemli-Opitz subjects.

Smith-Lemli-Opitz syndrome (SLOS) is a hereditary disorder in which a defective gene encoding 7-dehydrocholesterol reductase causes the accumulation of noncholesterol sterols, such as 7- and 8-dehydrocholesterol. Using rigorous analytical methods in conjunction with a large collection of authentic standards, we unequivocally identified numerous noncholesterol sterols in 6 normal and 17 SLOS blood samples. Plasma or erythrocytes were saponified under oxygen-free conditions, followed by multiple chromatographic separations.

Progestins block cholesterol synthesis to produce meiosis-activating sterols.

The resumption of meiosis is regulated by meiosis-preventing and meiosis-activating substances in testes and ovaries. Certain C29 precursors of cholesterol are present at elevated levels in gonadal tissue, but the mechanism by which these meiosis-activating sterols (MAS) accumulate has remained an unresolved question. Here we report that progestins alter cholesterol synthesis in HepG2 cells and rat testes to increase levels of major MAS (FF-MAS and T-MAS).

Meiosis-activating sterol and the maturation of isolated mouse oocytes.

This study was carried out to examine the effects of the meiosis-activating C(29) sterol, 4,4-dimethyl-5 alpha-cholesta-8,14, 24-trien-3 beta-ol (FF-MAS), on mouse oocyte maturation in vitro. Cumulus cell-enclosed oocytes (CEO) and denuded oocytes (DO) from hormonally primed, immature mice were cultured 17-18 h in minimum essential medium (MEM) containing 4 mM hypoxanthine plus increasing concentrations of FF-MAS. The sterol induced maturation in DO with an optimal concentration of 3 microg/ml but was without effect in CEO, even at concentrations as high as 10 microg/ml.

Aberrant pathways in the late stages of cholesterol biosynthesis in the rat. Origin and metabolic fate of unsaturated sterols relevant to the Smith-Lemli-Opitz syndrome.

Minor aberrant pathways of cholesterol biosynthesis normally produce only trace levels of abnormal sterol metabolites but may assume major importance when an essential biosynthetic step is blocked. Cholesta-5,8-dien-3beta-ol, its Delta(5,7) isomer, and other noncholesterol sterols accumulate in subjects with the Smith-Lemli-Opitz syndrome (SLOS), a severe developmental disorder caused by a defective Delta(7) sterol reductase gene. We have explored the formation and metabolism of unsaturated sterols relevant to SLOS by incubating tritium-labeled Delta(5,8), Delta(6, 8), Delta(6,8(14)), Delta(5,8(14)), and Delta(8) sterols with rat liver preparations.

Kinetics and plasma concentrations of 26-hydroxycholesterol in baboons.

26-Hydroxycholesterol (26OHC), a major oxysterol in human blood, is believed to play an important role in reverse cholesterol transport, bile acid formation, and regulation of various cellular processes. Using isotope dilution mass spectrometry, we measured plasma 26OHC concentrations in baboons fed either a high cholesterol/saturated fat (HC-SF) or normal chow diet. Plasma 26OHC levels in baboons were comparable to those reported for humans and were positively correlated with plasma cholesterol concentrations.

Inhibition of Ca2+ release channel (ryanodine receptor) activity by sphingolipid bases: mechanism of action.

Sphingosine inhibits the activity of the skeletal muscle Ca2+ release channel (ryanodine receptor) and is a noncompetitive inhibitor of [3H]ryanodine binding (Needleman et al., Am. J.

Synthesis of [3alpha-3H]cholesta-5,8-dien-3beta-ol and tritium-labeled forms of other sterols of potential importance in the Smith-Lemli-Optiz syndrome.

Five unsaturated sterols relevant to the Smith-Lemli-Opitz syndrome have been prepared in high radiochemical purity with a tritium label at the 3alpha position. Swern oxidation of cholesta-5,8-dien-3beta-ol and other unlabeled C27 sterols afforded the corresponding 3-ketosteroids, and reduction with tritiated NaBH4 gave the desired 3alpha-3H sterols, with double bonds at the delta(5,8), delta(5,8(14)), delta(6,8), delta(6,8(14)), and delta8 positions. High radiochemical purity of the tritiated sterols was demonstrated by normal phase, reversed phase, and silver-ion (Ag+) high-performance liquid chromatography (HPLC).

Oxysterols: modulators of cholesterol metabolism and other processes.

Oxygenated derivatives of cholesterol (oxysterols) present a remarkably diverse profile of biological activities, including effects on sphingolipid metabolism, platelet aggregation, apoptosis, and protein prenylation. The most notable oxysterol activities center around the regulation of cholesterol homeostasis, which appears to be controlled in part by a complex series of interactions of oxysterol ligands with various receptors, such as the oxysterol binding protein, the cellular nucleic acid binding protein, the sterol regulatory element binding protein, the LXR nuclear orphan receptors, and the low-density lipoprotein receptor. Identification of the endogenous oxysterol ligands and elucidation of their enzymatic origins are topics of active investigation.

An improved synthesis of (20R,22R)-cholest-5-ene-3beta,20,22-triol, an intermediate in steroid hormone formation and an activator of nuclear orphan receptor LXR alpha.

Asymmetric dihydroxylation of (20(22)E)-cholesta-5,20(22)-dien-3beta-ol acetate (2a), prepared from pregnenolone, gave a 1:1 mixture (67% yield) of (20R,22R)-cholest-5-ene-3beta,20,22-triol 3-acetate (3a) and its 20S,22S isomer 3b. Highly purified 3a and 3b were obtained by semipreparative silver ion high performance liquid chromatography. Saponification of 3a and 3b gave (20R,22R)-cholest-5-ene-3beta,20,22-triol (4a) and its 20S,22S isomer 4b.

Sterol synthesis. Preparation and characterization of fluorinated and deuterated analogs of oxygenated derivatives of cholesterol.

Oxygenated sterols, including both autoxidation products and sterol metabolites, have many important biological activities. Identification and quantitation of oxysterols by chromatographic and spectroscopic methods is greatly facilitated by the availability of authentic standards, and deuterated and fluorinated analogs are valuable as internal standards for quantitation. We describe the preparation, purification and characterization of 43 oxygenated sterols, including the 4 beta-hydroxy, 7 alpha-hydroxy, 7 beta-hydroxy, 7-keto, and 19-hydroxy derivatives of cholesterol and their analogs with 25,26,26,26,27,27,27-heptafluoro (F7) and 26,26,26,27,27,27-hexadeuterio (d6) substitution.

New methods for determining the enantiomeric purity of erythro -sphingosine.

The enantiomeric purity of erythro -sphingosine samples can be determined simply, reliably, and accurately from 1H or 19F nuclear magnetic resonance spectra of the alpha-methoxy-alpha-(trifluoromethyl)phenylacetate (MTPA) derivative. As little as 0.1% of the minor enantiomer could be observed in a 1-mg sample, and detection limits of 1% and 5% were estimated for samples of 100 microg and 10 microg.

An alternative synthesis of 4,4-dimethyl-5 alpha-cholesta-8,14,24-trien-3 beta-ol, an intermediate in sterol biosynthesis and a reported activator of meiosis and of nuclear orphan receptor LXR alpha.

4,4-dimethyl-5 alpha-cholesta-8,14,24-trien-3 beta-ol, a sterol of current biological interest, has been synthesized in six steps from 3 beta-acetoxy-4,4-dimethyl-5 alpha-cholest-8(14)-en-15-one.

Chemical synthesis of D-ribo-phytosphingosine-1-phosphate, a potential modulator of cellular processes.

d-erythro -Sphingosine-1-phosphate (2), an intermediate in sphingosine metabolism, shows a diversity of biological activities. Comparable roles might be anticipated for d-ribo -phytosphingosine-1-phosphate (1). We describe an efficient three-step chemical synthesis of 1 from d-ribo -phytosphingosine.

Sterols affecting meiosis: novel chemical syntheses and the biological activity and spectral properties of the synthetic sterols.

4,4-Dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (I) from human follicular fluid and 4,4-dimethyl-5alpha-cholesta-8,24-dien-3beta-ol (II) from bull testes have been reported to activate meiosis in mouse oocytes (Byskov et al., 1995. Nature.

Sterol synthesis. Synthesis of 3 beta-hydroxy-25,26,26,26,27,27,27-heptafluorocholest-5-en-7-one and its effects on HMG-CoA reductase activity in Chinese hamster ovary cells, on ACAT activity in rat jejunal microsomes, and serum cholesterol levels in rats.

3 beta-Hydroxycholest-5-en-7-one (I; 7-ketocholesterol) is an oxysterol of continuing interest in biology and medicine. In the present study, we have prepared a side-chain fluorinated analog, 3 beta-hydroxy-25,26,26,26,27,27,27-heptafluorocholest-5-en-7-one (VI), with the anticipation that the F7 substitution would block major metabolism of the 7-ketosterol, and thereby enhance its potential in vivo effects on serum cholesterol levels and other parameters. Chromium trioxide/dimethyl pyrazole oxidation of the acetate derivative of the previously described 25,26,26,26,27,27,27-heptafluorocholest-5-en-3 beta-ol (Swaminathan et al.

Sterol synthesis. A timely look at the capabilities of conventional and silver ion high performance liquid chromatography for the separation of C27 sterols related to cholesterol biosynthesis.

Sterol intermediates in the biosynthesis of cholesterol have recently assumed a very prominent position in a number of important problems in medicine and biology. In studies of these matters, the separation and identification of the sterol intermediates present formidable challenges, a situation which does not appear to be generally appreciated. High performance liquid chromatography (HPLC) is a simple and rapid approach for the separation of the concerned compounds.

The orphan nuclear receptor LXRalpha is positively and negatively regulated by distinct products of mevalonate metabolism.

LXRalpha is an orphan member of the nuclear hormone receptor superfamily that displays constitutive transcriptional activity. We reasoned that this activity may result from the production of an endogenous activator that is a component of intermediary metabolism. The use of metabolic inhibitors revealed that mevalonic acid biosynthesis is required for LXRalpha activity.

An updated look at the analysis of unsaturated C27 sterols by gas chromatography and mass spectrometry.

Gas chromatography-mass spectrometry (GC-MS) and GC are commonly used methods for the identification and quantitation of sterols from samples of biological origin. To investigate the utility and limitations of these methods, we have determined gas chromatographic mobilities and mass spectral properties of 5alpha-cholestan-3beta-ol and 26 unsaturated C27 sterols as their acetate and trimethylsilyl (TMS) ether derivatives by GC and GC-MS. The GC retention data showed that numerous sterols were essentially coeluted on capillary GC columns coated with either 5% phenyl-95% methyl polysiloxane or polyethylene glycol, although the peaks were more widely dispersed on the latter column.

Modulation of skeletal muscle Ca2(+)-release channel activity by sphingosine.

The effect of D-erythro-C18-sphingosine (sphingosine) and related compounds on the Ca(2+)-release channel (ryanodine binding protein) was examined on rabbit skeletal muscle membranes, on the purified ryanodine binding protein, and on the channel reconstituted into planar lipid bilayers. Sphingosine inhibited [3H]ryanodine binding to sarcoplasmic reticulum (SR) membranes in a dose-dependent manner similar to published results (R. A.

Inhibitors of sterol synthesis. Synthesis and spectral properties of 3 beta-hydroxy-25,26,26,26,27,27,27-heptafluoro-5 alpha-cholestan-15-one.

3 beta-Hydroxy-25,26,26,26,27,27,27-heptafluoro-5 alpha-cholestan-15-one (4) has been prepared as part of a program to synthesize 15-ketosterols that are not readily metabolized to cholesterol or side-chain oxygenated species. Saponification of 3 beta-acetoxy-5 alpha-chola-8(14),23-dien-15-one (5) followed by lithium-ammonia reduction with a bromobenzene quench gave 3 beta-hydroxy-5 alpha-chol-23-en-15-one (6). Addition of (CF3)2CFI to 6 in the presence of triethylborane gave an iodide preparation, which was reduced to 4 with tributyltin hydride (71% overall yield of 4 from 5).

Silver ion high pressure liquid chromatography provides unprecedented separation of sterols: application to the enzymatic formation of cholesta-5,8-dien-3 beta-ol.

We report that silver ion HPLC provides remarkable separations of C27 sterols differing only in the number or location of olefinic double bonds. This technique has been extended to LC-MS, analysis of purified components by GC, GC-MS, and 1H NMR, and to its use on a semipreparative scale. The application of this methodology for the demonstration of the catalysis, by rat liver microsomes, of the conversion of 7-dehydrocholesterol to cholesta-5,8-dien-3 beta-ol is also presented.

Analysis of unsaturated C27 sterols by nuclear magnetic resonance spectroscopy.

Complete 1H and 13C nuclear magnetic resonance (NMR) signal assignments have been established for 5 alpha-cholestan-3 beta-ol, 22 unsaturated C27 sterols, and their acetate derivatives. Assignments were made from a combination of 1D and 2D spectra and include stereochemical 1H assignments for the C-22 and C-23 protons of 5 alpha-cholesta-8,24-dien-3 beta-ol and other delta 24 sterols with a C8 side chain. At the temperature and concentration range described, chemical shifts were generally reproducible to +/- 0.