Online solid-phase extraction-liquid chromatography-mass spectrometry to determine free sterols in human serum.

An automated method for analyzing free non-cholesterol sterols in human serum using online solid phase extraction-liquid chromatography-mass spectrometry is proposed herein. The method allows the determination of three phytosterols (sitosterol, stigmasterol and campesterol) and two cholesterol precursors (desmosterol and lanosterol). The analysis of sterols in human serum is critical in the study of cholesterol-related disorders, such as inherited familial hypercholesterolemias. Special effort was made to isolate the analytes from the serum lipoproteins, their natural conveyance through the bloodstream. The sample treatment consisted of a Bligh-Dyer extraction followed by dilution of the extract. This treatment allowed the sample to be injected into the online system and ensured the correct detection of the analytes, while avoiding the matrix effects commonly related to serum samples. The analytical performance showed linear ranges that covered two orders of magnitude, with correlation coefficients above 0.99. Limits of detection and quantification ranged from 0.2 ng/mL to 13 ng/mL and from 1.0 ng/mL to 43 ng/mL, respectively. Recovery when spiking serum with a half or a tenth of the average concentration reported in human serum ranged from 99% to 111% and from 102% to 120%, respectively. Intra-day precision and inter-day precision were below 20%.

HPLC method for quantification and characterization of cholesterol and its oxidation products in eggs.

A new method was developed for the simultaneous determination of cholesterol and its oxidation products in eggs, using HPLC with UV and refractive index (RI) detectors, and HPLC interfaced with atmospheric pressure chemical ionization coupled to MS (HPLC-APCI-MS). The best conditions for direct saponification of the sample and extraction of the non-saponifiable material were defined using complete factorial designs with central points. The method showed accuracy and precision with a detection limit between 0.002 and 0.079 microg/g. The oxides cholest-5-ene-3beta,20alpha-diol and cholest-5-ene-3beta,25-diol identified by HPLC-UV-RI were not confirmed by HPLC-APCI-MS.

Cytotoxic oxygenated desmosterols of the red alga Galaxaura marginata.

Desmosterol (1), 24,25-epoxycholesterol (2), 24-hydroperoxycholesta-5,25-dien-3 beta-ol (3), 25-hydroperoxycholesta-5,23(E)-dien-3 beta-ol (4), cholesta-5,25-diene-3 beta,24-diol (5), and 24,25-epoxy-6 beta-hydroxycholest-4-en-3-one (7) were isolated from the marine red alga Galaxaura marginata; sterols 3, 4, and 7 were isolated for the first time from a natural source. Sterols 3-7 exhibited significant cytotoxicity toward several cancer cell lines.

Enzyme inhibition during the conversion of squalene to cholesterol.

Two separate enzymatic assays were developed in order to test the selectivity of inhibitors in cholesterol biosynthesis. One assay detects inhibition of delta 5.7-sterol delta 7-reductase, the enzyme involved in the conversion of 7-dehydrocholesterol to cholesterol. Delta 5.7-Sterol delta 7-reductase was inhibited by both RPR 101821, a protonated cyclohexylamine, and BM 15.766, a piperazine derivative, with IC50 values of 1 microM. The second assay detects accumulation of any of five intermediates (squalene oxide, squalene dioxide, lanosterol, desmosterol, and 7-dehydrocholesterol) upon inhibition of enzymes catalyzing reactions in the conversion of squalene to cholesterol. In this assay, inhibition data were most accurate when control assays exhibited a conversion of squalene to cholesterol in the order of 50%. The time required to attain 50% conversion of squalene to cholesterol was 6 h. Given a high inhibitor to substrate concentration ratio and the possible values of Ki, kon, and koff for the reaction between enzymes and inhibitor to form enzyme-inhibitor complexes, it was predicted that in the presence of inhibitors, intermediate accumulation could still be observed after 6 h incubation. The experimental results were in agreement with this prediction.

Sex pheromones in marine polychaetes: steroids from ripe Nereis succinea.

A number of neutral marine steroids such as desmosterol, campesterol, brassicasterol, gorgosterol, and other trace steroids were isolated from the coelomic fluid of ripe Nereis succinea and checked for biological activity as sex pheromones on swarming specimens of Platynereis dumerilii and Nereis succinea. No significant influence of synthetic gorgosterol or a natural extract of gorgosterol or the other identified steroids on the swarming behavior was observed.

Desmosterol in human milk.

Milk samples were collected from mothers at 2, 6, 12 and 16 weeks postpartum. Desmosterol was found to be present in all the milk samples. Identification of desmosterol was based on retention times with two gas liquid chromatography (GLC) columns and verified by GC-mass spectrometry. The concentration of desmosterol in breast milk increased significantly (P less than .05) from 0.6 mg/100 ml at 2 weeks to 1.3 mg/100 ml at 16 weeks postpartum. Desmosterol was not significantly correlated with total lipid, total cholesterol or free cholesterol in the milk.

Resolution of desmosterol, cholesterol, and other sterol intermediates by reverse-phase high-pressure liquid chromatography.

A highly efficient technique has been developed for the resolution of several sterols that are intermediates in the biosynthesis of cholesterol and that differ only by one carbon-carbon double bond or by one methyl group. The technique described utilizes reverse-phase high-pressue liquid chromatography on a micronBondapak-C18 column with acetonitrile as eluting solvent. This procedure is capable of measuring the enzymatic conversion of desmosterol to cholesterol. This chromatographic separation can be conducted by reverse-phase high-pressure liquid chromatography in approximately 10 min, whereas other procedures can require several days.

[Isolation and identification of desmosterol and its concentration in animal brain tissue]. / Vydilennia ta identyfikatsiia desmosterynu i ioho vmist u tkanyni mozku tvaryn

A method is developed for isolating desmosterol fro the brain of some animals. Sterols with Rf 0.37 and 0.51 are identified. Desmosterol is found in the brain unsaponifiable fraction of calves, piggies, chickens and rats. Its content in the mentioned animals brain is compared. Desmosterol content is shown to rise sharply in the rat brain during the first 12 days after birth. Interrelation between the level of sterols in the brain and formation of the nerve tissue myelin sheath is discussed.

Separation of 24- and 25-dehydrocholesterols; and the impure state of commercial desmosterol preparations.

Chromatographic methods for the separation of 24- and 25-dehydrocholesterols are described. The purities of three commercial samples of 24-dehydrocholesterol examined by thin-layer and gas-liquid chromatography were only 42, 79, and 80%, respectively; a commercial sample of radioactive 24-dehydrocholesterol was shown to contain 40% 25-dehydrocholesterol.