Non-aqueous capillary electrophoretic separation of cholesterol and 25-hydroxycholesterol after derivatization with Girard P reagent.

Capillary electrophoresis (CE) can provide high separation efficiency with very simple instrumentation, but has yet to be explored regarding oxysterols/cholesterol. Cholesterol and 25-hydroxycholesterol (both are 4-ene-3-ketosteroids) were quantitatively transformed into hydrazones using Girard P reagent after enzymatic oxidation by cholesterol oxidase. Separation was achieved using non-aqueous capillary electrophoresis with UV detection at 280nm; the "charge-tagging" Girard P reagent ensured both charge and chromophore (which are requirements for CE-UV). Excess reagent was also separated from the two analytes, eliminating the need for removal prior to the analysis. The compounds were separated in less than 5min with excellent separation efficiency, using separation electrolytes fully compatible with mass spectrometry. The CE-UV method was used to optimize steps for charge-tagging, revealing that the procedure is affected by the analyte/reagent ratio and reaction time, but also the analyte structure.

Purification and identification of 25-hydroxycholesterol in a reptile: Seasonal variation and hormonal regulation.

The present in vitro study, for the first time, demonstrates the production of 25-hydroxycholestrol (25-HC) by testicular macrophages of a non-mammalian vertebrate. The ether extracts of testicular macrophage-conditioned medium (TMCM) were fractionated on a C18 reversed phase high-performance liquid chromatography (HPLC) column using methanol as the mobile phase. The mass spectrometry (MS) fragmentation pattern of HPLC-purified 25-HC was found to be identical to that of authentic 25-HC. Further, a significant seasonal variation in 25-HC concentration was observed with maximal level in regressed and minimal during breeding phase. To understand the hormonal control of 25-HC production, testicular macrophages from regressed phase testes were incubated with 0.5µg/ml of ovine follicle stimulating hormone (FSH) and 0.1, 1 and 10µg/ml of testosterone (T). FSH considerably enhanced 25-HC production by testicular macrophages. In contrast, T markedly inhibited 25-HC production in a dose-dependent manner. In addition, T significantly inhibited FSH-induced 25-HC production, though pre-treatment with T was more effective as compared to post-treatment with T to FSH. Our findings on production, seasonal variation and hormonal control of 25-HC suggest the functional significance of 25-HC in the testis of reptiles.

Versatile, sensitive liquid chromatography mass spectrometry - Implementation of 10 µm OT columns suitable for small molecules, peptides and proteins.

We have designed a versatile and sensitive liquid chromatographic (LC) system, featuring a monolithic trap column and a very narrow (10 µm ID) fused silica open tubular liquid chromatography (OTLC) separation column functionalized with C18-groups, for separating a wide range of molecules (from small metabolites to intact proteins). Compared to today's capillary/nanoLC approaches, our system provides significantly enhanced sensitivity (up to several orders) with matching or improved separation efficiency, and highly repeatable chromatographic performance. The chemical properties of the trap column and the analytical column were fine-tuned to obtain practical sample loading capacities (above 2 µg), an earlier bottleneck of OTLC. Using the OTLC system (combined with Orbitrap mass spectrometry), we could perform targeted metabolomics of sub-µg amounts of exosomes with 25 attogram detection limit of a breast cancer-related hydroxylated cholesterol. With the same set-up, sensitive bottom-up proteomics (targeted and untargeted) was possible, and high-resolving intact protein analysis. In contrast to state-of-the-art packed columns, our platform performs chromatography with very little dilution and is "fit-for-all", well suited for comprehensive analysis of limited samples, and has potential as a tool for challenges in diagnostics.

Isolation and identification of antitrypanosomal and antimycobacterial active steroids from the sponge Haliclona simulans.

The marine sponge Haliclona simulans collected from the Irish Sea yielded two new steroids: 24-vinyl-cholest-9-ene-3ß,24-diol and 20-methyl-pregn-6-en-3ß-ol,5a,8a-epidioxy, along with the widely distributed 24-methylenecholesterol. One of the steroids possesses an unusually short hydrocarbon side chain. The structures were elucidated using nuclear magnetic resonance spectroscopy and confirmed using electron impact- and high resolution electrospray-mass spectrometry. All three steroids possess antitrypanosomal and anti-mycobacterial activity. All the steroids were found to possess low cytotoxicity against Hs27 which was above their detected antitrypanosomal potent concentrations.

A new cytotoxic steroid from co-fermentation of two marine alga-derived micro-organisms.

Bioactivity-guided chemical investigation of a co-culture of marine-derived micro-organisms has yielded one new steroid, 7ß-hydroxycholesterol-1ß-carboxylic acid (1) with an unprecedented carboxylic acid group at C-1, together with three known steroidal metabolites (2-4). The chemical structures and stereochemistry of the isolated compounds were unambiguously determined based on extensive 1D, 2D NMR and HR-ESI-MS measurements. The isolated compounds were assessed for their cytotoxic activity against four different human tumour cell lines K562 (leukaemia), HCT116 (colon), A2780 (ovary) and its cisplatin-resistant mutant (A2780 CisR), and they revealed moderate activities with IC50 values ranging from 10.0 to 100.0 µM.

Sample preparation: a critical step in the analysis of cholesterol oxidation products.

In recent years, cholesterol oxidation products (COPs) have drawn scientific interest, particularly due to their implications on human health. A big number of these compounds have been demonstrated to be cytotoxic, mutagenic, and carcinogenic. The main source of COPs is through diet, and particularly from the consumption of cholesterol-rich foods. This raises questions about the safety of consumers, and it suggests the necessity for the development of a sensitive and a reliable analytical method in order to identify and quantify these components in food samples. Sample preparation is a necessary step in the analysis of COPs in order to eliminate interferences and increase sensitivity. Numerous publications have, over the years, reported the use of different methods for the extraction and purification of COPs. However, no method has, so far, been established as a routine method for the analysis of COPs in foods. Therefore, it was considered important to overview different sample preparation procedures and evaluate the different preparative parameters, such as time of saponification, the type of organic solvents for fat extraction, the stationary phase in solid phase extraction, etc., according to recovery, precision and simplicity.

Fast LC-MS/MS analysis of free oxysterols derived from reactive oxygen species in human plasma and carotid plaque.

BACKGROUND: A rapid liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the quantification of reactive oxygen species (ROS) derived free oxysterols and cholesterol in human plasma and atherosclerotic plaque. METHOD: In vitro autoxidation of cholesterol during sample pretreatment was avoided by applying only one protein precipitation and re-concentration step using 80 µl plasma. For preparation of 10mg atherosclerotic plaques an additional liquid-liquid extraction was included. Free 7-keto-, 7-α/ß-hydroxy-, 5,6-α-epoxy-, 5,6-ß-epoxycholesterol, cholestane-3ß,5α,6ß-triol and cholesterol were separated within 7 min on a monolithic column. An API 4000 tandem mass spectrometer was applied in positive ionization mode using atmospheric pressure chemical ionization. RESULTS: The detection limit was 0.1 ng/ml and the linearity ranged from 0.5 to 0.75 to 2000 ng/ml for the oxysterols and from 50 to 1000 µg/ml for cholesterol. Recovery was between 80.9 and 107.9%. Between-run imprecision ranged from 7.9 to 11.7%. Analysis of plasma samples from additional 50 middle-aged volunteers revealed a large inter-individual variability (e.g. 7-ketocholesterol 2.63-30.47 ng/ml). Oxysterol concentrations normalized to cholesterol were about 43 times higher in carotid plaque compared to plasma (n=5). CONCLUSION: This rapid LC-MS/MS method enables reliable quantification focused on especially ROS-derived oxysterols in human plasma and atherosclerotic plaque samples under high-throughput conditions.

Chromatographic separation of bioactive oxycholesterols by GC, HPLC and SFC.

In this paper we report the development of chromatographic methods for the separation of 8 biologically active hydroxycholesterols (OHC's) which include the single-hydroxyl addition species 7α-OHC, 7ß-OHC, 25-OHC and 27- OHC, together with the double-hydroxyl addition species 7α, 25-OHC, 7ß, 25-OHC, 7α, 27-OHC, and 7ß, 27-OHC. Four complementary techniques were employed (gas chromatography, normal phase and reversed phase high performance liquid chromatography, and supercritical fluid chromatography), and for each of the techniques, an optimized method for the separation of all eight compounds in a mixture is presented.

High sensitivity measurements of active oxysterols with automated filtration/filter backflush-solid phase extraction-liquid chromatography-mass spectrometry.

Oxysterols are important in numerous biological processes, including cell signaling. Here we present an automated filtration/filter backflush-solid phase extraction-liquid chromatography-tandem mass spectrometry (AFFL-SPE-LC-MS/MS) method for determining 24-hydroxysterol and the isomers 25-hydroxycholesterol and 22S-hydroxycholesterol that enables simplified sample preparation, high sensitivity (~25 pg/mL cell lysis sample) and low sample variability. Only one sample transfer step was required for the entire process of cell lysis, derivatization and determination of selected oxysterols. During the procedure, autoxidation of cholesterol, a potential/common problem using standard analytical methods, was found to be negligible. The reversed phase AFFL-SPE-LC-MS/MS method utilizing a 1mm inner diameter column was validated, and used to determine levels of the oxysterol analytes in mouse fibroblast cell lines SSh-LII and NIH-3T3, and human cancer cell lines, BxPC3, HCT-15 and HCT-116. In BxPC3 cells, the AFFL-SPE-LC-MS/MS method was used to detect significant differences in 24S-OHC levels between vimentin+ and vimentin- heterogenous sub-populations. The methodology also allowed monitoring of significant alterations in 24S-OHC levels upon delivery of the Hedgehog (Hh) antagonist MS-0022 in HCT-116 colorectal carcinoma cell lines.

Isolation and identification of two steroid compounds from Oviductus Ranae.

In this study, cholest-5-en-3ß,7ß-diol and 3ß-hydroxy-cholest-5-en-7-one were isolated from Oviductus Ranae by column chromatographies on Sephadex LH-20, octadecylsilyl (ODS) and pre high-performance liquid chromatography (HPLC). Their structures were elucidated on the basis of chemical and spectral analyses, including electrospray ionisation-mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). The above two compounds were obtained from Oviductus Ranae for the first time.

Liquid chromatography-tandem mass spectrometry determination of plasma 24S-hydroxycholesterol with chromatographic separation of 25-hydroxycholesterol.

Concentrations of circulating 24S-hydroxycholesterol (24SOHChol) are of interest as a practical measure of cholesterol efflux from the human brain. The current method of choice for 24SOHChol quantification is with gas chromatography-mass spectrometry (GC-MS). Liquid chromatography-mass spectrometry (LC-MS) methods to detect 24SOHChol have been described, but they lack rigorous high-performance liquid chromatography (HPLC) separation of a closely eluting isomeric oxysterol, 25-hydroxycholesterol. This is important because 25-hydroxycholesterol can be present in significant amounts and tandem mass spectrometry (MS/MS) cannot completely differentiate 24SOHChol. We describe an LC-MS method with rapid chromatographic separation of the oxysterols to permit accurate determination of plasma 24SOHChol. The availability of an LC-MS method offers advantages such as simplified sample work-up and analysis.

Sinugrandisterols A-D, trihydroxysteroids from the soft coral Sinularia grandilobata.

Four new trihydroxysteroids, sinugrandisterols A-D (1-4), have been isolated from the CH(2)Cl(2)-soluble fraction of the EtOH extract of Sinularia grandilobata. The structures of these metabolites were determined on the basis of spectroscopic (IR, MS, and 1D and 2D NMR) analysis. The cytotoxicity of 1-4 toward a limited panel of cancer cell lines is also reported.

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 constituents of the twigs and leaves of Aglaia rubiginosa.

Activity-guided fractionation of a CHCl(3)-soluble extract of the twigs of Aglaia rubiginosa, using human oral epidermoid carcinoma (KB) cells as a monitor, led to the isolation of a new naturally occurring cyclopenta[b]benzofuran, 1-O-acetylrocaglaol (1), along with seven known compounds, methyl rocaglate (2), rocagloic acid (3), 1-O-acetylmethyl rocaglate (4), desyclamide, eryodictiol, 5-hydroxy-3,7,4'-trimethoxyflavone, and naringenin. A CHCl(3) extract of the leaves of A. rubiginosa yielded the new compound (3S,4R,22R)-cholest-7,24-diene-3,4,22-triol (5), as well as 11 known compounds, including 2 and 4 and cabraleone, dammarelonic acid, (20S,23E)-20,25-dihydroxy-3,4-secodammara-4(28),23-dienoic acid, (20S,23E)-20,25-dihydroxy-3,4-secodammara-4(28),23-dienoic acid methyl ester, (3beta,4beta,22R)-ergosta-5,24(24')-diene-3,4,22-triol, ocotillone, shoreic acid, beta-sitosterol, and beta-sitosterol glycoside. The structures of 1 and 5 were elucidated by spectral and chemical methods. Isolates were evaluated with a human cancer cell panel, and compounds 1-4 were found to exhibit potent cytotoxic activity.

Determination of 24S- and 27-hydroxycholesterol in plasma by high-performance liquid chromatography-mass spectrometry.

24S-Hydroxycholesterol (24S-OH-Chol) and 27-hydroxycholesterol (27-OH-Chol) are oxidized derivatives of cholesterol and of potential diagnostic interest because their circulating levels may reflect the cholesterol metabolism of the brain and macrophages, respectively. We developed a sensitive and specific HPLC-MS method for the quantification of 24S-OH-Chol and 27-OH-Chol in human plasma. In contrast to currently available procedures based on gas chromatography-mass spectrometry, this methodology offers the advantage that no time-consuming derivatization is needed. After saponification, solid-phase extraction, and HPLC separation under reversed-phase column conditions, detection by MS was performed using atmospheric pressure chemical ionization and selected ion monitoring mode. The standard curves were linear throughout the calibration range for both oxysterols. Within-day and between-day coefficients of variation were less than 9%, and the recoveries ranged between 98% and 103%. The quantification limits were 40 and 25 microg/l for 24S-OH-Chol and 27-OH-Chol, respectively. Mean values for both oxysterols were determined in plasma from 22 healthy volunteers. The sensitive and selective HPLC-MS method described here combined with the appropriate workup procedure allow the quantification of 24S-OH-Chol and 27-OH-Chol in plasma samples, for example in clinical studies to elaborate the clinical usefulness of these two oxysterols.

The detection of 20S-hydroxycholesterol in extracts of rat brains and human placenta by a gas chromatograph/mass spectrometry technique.

The presence of 20(S)-hydroxycholesterol in rat brains and human placenta has been established using the gas chromatography/mass spectrometry (GC/MS) select ion monitoring (SIM) technique. Identification was ensured by three criteria: the specific retention time when the compound emerges from the gas chromatogram and the two m/z ions (201 and 461amu) which are characteristic of its mass spectrum. The possible role of 20(S)-hydroxycholesterol in steroid hormone biosynthesis and in other biological processes is discussed.

Effect of chronic ethanol feeding on oxysterols in rat liver.

It was our hypothesis that, as a consequence of increased oxidative stress, cholesterol-derived hydroperoxides and oxysterols are increased in livers of rats exposed to ethanol. To test this we dosed Wistar rats (approximately 0.1 kg initial body weight) with ethanol chronically (rats fed a nutritionally complete liquid diet containing ethanol as 35% of total calories; sampled liver at approximately 6-7 weeks). We measured concentrations of 7 alpha- and 7 beta-hydroperoxycholest-5-en-3 beta-ol (7 alpha-OOH and 7 beta-OOH) as well as 7 alpha- and 7 beta-hydroxycholesterol (7 alpha-OH and 7 beta-OH), and 3 beta-hydroxycholest-5-en-7-one (also termed 7-ketocholesterol; 7-keto). In response to chronic alcohol feeding, there were significant elevations in the concentrations of 7 alpha-OOH (+169%, P = 0.005) and 7 beta-OOH (+199%, P = 0.011). Increases in the concentrations of hepatic 7-keto (+74%, P = 0.01) and decreases in cholesterol (-43%; P = 0.03) also occurred. In contrast, the concentrations of both 7 alpha-OH and 7 beta-OH were not significant (NS). However, when oxysterols in chronic ethanol-fed rats were expressed relative to cholesterol there were significant increases in 7-keto/cholesterol (P = 0.0006), 7 alpha-OH/cholesterol (P = 0.0018) and 7 beta-OH/cholesterol (P = 0.0047). In conclusion, this is the first report of increased 7 alpha-OOH, 7 beta-OOH, and 7-keto in liver of rats and their elevation in chronic experimental alcoholism represent evidence of increased oxidative stress.

Cholesterol and its derivatives, are the principal steroids isolated from the leech species Hirudo medicinalis.

Steroids were isolated from the blood-sucking leech species Hirudo medicinalis and their structure was studied with one- and two-dimensional NMR spectroscopy (DQF-COSY and HMQC), GC-MS and ESI-MS spectrometry. Fractionating leech lipid using silicic acid chromatography led to the isolation of cholesterol in an early chloroform-eluted peak. Only minor traces of cholest-4-en-3-one, 4 beta-methylcholesterol, and sitosterol were present. The subsequent acetone-eluted fraction contained steroidtriols that were further purified by preparative TLC; these included cholest-7-ene-3,5,6 triol, cholest-4,7-diene-3,6,15 triol and to a lesser amount, cholestane-3,5,6 triol. A developmental study on cholesterol content in the leech showed that it is also the principal steroid in embryonic and freshly hatched leeches prior to feeding. The abundance of cholesterol, comprising approximately 5% of the total leech lipid, suggests that H. medicinalis, a blood sucking leech, has adapted itself fully to its mammalian host in terms of its steroid content. It remains to be seen whether lipids are directly transferred from the host to the parasite or whether leeches have evolved mechanisms to synthesize their own steroids.

Determination of 7alpha-hydroxycholesterol in dog plasma by high-performance liquid chromatography with fluorescence detection.

A new method was developed for the determination of 7alpha-hydroxycholesterol (7-HC) in dog plasma by means of high-performance liquid chromatography (HPLC) with fluorescence detection. 7-HC extracted with organic solvent from plasma was purified with Bond Elut 2OH and converted to a sensitive fluorescent derivative containing double coumarin groups at the C-3 and C-7 positions of the steroid nucleus with 7-methoxycoumarin-3-carbonyl azide. After removal of the excess reagent with Bond Elut NH2, the 7-HC derivative was separated by reverse-phase HPLC method. The detection limit of the authentic 7-HC-3,7-coumarin derivative was 4 pg (S/N = 5), approximately four times less than that of the 7-HC-3-anthroyl derivative yielded by reaction of 7-HC with 1-anthroylcyanide. The newly developed method was used to investigate the effects of consecutive oral administrations of cholestyramine (CA) on 7-HC levels in dog plasma. The plasma 7-HC levels of the CA-treated group were two times greater than those of the control group.

[Studies on the isolation, structure identification and biological function of two tumor cell suppressors of human fetal liver origin].

Our previous work showed the existence of low molecular weight tumor suppressors in human fetal tissues. In this paper, two tumor cell suppressors were isolated and purified from methanol extract of human fetal liver by C18 reversed-phase medium pressure chromatography, gel filtration on Sephadex LH-20, and high-performance liquid chromatography, directed by suppression of growth of HL-60 cells. The structures of the suppressors were identified to be 7-ketocholesterol and 7-beta-hydroxycholesterol. Under the condition of in vitro agar plate culture, 7-ketocholesterol and 7-beta-hydroxycholesterol showed preferentially inhibitory effects on the growth of both human and murine leukemic cell lines including human HL-60 and murine S-180 cells, but less effective on the growth of normal human and murine bone marrow granulocyte-macrophage progenitors (CFU-GM).