phytosterols and brassicasterol

Brassinosteroids (BRs) are plant steroids essential for normal growth and development and can be defined as steroids that carry an oxygen moiety at C-3 and additional ones at one or more of the C-2, C-6, C-22 and C-23 carbon atoms. BR biosynthesis and metabolism mutants have been obtained and the corresponding genes cloned. These include genes encoding 5alpha-reductase and cytochrome P450 enzymes, that are similar to enzymes associated with mammalian steroid synthesis. Perception and/or response mutants have also been identified via screening for altered sensitivity to BRs. Some of these mutants have been found to be defective in a leucine-rich repeat receptor kinase and in a component of a vacuolar ATPase. This review highlights the recent advances in unraveling BR synthesis/metabolism, transport, perception and response through the analysis of BR mutants.

Topics: Biological Transport; Cholestadienols; Light; Phytosterols; Plant Growth Regulators

Topics: Cholestadienols; Cholestanols; Cholesterol; Food Handling; Oils; Phytosterols; Vegetables

Plant sterols (PS) lower plasma LDL-cholesterol through partial inhibition of intestinal cholesterol absorption. Although PS themselves are poorly absorbed, increased intakes of PS result in elevated plasma concentrations. In this paper, we report time curves of changes in plasma PS during 12 weeks of PS intake. Furthermore, the impact of cholesterol synthesis and absorption on changes in plasma PS is explored.. The study was a double-blind, randomized, placebo-controlled, parallel-group study with the main aim to investigate the effects of PS on vascular function (clinicaltrials.gov: NCT01803178). Hypercholesterolemic but otherwise healthy men and women (n = 240) consumed low-fat spreads without or with added PS (3 g/d) for 12 weeks after a 4-week run-in period. Blood sampling was performed at week 0, 4, 8 and 12. Basal cholesterol-standardized concentrations of lathosterol and sitosterol + campesterol were used as markers of cholesterol synthesis and absorption, respectively. In the PS group, plasma sitosterol and campesterol concentrations increased within the first 4 weeks of intervention by 69% (95%CI: 58; 82) starting at 7.2 μmol/L and by 28% (95%CI: 19; 39) starting at 11.4 μmol/L, respectively, and remained stable during the following 8 weeks. Placebo-corrected increases in plasma PS were not significantly different between high and low cholesterol synthesizers (P-values >0.05). Between high and low cholesterol absorbers, no significant differences were observed, except for the cholesterol-standardized sum of four major plasma PS (sitosterol, campesterol, brassicasterol and stigmasterol) showing larger increases in low absorbers (78.3% (95%CI: 51.7; 109.5)) compared to high absorbers (40.8% (95%CI: 19.9; 65.5)).. Increases in plasma PS stabilize within 4 weeks of PS intake and do not seem impacted by basal cholesterol synthesis or absorption efficiency. This study was registered at clinicaltrials.gov (NCT01803178).

Topics: Adult; Aged; Cholestadienols; Cholesterol; Cholesterol, LDL; Double-Blind Method; Female; Humans; Hypercholesterolemia; Intestinal Absorption; Lipid Metabolism; Male; Middle Aged; Phytosterols; Prospective Studies; Sitosterols; Stigmasterol

The sterol profile of rapeseed oil differs from that of tall oil with higher contents of campesterol and brassicasterol. We previously found that margarines providing 2 g/day of sterols from rapeseed or tall oil resulted in similar reductions in LDL cholesterol of 8-9%. The aim of the present study was to investigate whether the consumption of these margarines affected markers of endothelial function, inflammation and hemostasis.. Blood samples were collected from 58 hypercholesterolemic volunteers who completed a double-blinded, randomized, crossover trial. Subjects consumed each of the two sterol margarines and a control non-sterol margarine for 4 weeks separated by one-week washout periods. All the margarines had the same fatty acid composition. Concentrations of vascular cell adhesion molecule-l (VCAM-1), E-selection, circulating tumor necrosis factor α (TNFα) and plasminogen activator inhibitor-1 (total, tPAI-1; active, PAI-1) were quantified.. Rapeseed-sterol margarine reduced E-selection concentrations compared to the control margarine (p = 0.012) while tall-sterol margarine had no effect. The rapeseed-sterol margarine also reduced tPAI-1 (p = 0.008) compared to the tall-sterol margarine. No significant changes were observed in TNFα and VCAM-1. No association was found between LDL reduction and changes in E-selection and tPAI-1.. Rapeseed-sterol margarine demonstrated favorable effects on vascular risk markers.

Topics: Adult; Aged; Biomarkers; Brassica rapa; Cholestadienols; Cholesterol, LDL; Cytokines; E-Selectin; Endothelium, Vascular; Female; Hemostasis; Humans; Hypercholesterolemia; Inflammation; Male; Margarine; Middle Aged; Phytosterols; Plant Oils; Vascular Cell Adhesion Molecule-1

In the present work, a dispersive liquid-liquid microextraction approach has been developed for extraction of four phytosterols (stigmasterol, β-sitosterol, campesterol, and brassicasterol) from cow milk samples using organic and deep eutectic solvents and the results were critically compared. The extracted analytes were determined using high performance liquid chromatography. In the developed method, carbon tetrachloride and choline chloride:p-chlorophenol deep eutectic solvent were selected to use as the best extraction solvent. Effective parameters and validation data were studied for both methods, independently. Under optimum conditions, limits of detection and quantification were within the ranges of 0.3-0.9 and 1.0-3.0 ng/mL for organic solvent based dispersive liquid-liquid microextraction and 0.09-0.32 and 0.3-1.0 ng/mL for deep eutectic solvent based dispersive liquid-liquid microextraction, respectively. Good coefficient of determinations and relative standard deviations obtained for the methods were ≥0.994 and ≤7.6%, respectively. The introduced method was performed on different milk samples for the determination of target analytes using both solvents and the results were analyzed statistically by the t-test.

Topics: Animals; Cholestadienols; Cholesterol; Chromatography, High Pressure Liquid; Deep Eutectic Solvents; Food Contamination; Limit of Detection; Liquid Phase Microextraction; Milk; Pesticide Residues; Phytosterols; Solvents

We investigated the main sterols, phytosterols, and the α- and γ-tocopherol content in donkey milk during the first 2 mo of lactation. Cholesterol was the main sterol in milk (mean ± standard deviation = 0.97 ± 0.443 g/100 g of fat). Lanosterol was the main minor sterol of animal origin, followed by desmosterol (0.003 ± 0.001 and 0.001 ± 0.001 g/100 g of fat, respectively). Of the phytosterols, β-sitosterol was the main sterol of vegetal origin in donkey milk (0.005 ± 0.002 g/100 g of fat), but lower levels of campesterol, brassicasterol, and stigmasterol were also recorded. Mean levels of α- and γ-tocopherol were 0.01 ± 0.007 and 0.003 ± 0.001 g/100 g of fat, respectively. We observed no significant changes in sterol or tocopherol content during the first 2 mo of lactation. The presence of lanosterol in donkey milk is of particular interest, because lanosterol is a potential drug and has important physiological effects. The presence of phytosterols, which are considered nutraceutical molecules, enhances the nutritional quality of donkey milk fat for consumers.

Topics: Animals; Cholestadienols; Cholesterol; Desmosterol; Equidae; Female; Lactation; Lanosterol; Milk; Nutritive Value; Phytosterols; Saponins; Sitosterols; Sterols; Tocopherols

A novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously quantify phytosterols (brassicasterol, campesterol, stigmasterol and β-sitosterol) and tocopherols (alpha, beta, gamma and delta) entrapped in the lipid bilayer of a liposomal formulation. Apart from liposomes (a pharmaceutical product), the developed method was able to quantify target analytes in agricultural products, thus showing wide applications. Atmospheric pressure chemical ionization (APCI) was employed due to the enhanced ionization of phytosterols and tocopherols in comparison to electrospray ionization. Unlike published work, the chromatographic conditions were modified to simplify the analytical approach. For the first time, a simple isocratic elution (acetonitrile:methanol 99:1 v/v) was utilized for the separation of four phytosterols and four tocopherols in a single run. A substantially better baseline separation of phytosterols were obtained in comparison to reported methods by using poroshell C18 column. The method has a total run time of 7 min, which is the shortest run time among all reported quantitative methods for the simultaneous determination of four phytosterols and four tocopherols. Calibration curves for all phytosterols were linear in the range of 0.05-10 μg/mL. In the case of tocopherols, alpha tocopherol showed linear response in the range of 0.25-10 μg/mL. However, gamma and delta tocopherols exhibited quadratic relationship in the same concentration range (0.25-10 μg/mL). Validation parameters met the International Conference on Harmonization (ICH) guidelines in terms of selectivity, accuracy, precision, repeatability, sensitivity, matrix effects, dilution integrity and stability. The method was, for the first time, successfully applied for the quantifying phytosterols and tocopherols entrapped inside liposomes. An interesting chromatographic phenomenon was observed during sample analysis. Alpha tocopherol (entrapped in the liposomal lipid bilayer) was found to elute at two retention times, 2.53 min and 3.60 min. Such dual separation was not observed in calibration standards and quality controls. It was concluded that the chiral recognition ability of liposomes made up of phosphatidylcholine separated the enantiomers of alpha tocopherol, giving rise to two peaks at two different retention time. To sum, the reported novel LC-MS/MS method addresses three major analytical shortcomings, namely i)longer run time,

Topics: Atmospheric Pressure; Calibration; Cholestadienols; Cholesterol; Chromatography, High Pressure Liquid; Chromatography, Liquid; Liposomes; Phytosterols; Reproducibility of Results; Sitosterols; Spectrometry, Mass, Electrospray Ionization; Stigmasterol; Tandem Mass Spectrometry; Tocopherols

This study investigated the effect of blanching pomegranate seeds (PS) on oil yield, refractive index (RI), yellowness index (YI), conjugated dienes (K232), conjugated trienes (K270), total carotenoid content (TCC), total phenolic compounds (TPC) and DPPH radical scavenging of the extracted oil. Furthermore, phytosterol and fatty acid compositions of the oil extracted under optimum blanching conditions were compared with those from the oil extracted from unblanched PS. Three different blanching temperature levels (80, 90, and 100 °C) were studied at a constant blanching time of 3 min. The blanching time was then increased to 5 min at the established optimum blanching temperature (90 °C). Blanching PS increased oil yield, K232, K270, stigmasterol, punicic acid, TPC and DPPH radical scavenging, whereas YI, β-sitosterol, palmitic acid and linoleic acid were decreased. The RI, TCC, brassicasterol, stearic acid, oleic acid and arachidic acid of the extracted oil were not significantly (

Topics: Antioxidants; Biphenyl Compounds; Carotenoids; Cholestadienols; Dietary Supplements; Eicosanoic Acids; Fatty Acids; Food Technology; Free Radical Scavengers; Linoleic Acid; Linolenic Acids; Oleic Acid; Phenol; Phenols; Phytosterols; Picrates; Plant Oils; Pomegranate; Refractometry; Seeds; Stearic Acids; Temperature

We previously revealed that Choreito, a traditional Kampo medicine, strongly inhibits bladder carcinogenesis promotion. We have also shown that Polyporus sclerotium, which is one of the crude drugs in Choreito, has the strongest bladder carcinogenesis inhibitory effect and that the ergosterol contained in Polyporus sclerotium is the main active component. In this study, we analyzed the mechanism by which ergosterol inhibits bladder carcinogenesis. Rats were given an N-butyl-N-(4-hydroxybutyl) nitrosamine (BHBN) solution ad libitum, and then a promoter [saccharin sodium (SS), DL-tryptophan, or BHBN] was administered together with ergosterol or its metabolite, brassicasterol. The bladders were removed from rats, and the inhibitory effect on carcinogenesis promotion was evaluated by an agglutination assay with concanavalin A (Con A). Although the oral administration of ergosterol inhibited the promotion of bladder carcinogenesis with SS, the intraperitoneal administration of brassicasterol showed a stronger effect. The effect of brassicasterol on carcinogenesis promotion was observed regardless of the type of promoter. Administration of testosterone to castrated rats increased the number of cell aggregates caused by Con A. In contrast, intraperitoneal administration of brassicasterol to castrated rats treated with testosterone significantly decreased the number of cell aggregates, confirming the inhibition of bladder carcinogenesis promotion. The inhibitory effect of ergosterol on bladder carcinogenesis is due to brassicasterol, a metabolite of ergosterol. The action of brassicasterol via androgen signaling may play a role in the inhibitory effect on bladder carcinogenesis promotion.

Topics: Animals; Cholestadienols; Ergosterol; Humans; Male; Medicine, Kampo; Phytosterols; Rats; Rats, Wistar; Urinary Bladder Neoplasms

The objective of this study is to review the compliance of fatty acid compositions of Thai and India rice bran oil and level of desmethylsterols of Thai crude rice bran oil with the Codex Standard for Named Vegetable Oil (Codex Stan 210-1999). Fatty acid compositions of 90 samples of Thai and India refined rice bran oil were analyzed by capillary gas liquid chromatography. The results indicated that the contents of the C14:0, C18:2, C22:0 and C24:0 possible fall outside the range of Codex Stan 210-1999. In addition, sterol profile content of 40 samples of crude rice bran oil from Thai refinery plants were studied. The test results of major compositions of desmethylsterols are in good agreement with CODEX STAN 210-1999 except for Brassicasterol and other desmethylsterols. Accordingly, these data were proposed to corporate into the codex standard. Consequently, Codex agreed to amend the fatty acid composition of C14:0, C18:2, C22:0 and C24:0 from ND to 1.0, 21 to 42, ND to 1.0 and ND to 0.9 % and broaden level of Brassicasterol and other desmethylsterols to "ND-0.3" and to "7.5-12.8" accordingly.

Topics: Asian People; Cholestadienols; Chromatography, Gas; Fatty Acids; Food Analysis; Food Quality; Humans; India; Phytosterols; Plant Oils; Rice Bran Oil

Sewage pollution is a principal factor of decreasing water quality, although it has not been considered a real impact in Amazonia that is still considered a pristine environment around the world. Thus, this study aimed to assess the levels of sewage contamination in sediments from three streams crossing Manaus - a Brazilian city of 2,403,796 inhabitants in the heart of the Amazon rain forest. Cholesterol, cholestanol, brassicasterol, ergosterol, stigmasterol, β-sitosterol, campesterol, stigmastanol, coprostanol, and epicoprostanol levels were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). The fecal indicator, coprostanol, was found in high concentrations (509-12 830 ng g

Topics: Biomarkers; Brazil; Cholestadienols; Cholestanol; Cholestanols; Cholesterol; Chromatography, Liquid; Drug Contamination; Environmental Monitoring; Feces; Geologic Sediments; Phytosterols; Rivers; Sewage; Sitosterols; Sterols; Tandem Mass Spectrometry; Water Pollutants; Water Pollution; Water Quality

The seed of five fruits and vegetables, which are often eaten by Chinese people, were selected as research objects to study the physicochemical properties, nutritional ingredients and antioxidant capacity of their seed oils. The fatty acid results indicated that the oleic acid was the main unsaturated fatty acid in almond oil and celery seed oil (content of 64.10% and 62.96%, respectively), and the wax gourd seed oil, watermelon seed oil and pumpkin seed oil were linoleic acid as the main unsaturated fatty acid (content of 72.45%, 76.77% and 47.35%, respectively). Unsaturated fatty acids are mainly located at the sn-2 position of the triacylglycerol (TAG), whereas saturated fatty acids are mainly located at the sn-1, 3 positions for the five seed oils. The pumpkin seed oil had certain advantages in terms of phytosterols and squalene (3716 and 2732 mg/kg, respectively). The high content of polyphenol for celery seed oil exhibits higher medicinal value. Polyphenols, and brassicasterols were have significant correlation with antioxidant capacity (p < 0.05, r = 0.890-0.998). The significant differences in nutrient composition between these fruits and vegetables seed oils indicate their unique value as food.

Topics: Antioxidants; Chemical Phenomena; China; Cholestadienols; Fatty Acids; Fatty Acids, Unsaturated; Fruit; Linoleic Acids; Oleic Acid; Phytosterols; Plant Oils; Polyphenols; Squalene; Triglycerides; Vegetables

Milk fat adulteration is a common issue in Central Asia. To assess the current situation in the commercial milk market, 17 milk samples were checked for fatty acid (FA) and sterol profiles to detect potential adulteration using multivariate analysis. Analysis of FA and sterols was performed using gas chromatography with flame ionization detection and gas chromatography with mass-spectrometric detection, respectively. Cluster analysis of FA profiles revealed 3 types of milk samples: (1) samples containing a higher proportion of short-chain FA, (2) samples containing a higher proportion of long-chain FA, and (3) samples with significant amounts of C18 FA. Analysis of sterols showed that samples included (1) milk fat containing 100% cholesterol, sometimes with traces of phytosterols, (2) milk fat with high proportions of β-sitosterol and campesterol, and (3) milk fat containing high proportions of brassicasterol. We found significant relationships between FA profiles and sterol profiles. The profiles were compared with vegetable oil patterns reported in the literature. More than 50% of the samples appeared to be counterfeited. We conclude that identification of adulteration in milk can be based solely on determination of sterol patterns.

Topics: Animals; Cholestadienols; Cholesterol; Fatty Acids; Food Contamination; Gas Chromatography-Mass Spectrometry; Milk; Phytosterols; Plant Oils; Sitosterols; Sterols

The aim of the study was to investigate how the dietary sterol composition, including cholesterol, phytosterol:cholesterol ratio and phytosterols, affect the absorption, biliary excretion, retention, tissue storage and distribution of cholesterol and individual phytosterols in Atlantic salmon (Salmo salar L.). A feeding trial was conducted at two different temperatures (6 and 12°C), using nine different diets with varying contents of phytosterols, cholesterol and phytosterol:cholesterol ratio. Cholesterol retention values were clearly dependent on dietary cholesterol, and showed that fish fed cholesterol levels <1000 mg/kg feed produced considerable quantities of cholesterol de novo. Despite this production, cholesterol content increased with increasing dietary cholesterol in liver, plasma, bile, muscle, adipose tissue and whole fish at 12°C, and in plasma, bile and whole fish at 6°C. The tissue sterol composition generally depended on the dietary cholesterol content and on the dietary phytosterol:cholesterol ratio, but not on the dietary phytosterol content in itself. Campesterol and brassicasterol appeared to be the phytosterols with the highest intestinal absorption in Atlantic salmon. There was a high biliary excretion of campesterol, but not of brassicasterol, which accumulated in tissues and particularly in adipose tissue, with 2-fold-higher retention at 12°C compared with 6°C. Campesterol had the second highest retention of the phytosterols in the fish, but with no difference between the two temperatures. Other phytosterols had very low retention. Although brassicasterol retention decreased with increasing dietary phytosterols, campesterol retention decreased with increasing dietary cholesterol, indicating differences in the uptake mechanisms for these two sterols.

Topics: Animals; Cholestadienols; Cholesterol; Diet; Intestinal Absorption; Liver; Phytosterols; Salmo salar; Sterols

Several feeding trials with Atlantic salmon fed naturally high phytosterol concentrations due to dietary rapeseed oil inclusion have shown changes in lipid metabolism and increased hepatic lipid storage in the fish. An in vitro trial with Atlantic salmon hepatocytes was, therefore, performed to study the possible direct effects of phytosterols on lipid storage and metabolism. The isolated hepatocytes were exposed to seven different sterol treatments and gene expression, as well as lipid accumulation by Oil Red O dyeing, was assessed. Fucosterol, a sterol found in many algae species, had an effect on the size of individual lipid droplets, leading to smaller lipid droplets than in the control without added sterols. A sterol extract from soybean/rapeseed led to an increase in the percentage of hepatocytes with visible lipid droplets at 20× magnification, while hepatocytes of both the sterol extract-treated groups and fucosterol-treated groups had a larger proportion of their area covered with lipids compared to control cells. Brassicasterol, a sterol characteristic of rapeseed oil, was the only sterol treatment leading to a change in gene expression, affecting the expression of the nuclear receptors, peroxisome proliferator-activated receptor gamma (pparg) and retinoid X receptor (rxr). The current study thus shows that phytosterols can have direct, although subtle, effects on both hepatic lipid storage and gene expression of Atlantic salmon in vitro.

Topics: Animals; Cholestadienols; Hepatocytes; Lipid Metabolism; Lipids; Phytosterols; Salmon; Stigmasterol

This study aimed to investigate the effect of metal ions on the degradation of phytosterols in oils. The oil was heated at 180°C for 1h with/without addition of Fe

Topics: Antioxidants; Cholestadienols; Cholesterol; Food Analysis; Food Handling; Gas Chromatography-Mass Spectrometry; Hot Temperature; Nutritive Value; Oils; Phytosterols; Sitosterols; Steroids; Stigmasterol

Cell mortality is a key mechanism that shapes phytoplankton blooms and species dynamics in aquatic environments. Here we show that sterol sulfates (StS) are regulatory molecules of a cell death program in Skeletonema marinoi, a marine diatom-blooming species in temperate coastal waters. The molecules trigger an oxidative burst and production of nitric oxide in a dose-dependent manner. The intracellular level of StS increases with cell ageing and ultimately leads to a mechanism of apoptosis-like death. Disrupting StS biosynthesis by inhibition of the sulfonation step significantly delays the onset of this fatal process and maintains steady growth in algal cells for several days. The autoinhibitory activity of StS demonstrates the functional significance of small metabolites in diatoms. The StS pathway provides another view on cell regulation during bloom dynamics in marine habitats and opens new opportunities for the biochemical control of mass-cultivation of microalgae.

Topics: Cholestadienols; Cholesterol Esters; Diatoms; Ecosystem; Eutrophication; Microalgae; Phylogeny; Phytoplankton; Phytosterols; Signal Transduction; Sitosterols; Sterols; Sulfates; Sulfotransferases

Compounds with estrogenic potencies and their adverse effects in surface waters have received much attention. Both anthropogenic and natural compounds contribute to overall estrogenic activity in freshwaters. Recently, estrogenic potencies were also found to be associated with cyanobacteria and their blooms in surface waters. The present study developed and compared the solid phase extraction and LC-MS/MS analytical approaches for determination of phytoestrogens (8 flavonoids - biochanin A, coumestrol, daidzein, equol, formononetin, genistein, naringenin, apigenin - and 5 sterols - ergosterol, β-sitosterol, stigmasterol, campesterol, brassicasterol) and cholesterol in water. The method was used for analyses of samples collected in stagnant water bodies dominated by different cyanobacterial species. Concentrations of individual flavonoids ranged from below the limit of detection to 3.58 ng/L. Sterols were present in higher amounts up to 2.25 μg/L. Biological potencies of these phytoestrogens in vitro were characterized using the hERα-HeLa-9903 cell line. The relative estrogenic potencies (compared to model estrogen - 17β-estradiol) of flavonoids ranged from 2.25E-05 to 1.26E-03 with coumestrol being the most potent. None of the sterols elicited estrogenic response in the used bioassay. Estrogenic activity was detected in collected field water samples (maximum effect corresponding to 2.07 ng/L of 17β-estradiol equivalents, transcriptional assay). At maximum phytoestrogens accounted for only 1.56 pg/L of 17β-estradiol equivalents, contributing maximally 8.5% of the total estrogenicity of the water samples. Other compounds therefore, most likely of anthropogenic origin such as steroid estrogens, are probably the major drivers of total estrogenic effects in these surface waters.

Topics: Cholestadienols; Cholesterol; Cyanobacteria; Estradiol; Estrogens; Estrone; Fresh Water; Genistein; HeLa Cells; Humans; Isoflavones; Phytoestrogens; Phytosterols; Receptors, Estrogen; Sitosterols; Sterols; Tandem Mass Spectrometry; Water; Water Pollutants, Chemical

Pneumolysin is the one of the major virulence factor of the bacterium Streptococcus pneumoniae. In previous report, it is shown that β-sitosterol, a natural compound without antimicrobial activity, is a potent antagonist of pneumolysin. Here, two new pneumolysin natural compound inhibitors, with differential activity, were discovered via haemolysis assay. To explore the key factor of the conformation for the inhibition activity, the interactions between five natural compound inhibitors with differential activity and pneumolysin were reported using molecular modelling, the potential of mean force profiles. Interestingly, it is found that incorporation of the single bond (C22-C23-C24-C25) to replace the double bond (hydrocarbon sidechain) improved the anti-haemolytic activity. In view of the molecular modelling, binding of the five inhibitors to the conserved loop region (Val372, Leu460, and Tyr461) of the cholesterol binding sites led to stable complex systems, which was consistent with the result of β-sitosterol. Owing to the single bond (C22-C23-C24-C25), campesterol and brassicasterol could form strong interactions with Val372 and show higher anti-haemolytic activity, which indicated that the single bond (C22-C23-C24-C25) in inhibitors was required for the anti-haemolytic activity. Overall, the current molecular modelling work provides a starting point for the development of rational design and higher activity pneumolysin inhibitors.

Topics: Animals; Bacterial Proteins; Cholestadienols; Cholesterol; Hemolysis; Models, Molecular; Molecular Dynamics Simulation; Phytosterols; Sheep; Streptococcus pneumoniae; Streptolysins; Structure-Activity Relationship

Sterols are components present in the fat fraction of infant formulas (IFs). Their characterization is therefore of interest, though there are no official reference methods for their analysis in these matrices.. To validate a gas chromatographic method with flame ionization detection for the determination of animal (cholesterol and desmosterol) and plant sterols (brassicasterol, campesterol, stigmasterol, β-sitosterol and sitostanol) found in IFs. All correlation coefficients obtained for the calibration curves of sterols studied were >0.99. Limits of detection (<1 μg/100 mL) and quantification (<4 μg/100 mL) are suitable for sterols determination in IFs. The within-assay precision ranged from 1.6% to 8.8%, while the between-assay precision was <10% for most of sterols. Accuracy was satisfactory and was calculated by recovery assays (ranging 93-108%). The analytical parameters obtained showed the suitability of the proposed method for the determination of sterols in IFs.

Topics: Calibration; Cholestadienols; Cholesterol; Chromatography, Gas; Desmosterol; Flame Ionization; Infant Formula; Limit of Detection; Phytosterols; Reproducibility of Results; Sitosterols; Stigmasterol

Topics: Cholestadienols; Cholesterol; Chromatography, High Pressure Liquid; Edible Grain; Factor Analysis, Statistical; Fruit and Vegetable Juices; Humans; Limit of Detection; Liquid Phase Microextraction; Phytosterols; Pisum sativum; Poaceae; Reproducibility of Results; Sitosterols; Stigmasterol; Tandem Mass Spectrometry

We performed comparative DSC and FTIR spectroscopic measurements of the effects of β-sitosterol (Sito) and stigmasterol (Stig) on the thermotropic phase behavior and organization of DPPC bilayers. Sito and Stig are the major sterols in the biological membranes of higher plants, whereas cholesterol (Chol) is the major sterol in mammalian membranes. Sito differs in structure from Chol in having an ethyl group at C24 of the alkyl side-chain, and Stig in having both the C24 ethyl group and trans-double bond at C22. Our DSC studies indicate that the progressive incorporation of Sito and Stig decrease the temperature and cooperativity of the pretransition of DPPC to a slightly lesser and greater extent than Chol, respectively, but the pretransition persists to 10 mol % sterol concentration in all cases. All three sterols produce essentially identical effects on the thermodynamic parameters of the sharp component of the DPPC main phase transition. However, the ability to increase the temperature and decrease the cooperativity and enthalpy of the broad component decreases in the order Chol>Sito>Stig. Nevertheless, at higher Sito/Stig concentrations, there is no evidence of sterol crystallites. Our FTIR spectroscopic studies demonstrate that Sito and especially Stig incorporation produces a smaller ordering of the hydrocarbon chains of fluid DPPC bilayers than does Chol. In general, the presence of a C24 ethyl group in the alkyl side-chain reduces the characteristic effects of Chol on the thermotropic phase behavior and organization of DPPC bilayer membranes, and a trans-double bond at C22 magnifies this effect.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Calorimetry, Differential Scanning; Cholestadienols; Cholesterol; Lipid Bilayers; Molecular Structure; Phase Transition; Phytosterols; Sitosterols; Spectroscopy, Fourier Transform Infrared; Stigmasterol; Temperature

The addition of plant sterols/stanols (sterols or stanols) can reduce the solubilization of cholesterol in a model intestinal solution system. We studied the molecular structure of seven different sterols/stanols and the effect they had on the solubilization of cholesterol or cholesterol ester in a model intestinal solution. The differences in the molecular structures of the sterol/stanol species influenced their abilities to reduce the solubility of cholesterol in the competitive solubilization experiments. Cholestanol whose molecular structure resembled cholesterol was the most effective at reducing the solubilization of cholesterol and cholesterol ester, with the solubilities of cholesterol and cholesteryl oleate being 41% and 39% respectively of the values observed for the single solubilizate systems. β-Sitosterol was also able to reduce the solubilities of cholesterol and cholesteryl oleate to 43% and 45% of those observed in a single solubilizate system. Both, stigmasterol and brassicasterol have an unsaturated double bond in a steroid side chain and did not exhibit major cholesterol-lowering effects. These results were reflected by the Gibbs free energy change values (ΔG(0)) for solubilization, where the sterol/stanol species with cholesterol-lowering effects had similar or larger negative ΔG(0) values than those observed for cholesterol.

Topics: Body Fluids; Cholestadienols; Cholestanol; Cholesterol; Cholesterol Esters; Intestines; Models, Biological; Molecular Structure; Phytosterols; Sitosterols; Solubility; Stigmasterol; Structure-Activity Relationship

The hydrogen isotope (2H/1H) ratio of lipids from phytoplankton is a powerful new tool for reconstructing hydroclimate variations in the geologic past from marine and lacustrine sediments. Water 2H/1H changes are reflected in lipid 2H/1H changes with R2 > 0.99, and salinity variations have been shown to cause about a 1‰ change in lipid δ2H values per unit (ppt) change in salinity. Less understood are the effects of growth rate, nutrient limitation and light on 2H/1H fractionation in phytoplankton. Here we present the first published study of growth rate effects on 2H/1H fractionation in the lipids of coccolithophorids grown in continuous cultures. Emiliania huxleyi was cultivated in steady state at four growth rates and the δ2H value of individual alkenones (C37:2, C37:3, C38:2, C38:3), fatty acids (C14:0, C16:0, C18:0), and 24-methyl cholest-5,22-dien-3β-ol (brassicasterol) were measured. 2H/1H fractionation increased in all lipids as growth rate increased by 24‰ to 79‰ (div d-1)-1. We attribute this response to a proportional increase in the fraction of NADPH from Photosystem I (PS1) of photosynthesis relative to NADPH from the cytosolic oxidative pentose phosphate (OPP) pathway in the synthesis of lipids as growth rate increases. A 3-endmember model is presented in which lipid hydrogen comes from NADPH produced in PS1, NADPH produced by OPP, and intracellular water. With published values or best estimates of the fractionation factors for these sources (αPS1 = 0.4, αOPP = 0.75, and αH2O = 0) and half of the hydrogen in a lipid derived from water the model indicates αlipid = 0.79. This value is within the range measured for alkenones (αalkenone = 0.77 to 0.81) and fatty acids (αFA = 0.75 to 0.82) in the chemostat cultures, but is greater than the range for brassicasterol (αbrassicasterol = 0.68 to 0.72). The latter is attributed to a greater proportion of hydrogen from NADPH relative to water in isoprenoid lipids. The model successfully explains the increase in 2H/1H fractionation in the sterol 24-methyl-cholesta-5,24(28)-dien-3β-ol from marine centric diatom T. pseudonana chemostat cultures as growth rate increases. Insensitivity of αFA in those same cultures may be attributable to a larger fraction of hydrogen in fatty acids sourced from intracellular water at the expense of NADPH as growth rate increases. The high sensitivity of α to growth rate in E. huxleyi lipids and a T. pseudonana sterol implies that any change in growth rate larger than ~0.15 di

Topics: Cholestadienols; Deuterium; Diatoms; Haptophyta; Lipid Metabolism; Phytosterols; Salinity

New and sustainable sources of long-chain (LC, ≥C₂₀) omega-3 oils containing DHA (docosahexaenoic acid, 22:6ω3) are required to meet increasing demands. The lipid content of the oilseed of a novel transgenic, DHA-producing land plant, Camelina sativa, containing microalgal genes able to produce LC omega-3 oils, contained 36% lipid by weight with triacylglycerols (TAG) as the major lipid class in hexane extracts (96% of total lipid). Subsequent chloroform-methanol (CM) extraction recovered further lipid (~50% polar lipid, comprising glycolipids and phospholipids) and residual TAG. The main phospholipid species were phosphatidyl choline and phosphatidyl ethanolamine. The % DHA was: 6.8% (of total fatty acids) in the TAG-rich hexane extract and 4.2% in the polar lipid-rich CM extract. The relative level of ALA (α-linolenic acid, 18:3ω3) in DHA-camelina seed was higher than the control. Major sterols in both DHA- and control camelina seeds were: sitosterol, campesterol, cholesterol, brassicasterol and isofucosterol. C₁₆-C₂₂ fatty alcohols, including iso-branched and odd-chain alcohols were present, including high levels of iso-17:0, 17:0 and 19:0. Other alcohols present were: 16:0, iso-18:0, 18:0 and 18:1 and the proportions varied between the hexane and CM extracts. These iso-branched odd-chain fatty alcohols, to our knowledge, have not been previously reported. These components may be derived from wax esters, or free fatty alcohols.

Topics: Brassicaceae; Cholestadienols; Cholesterol; Fatty Acids, Omega-3; Gas Chromatography-Mass Spectrometry; Phospholipids; Phytosterols; Plant Oils; Plants, Genetically Modified; Seeds; Sitosterols; Stigmasterol; Triglycerides

We present a comparative differential scanning calorimetric study of the effects of the animal sterol cholesterol (Chol) and the plant sterols campesterol (Camp) and brassicasterol (Bras) on the thermotropic phase behavior of dipalmitoylphosphatidylcholine (DPPC) bilayers. Camp and Bras differ from Chol in having a C24 methyl group and, additionally for Bras, a C22 trans-double bond. Camp and especially Bras decrease the temperature, cooperativity and enthalpy of the DPPC pretransition more than Chol, although these effects are attenuated at higher sterol levels. This indicates that they destabilize gel-state DPPC bilayers to a greater extent, but are less soluble, than Chol. Not surprisingly, all three sterols have similar effects on the sterol-poor sharp component of the DPPC main phase transition. However, Camp and especially Bras less effectively increase the temperature and decrease the cooperativity and enthalpy of the broad component of the main transition than Chol. This indicates that at higher sterol concentrations, Camp and Bras are less miscible and less effective than Chol at ordering the hydrocarbon chains of the sterol-enriched fluid DPPC bilayers. Overall, these alkyl side chain modifications generally reduce the ability of Chol to produce its characteristic effects on DPPC bilayer physical properties. These differences are likely due to the less extended and more bent conformations of the alkyl side chains of Camp and Bras, producing sterols with a greater effective cross-sectional area and reduced length than Chol. Hence, the structure of Chol is likely optimized for maximum solubility in, as opposed to maximum ordering of, phospholipid bilayers.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Calorimetry, Differential Scanning; Cholestadienols; Cholesterol; Lipid Bilayers; Membranes; Models, Molecular; Phase Transition; Phytosterols; Temperature

A new method, reversed phase liquid chromatography with off-line surface-assisted laser desorption/ionization mass spectrometry (RPLC-SALDI MS) for the determination of brassicasterol (BR), cholesterol (CH), stigmasterol (ST), campesterol (CA) and β-sitosterol (SI) in oil samples has been developed. The sample preparation consisted of alkaline saponification followed by extraction of the unsaponificable fraction with diethyl ether. The recovery of the sterols ranged from 91 to 95% with RSD less than 4%. Separation of the five major sterols on a C18 column using methanol-water gradient was achieved in about 10min. An on-line UV detector was employed for the initial sterol detection prior to effluent deposition using a laboratory-built spotter with 1:73 splitter. Off-line SALDI MS was then applied for mass determination/identification and quantification of the separated sterols. Ionization of the nonpolar analytes was achieved by silver ion cationization with silver nanoparticles used as the SALDI matrix providing limits of detection 12, 6 and 11fmol for CH, ST and SI, respectively. Because of the incorporated splitter, the effective limits of detection of the RPLC-SALDI MS analysis were 4, 3 and 4pmol (or 0.08, 0.06 and 0.08μg/mL) for CH, ST and SI, respectively. For quantification, 6-ketocholestanol (KE) was used as the internal standard. The method has been applied for the identification and quantification of sterols in olive, linseed and sunflower oil samples. The described off-line coupling of RPLC to SALDI MS represents an alternative to GC-MS for analysis of nonpolar compounds.

Topics: Cholestadienols; Cholesterol; Chromatography, Reverse-Phase; Ketocholesterols; Linseed Oil; Olive Oil; Phytosterols; Plant Oils; Reference Standards; Silver; Sitosterols; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stigmasterol; Sunflower Oil

Previous methods for the quantitative analysis of phytosterols have usually used GC-MS and require elaborate sample preparation including chemical derivatization. Other common methods such as HPLC with absorbance detection do not provide information regarding the identity of the analytes. To address the need for an assay that utilizes mass selectivity while avoiding derivatization, a quantitative method based on LC-tandem mass spectrometry (LC-MS-MS) was developed and validated for the measurement of six abundant dietary phytosterols and structurally related triterpene alcohols including brassicasterol, campesterol, cycloartenol, β-sitosterol, stigmasterol, and lupeol in edible oils. Samples were saponified, extracted with hexane and then analyzed using reversed phase HPLC with positive ion atmospheric pressure chemical ionization tandem mass spectrometry and selected reaction monitoring. The utility of the LC-MS-MS method was demonstrated by analyzing 14 edible oils. All six compounds were present in at least some of the edible oils. The most abundant phytosterol in all samples was β-sitosterol, which was highest in corn oil at 4.35 ± 0.03 mg/g, followed by campesterol in canola oil at 1.84 ± 0.01 mg/g. The new LC-MS-MS method for the quantitative analysis of phytosterols provides a combination of speed, selectivity and sensitivity that exceed those of previous assays.

Topics: Cholestadienols; Cholesterol; Chromatography, Liquid; Molecular Structure; Pentacyclic Triterpenes; Phytosterols; Plant Oils; Reproducibility of Results; Sitosterols; Stigmasterol; Tandem Mass Spectrometry; Triterpenes

Ethyl acetate extracts of Armillaria tabescens (strain JNB-OZ344) showed significant fungistatic and bacteristatic activities against several major human pathogens including Candida albicans, Cryptococcus neoformans, Escherichia coli and Mycobacterium intracellulare. Chemical analysis of these extracts led to the isolation and identification of four new compounds, emestrin-F (1), emestrin-G (2), 6-O-(4-O-methyl-β-D-glucopyranosyl)-8-hydroxy-2,7-dimethyl-4H-benzopyran-4-one (3) and cephalosporolide-J (4), along with five other previously known compounds, emestrin (5), cephalosporolide-E (6), decarestrictine-C2 (7), ergosterol and brassicasterol. Structural elucidation of all compounds was carried out by NMR and MS analyses. Antimicrobial assays revealed that compounds 1 and 5 were responsible for the observed growth inhibitory activities of the fungal extracts against the human pathogens tested.

Topics: Anti-Bacterial Agents; Antifungal Agents; Armillaria; Candida albicans; Cholestadienols; Cryptococcus neoformans; Ergosterol; Escherichia coli; Magnetic Resonance Spectroscopy; Mass Spectrometry; Microbial Sensitivity Tests; Phytosterols

A simple method for the determination of cellular uptake of phytosterols by Caco-2 cells has been developed by ultra performance liquid chromatography with ultraviolet detection (UPLC-UV). UPLC-UV was established using an ODS column, acetonitrile/H(2)O (9:1, v/v) as a mobile phase, and a detection wavelength at 210 nm. As analytes, β-sitosterol, campesterol, stigmasterol, and brassicasterol were selected based on the abundance in foods and the similarity of their structures. A linear relation was observed between the peak area and the amount of sterol injected from 50 to 2000 pmol (r>0.999) with a relative standard deviation (RSD) of less than 2.5% (n=6). This method was applied to the determination of cellular uptake of phytosterols by Caco-2 cells. Recovery tests showed that phytosterols were extracted from the cell lysates by chloroform and determined by UPLC-UV with a recovery rate of more than 80.2% and an RSD of less than 11.3% (n=3). When Caco-2 cells were incubated with phytosterols at 37°C, their uptake was increased with time in a concentration-dependent manner. This method will be useful for the simultaneous determination of cellular phytosterols in an in vitro intestine model.

Topics: Biological Transport, Active; Caco-2 Cells; Cholestadienols; Cholesterol; Chromatography, Liquid; Humans; Kinetics; Phytosterols; Sitosterols; Stigmasterol

Orobanche and Phelipanche species (the broomrapes) are root parasitic plants, some of which represent serious weed problems causing severe yield losses on important crops. Control strategies have largely focused on agronomic practices, resistant crop varieties and herbicides, albeit with marginal success. An alternative control method is the induction of suicidal seed germination with natural substances isolated from root exudates of host and non-host plants.. Soyasapogenol B [olean-12-ene-3,22,24-triol(3β,4β,22β)] and trans-22-dehydrocampesterol [(ergosta-5,22-dien-3-ol, (3β,22E,24S)] were isolated from Vicia sativa root exudates. They were identified by comparing their spectroscopic and optical properties with those reported in the literature. Soyasapogenol B was very specific, stimulating the germination of O. minor seeds only, whereas trans-22-dehydrocampesterol stimulated P. aegyptiaca, O. crenata, O. foetida and O. minor.. Soyasapogenol B and trans-22-deydrocampesterol were isolated for the first time from Vicia sativa root exudates, and their biological activity as stimulants of Orobanche or Phelipanche sp. seed germination was reported.

Topics: Cholestadienols; Germination; Magnetic Resonance Spectroscopy; Molecular Conformation; Oleanolic Acid; Orobanche; Phytosterols; Plant Exudates; Plant Roots; Saponins; Spectrometry, Mass, Electrospray Ionization; Stereoisomerism; Vicia sativa

The first committed step in the formation of 24-alkylsterols in the ascomycetous fungus Paracoccidiodes brasiliensis (Pb) has been shown to involve C24-methylation of lanosterol to eburicol (24(28)-methylene-24,25-dihydro-lanosterol) on the basis of metabolite co-occurrence. A similarity-based cloning strategy was employed to obtain the cDNA clone corresponding to the sterol C24-methyltransferase (SMT) implicated in the C24-methylation reaction. The resulting catalyst, prepared as a recombinant fusion protein (His/Trx/S), was expressed in Escherichia coli BL21(C43) and shown to possess a substrate specificity for lanosterol and to generate a single exocyclic methylene product. The full-length cDNA has an open reading frame of 1131 base pairs and encodes a protein of 377 residues with a calculated molecular mass of 42,502Da. The enzymatic C24-methylation gave a K(mapp) of 38microM and k(catapp) of 0.14min(-1). Quite unexpectedly, "plant" cycloartenol was catalyzed in high yield to 24(28)-methylene cycloartanol consistent with conformational arguments that favor that both cycloartenol and lanosterol are bound pseudoplanar in the ternary complex. Incubation of [27-(13)C]- or [24-(2)H]cycloartenol with PbSMT and analysis of the enzyme-generated product by a combination of (1)H and (13)CNMR and mass spectroscopy established the regiospecific conversion of the pro-Z methyl group of the Delta(24(25))-substrate to the pro-R isopropyl methyl group of the product and the migration of H24 to C25 on the Re-face of the original substrate double bond undergoing C24-methylation. Inhibition kinetics and products formed from the substrate analogs 25-azalanosterol (K(i) 14nM) and 26,27-dehydrolanosterol (K(i) 54muM and k(inact) of 0.24min(-1)) provide direct evidence for distinct reaction channeling capitalized by structural differences in the C24- and C26-sterol acceptors. 25-Azalanosterol was a potent inhibitor of cell growth (IC(50), 30nM) promoting lanosterol accumulation and 24-alkyl sterol depletion. Phylogenetic analysis of PbSMT with related SMTs of diverse origin together with the results of the present study indicate that the enzyme may have a similar complement of active-site amino acid residues compared to related yeast SMTs affording monofunctional C(1)-transfer behavior, yet there are sufficient differences in its overall amino acid composition and substrate-dependent partitioning pathways to group PbSMT into a fourth and new class of SMT.

Topics: Alkylation; Amino Acid Sequence; Biocatalysis; Cholestadienols; Chromatography, Gas; Chromatography, High Pressure Liquid; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Lanosterol; Metabolic Networks and Pathways; Methyltransferases; Molecular Sequence Data; Mutagenesis, Site-Directed; Paracoccidioides; Phylogeny; Phytosterols; Recombinant Proteins; Reproducibility of Results; Sequence Alignment; Sequence Analysis, DNA; Substrate Specificity; Tyrosine

A comparative study was performed to determine the free sterols content and composition during the development of three varieties of linseed (H52, O116 and P129). Seed samples were collected at regular intervals from 7 to 60 days after flowering (DAF). Ten compounds were identified: cholesterol, campesterol, brassicasterol, stigmasterol, beta-sitosterol, Delta5-avenasterol, cycloartenol; 24-methylene cycloartanol, obtusifoliol, citrostadienol. The maximum level of 4-desmethylsterols (1,515 mg/100g oil) was reached at 7 DAF in P129 variety. H52 had the highest level of 4-4 dimethylsterols (355 mg/100g oil) at 28 DAF. The greatest amount of 4-monomethylsterols (35 mg/100g oil) was detected in H52 at 14 DAF. During linseed development, beta sitosterol (830 mg/100g oil) was the major 4-desmethylsterols, followed by campesterol (564 mg/100g oil) and stigmasterol (265 mg/100g oil). Some of these compounds followed nearly the same accumulation pattern during linseed maturation.

Topics: Cholestadienols; Cholesterol; Chromatography, Thin Layer; Flax; Flowers; Gas Chromatography-Mass Spectrometry; Phytosterols; Seeds; Sitosterols; Species Specificity; Stigmasterol; Time Factors; Triterpenes

Rapeseed oil (RSO) is a novel source of plant sterols, containing the unique brassicasterol in concentrations higher than allowed for plant sterol blends in food products in the European Union. Effects of RSO sterols and stanols on aortic atherosclerosis were studied in cholesterol-fed heterozygous Watanabe heritable hyperlipidaemic (Hh-WHHL) rabbits. Four groups (n 18 per group) received a cholesterol-added (2 g/kg) standard chow or this diet with added RSO stanol esters (17 g/kg), RSO stanol esters (34 g/kg) or RSO sterol esters (34 g/kg) for 18 weeks. Feeding RSO stanol esters increased plasma campestanol (P < 0.001) and sitostanol (P < 0.001) and aortic campestanol (P < 0.05) compared with controls. Feeding RSO sterol esters increased concentrations of plasma campesterol (P < 0.001), sitosterol (P < 0.001) and brassicasterol (P < 0.001) and aortic campesterol (P < 0.01). Significantly lower plasma cholesterol (P < 0.001) was recorded in the treated groups after 3 weeks and throughout the study. LDL-cholesterol was reduced 50 % in the high-dose RSO sterol ester (P < 0.01) and high-dose RSO stanol ester (P < 0.001) groups compared with controls. Atherosclerotic lesions were found in three rabbits in each of the RSO stanol ester groups and in one in the RSO sterol ester group. Aortic cholesterol was decreased in the treated groups (P < 0.001) in response to lowering of plasma cholesterol induced by RSO sterol and stanol esters. In conclusion, RSO stanol and sterol esters with a high concentration of brassicasterol were well tolerated. They were hypocholesterolaemic and inhibited experimental atherosclerosis in cholesterol-fed Hh-WHHL rabbits. A significant uptake of plant sterols into the blood and incorporation of campesterol and campestanol into aortic tissue was recorded.

Topics: Animals; Aorta; Atherosclerosis; Cholestadienols; Cholesterol; Cholesterol, Dietary; Fatty Acids, Monounsaturated; Female; Heterozygote; Hyperlipidemias; Lipids; Male; Phytosterols; Plant Oils; Rabbits; Rapeseed Oil; Sitosterols

We elucidated the molecular mechanism(s) underlying sterol trafficking by investigating alterations in gene expression in response to increased retention of dietary phytosterols and phytostanols in stroke-prone spontaneously hypertensive (SHRSP) and normotensive Wistar Kyoto (WKY) inbred rats.. SHRSP and WKY inbred rats were fed a control diet or a diet supplemented with phytosterols or phytostanols (2 g/kg diet).. Intake of phytosterols and phytostanols increased their incorporation in plasma, red blood cells, liver, aorta and kidney, but decreased cholesterol levels in liver and aorta in both rat strains. Phytosterol intake up-regulated mRNA expression of intestinal Npc1l1 and Abcg8, and hepatic Abcg5, Abca1, Cyp27a1 and Hmgcr. Phytostanol intake up-regulated Npc1l1 and Srebp2, but down-regulated Abcg5 mRNA expression in small intestine. Phytostanols also up-regulated Abca1 expression in SHRSP rats, but down-regulated Abca1 expression in WKY inbred rats. Compared to phytosterols, dietary phytostanols reduced phytosterol levels in plasma, red blood cells, and kidney, as well as altered mRNA levels of hepatic Abca1,Cyp27a1, and Hmgcr and intestinal Abcg5/8, Hmgcr and Srebp2.. Altered expression of multiple sterol-regulatory genes may contribute to the incorporation and cholesterol-lowering actions of phytosterols and phytostanols. Phytosterols and phytostanols may act through different mechanism(s) on cholesterol and phytosterol/phytostanol trafficking.

Topics: Animals; Anticholesteremic Agents; Cholestadienols; Cholesterol; Gene Expression Regulation; Hypolipidemic Agents; Jejunum; Liver; Male; Organ Specificity; Phytosterols; Rats; Rats, Inbred SHR; Rats, Inbred WKY; RNA, Messenger; Sitosterols; Sterols

Cholesterol and plant phytosterols are lipophilic compounds solubilized by intestinal micelles in a competitive manner. In this work, we used radioactive cholesterol- and phytosterol-loaded oil-in-water microemulsions to follow their incorporation and mutual competition in HaCaT keratinocytes, SZ95 sebocytes, and skin pieces in cultures. Dynamic light scattering showed homogenous nanostructures of 10.5+/-1.5 nm diameter and cryo-transmission electron microscopy confirmed the presence of uniform spherical droplets of 7.0+/-1.0 nm diameter. Up to 320 nmol/ml of cholesterol can be solubilized and transported into cells with minimal toxic effect by 0.5 wt% nanodroplets in a cell medium. Phytosterols inhibit incorporation of cholesterol into cells, in vitro, at molar ratios (phytosterols/cholesterol) of 4 and above. The loaded nanodroplets accumulate in intracellular vesicles (presumably endosomes). No metabolic conversion of cholesterol or phytosterols was found in these cells, in vitro, after 24 h, at 37 degrees C.

Topics: Cell Survival; Cells, Cultured; Cholestadienols; Cholesterol; Cryoelectron Microscopy; Emulsions; Humans; Keratinocytes; Microscopy, Electron, Transmission; Phytosterols; Sitosterols

The selectivity of the antibiotic nystatin towards ergosterol compared to cholesterol is believed to be a crucial factor in its specificity for fungi. In order to define the structural features of sterols that control this effect, nystatin interaction with ergosterol-, cholesterol-, brassicasterol- and 7-dehydrocholesterol-containing palmitoyloleoylphosphocholine vesicles was studied by fluorescence spectroscopy. Variations in sterol structure were correlated with their effect on nystatin photophysical and activity properties. Substitution of cholesterol by either 7-dehydrocholesterol or brassicasterol enhance nystatin ability to dissipate a transmembrane K+ gradient, showing that the presence of additional double bonds in these sterols-carbon C7 and C22, plus an additional methyl group on C-24, respectively-as compared to cholesterol, is fundamental for nystatin-sterol interaction. However, both modifications of the cholesterol molecule, like in the fungal sterol ergosterol, are critical for the formation of very compact nystatin oligomers in the lipid bilayer that present a long mean fluorescence lifetime and induce a very fast transmembrane dissipation. These observations are relevant to the molecular mechanism underlying the high selectivity presented by nystatin towards fungal cells (with ergosterol) as compared to mammalian cells (with cholesterol).

Topics: Cholestadienols; Cholesterol; Dehydrocholesterols; Ergosterol; Kinetics; Liposomes; Nystatin; Permeability; Phosphatidylcholines; Phytosterols; Spectrometry, Fluorescence; Sterols

Intestinal absorption of various plant sterols was investigated in thoracic duct-cannulated normal rats. Lymphatic recovery was the highest in campesterol, intermediate in brassicasterol and sitosterol, and the lowest in stigmasterol and sitostanol. Higher solubility in the bile salt micelle was observed in sitosterol, campesterol, and sitostanol than in brassicasterol and stigmasterol. The solubility of the latter two sterols was extremely low. When the affinity of plant sterols for the bile salt micelle was compared in an in vitro model system, which assessed sterol transfer from the micellar to the oil phase, the transfer rate was the highest in brassicasterol, intermediate in campesterol and stigmasterol, and lowest in sitosterol and sitostanol. Although no significant correlations between lymphatic recovery of plant sterols and their micellar solubility or transfer rate from the bile salt micelle were observed, highly positive correlation was obtained between the lymphatic recovery and the multiplication value of the micellar solubility and the transfer rate. These observations strongly suggest that both solubility in and affinity for the bile salt micelle of plant sterols are important determinants of their intestinal absorption in rats.

Topics: Animals; Bile Acids and Salts; Cholestadienols; Cholesterol; Intestinal Absorption; Lymph; Male; Micelles; Models, Biological; Phytosterols; Rats; Rats, Wistar; Sitosterols; Solubility; Stigmasterol; Triolein

Four sterols were isolated from a brown alga endophytic fungus NO. ZZF36 from the South China Sea. Their structures were identified as brassicaterol(2), ergosterol(2), ergosterol peroxide(3),7,22(E)-ergostadiene-beta,5alpha,6beta-triol(4) by spectroscopic methods.

Topics: Cholestadienols; Ergosterol; Fungi; Magnetic Resonance Spectroscopy; Molecular Structure; Phytosterols; Sargassum; Sterols

A novel analytical platform based on liquid chromatography and tandem mass spectrometry using atmospheric pressure photoionization was applied for the simultaneous quantification of free and esterified beta-sitosterol, campesterol, brassicasterol, and stigmasterol. The total time for sample pretreatment and analysis could be reduced from approximately 3 h [gas chromatography-mass spectrometry (GC-MS)] to 15 min. The detection limits of the different phytosterols ranged between 0.25 and 0.68 microg/l. Linear ranges were between 1 and 1,000 microg/l. The within-run and between-run variabilities ranged between 1.4% and 9.9%. The analytical sensitivity was at least 150-fold higher compared with GC-MS. Our new method allows a rapid and simultaneous determination of free and esterified phytosterols in serum.

Topics: Cholestadienols; Cholesterol; Chromatography, Liquid; Esters; Humans; Mass Spectrometry; Methods; Phytosterols; Reproducibility of Results; Sitosterols; Stigmasterol

The effect of Ganoderma total sterol (GS) and its main components(GS(1)) on rat cortical neuronal cultures exposed to hypoxia/reoxygenation (H/R) was studied in vitro. GS (0.01,0.1,1 microg/ml) increased neuron viability following H/R. GS also significantly reduced malondialdehyde content and reactive oxygen species production and increased manganese superoxide dismutase (Mn-SOD) activity; furthermore, the translocation of nuclear factor-kappa B and the production of interleukin-1beta and tumor necrosis factor alpha induced by H/R were also blocked. These findings suggest that GS might be useful in treating H/R-induced oxidative stress and inflammatory response. We also hypothesized that Mn-SOD might play a critical role in the neuroprotective effect of GS against H/R injury. In addition, pretreatment with GS(1) (0.01, 0.1, 1 microg/ml) significantly attenuated the decline of neuron viability and the formation of reactive oxygen species. Furthermore GS(1) possessed more potent protective effect on neurons compared with GS at the same dose. These findings demonstrated that GS(1) is the main component in GS; and play a critical role in the neuroprotective effect of GS against H/R.

Topics: Animals; Cell Hypoxia; Cell Survival; Cells, Cultured; Cerebral Cortex; Cholestadienols; Cytokines; Ganoderma; I-kappa B Proteins; Neuroprotective Agents; NF-kappa B; NF-KappaB Inhibitor alpha; Oxidative Stress; Phytosterols; Rats; Rats, Sprague-Dawley; Superoxide Dismutase

Sterol composition and content and their seasonal variations over 18 months were investigated in adductor muscle, digestive gland and gonads of Pecten maximus. Sterols were isolated by Silicagel 60 thin layer chromatography and identified by gas chromatography/mass spectrometry. Eleven sterols were identified, with cholesterol, brassicasterol, 24-methylenecholesterol and 22-trans-dehydrocholesterol being the principal components. The same sterols were found in all three tissues independent of season. The relative amounts of each sterol present in each tissue differed. Total sterol levels in gonad and muscle were higher than in digestive gland. Statistically significant differences (P<0.05) were found between the concentrations of each of the sterols isolated from the gonad or muscle and digestive gland. The seasonal variations in the sterol content of the gonad seem be related to the reproductive cycle, while the sterol content of the digestive gland appears to be linked to diet, mainly diatoms or dinoflagellates. The muscle sterol content showed minor changes throughout the year.

Topics: Animals; Cholestadienols; Cholesterol; Chromatography, Thin Layer; Dehydrocholesterols; Digestive System; Gas Chromatography-Mass Spectrometry; Gonads; Isomerism; Mollusca; Muscle, Skeletal; Phytosterols; Seasons; Spain; Sterols

Topics: Animals; Cholestadienols; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Female; Magnetic Resonance Spectroscopy; Phytosterols; Pneumocystis; Rats; Rats, Inbred Lew; Sterols; Stigmasterol

Isoprenoid biosynthesis was investigated in the two diatoms Phaeodactylum tricornutum and Nitzschia ovalis by labeling experiments performed in mixotrophic growth conditions with sodium [1-(13)C]acetate, 13CO2, [1-(13)C]glucose, sodium [3-(13)C]pyruvate and 1-deoxy-D-[5,5-(2)H2]xylulose. A clear dichotomy was found. Acetate was the preferred carbon source for the formation of the sterols in the cytoplasm via the mevalonate pathway. Carbon dioxide was the main source for phytol biosynthesis in the chloroplasts via the mevalonate-independent methylerythritol 4-phosphate pathway. The two diatoms showed the same compartmentation for isoprenoid biosynthesis as that previously found in higher plants, the red alga Porphyridium cruentum and the Chrysophyte Ochromonas danica.

Topics: Carbon Dioxide; Cholestadienols; Diatoms; Erythritol; Gas Chromatography-Mass Spectrometry; Mevalonic Acid; Phytol; Phytosterols; Sugar Phosphates

The 5alpha-hydroperoxides of beta-sitosterol, campesterol, stigmasterol, and brassicasterol were obtained by photooxidation of the respective sterols in pyridine in the presence of hematoporphyrine as sensitizer. The reduction of the hydroperoxides gives the corresponding 5alpha-hydroxy derivatives. The 7alpha- and 7beta-hydroperoxides of the sterols were obtained by allowing an aliquot of the 5alpha-hydroperoxides to isomerize to 7alpha-hydroperoxides, which in turn epimerize to 7beta-hydroperoxides. The reduction gave the corresponding 7alpha- and 7beta-hydroxy derivatives. The 5alpha-, 7alpha-, and 7beta-hydroxy derivatives of beta-sitosterol, campesterol, stigmasterol, and brassicasterol were identified by comparing thin-layer chromatography mobilities, specific color reactions, and mass spectral data with those of the corresponding hydroxy derivatives of cholesterol, which were synthesized in the same manner. The phytosterols had the same behavior to photooxidation as cholesterol and, moreover, the different phytosterols photooxidized at about the same rate. The mass spectra of the trimethylsilyl ethers of the hydroxy derivatives of the phytosterols investigated and of the corresponding hydroxy derivatives of cholesterol have the same fragmentation patterns and similar relative ion abundances.

Topics: Cholestadienols; Cholesterol; Gas Chromatography-Mass Spectrometry; Hydroxylation; Mass Spectrometry; Peroxides; Phytosterols; Sitosterols; Stigmasterol

The activity of ergosterol delta 7-reductase (3 beta-hydroxysteroid delta 7-reductase) was measured in cultured skin fibroblasts from 7 controls, 10 Smith-Lemli-Opitz syndrome (SLOS) patients, and 10 parents (obligate carriers). The fibroblasts were exposed to delipidated medium supplemented with lovastatin for 24 h and the enzyme activity was determined by incubating cell-free homogenate with ergosterol (ergosta-5,7,22-trien-3 beta-ol) and measuring the mass of brassicasterol (ergosta-5,22-dien-3 beta-ol) formed by gas chromatography-mass spectrometry with selected-ion monitoring. In carriers, the activity was significantly lower than in controls (22 +/- 2 vs 65 +/- 10 pmol/min per mg protein, p < 0.0005), and no overlap was observed. The mean activity in carriers' fibroblasts was more than 100 times higher than in patients' cells (0.2 pmol/min per mg protein). The use of ergosterol avoids the many problems caused by the instability and lack of availability of radiolabelled 7-dehydrocholesterol. The present method makes it possible to discriminate SLOS carriers from both controls and patients using a commercially available substrate and common analytical equipment.

Topics: Cells, Cultured; Cholestadienols; Ergosterol; Fibroblasts; Heterozygote; Humans; Oxidation-Reduction; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Phytosterols; Skin; Smith-Lemli-Opitz Syndrome; Stigmasterol

Rapeseed oil denatured with aniline was the vehicle of the causal agent of the toxic oil syndrome (TOS) epidemic that occurred in Spain in 1981. Although the precise aetiologic agent remains unknown, researchers established that increasing concentrations of oleyl anilide and other fatty acid anilides were associated with an increased risk for disease. To examine the hypothesis that 5-litre plastic containers of rapeseed oil associated with TOS, and which contained oleyl anilide had a characteristic shape, we measured fatty acid, sterol and fatty acid anilide levels in oil from containers of different shapes. We identified 1673 bottles of oil that had been collected during the Spanish Government's oil exchange programme and linked these bottles to people with TOS as reported in the official government census of patients with TOS. Although rapeseed oil (identified by the presence of brassicasterol) was found in 798 (47.7%) of the 1673 bottles examined, contamination with fatty acid anilide occurred in only 329 (19.6%) of the 1673 bottles and 319 (97%) of the 329 were oil containers of the shape sold by RAELCA, an oil company in Madrid. The first aniline-denatured oil that RAELCA had purchased to be refined specifically for distribution was refined at the ITH refinery of Seville, and this oil has been most directly associated with the epidemic. Previous work has shown that the only toxic oil linked to a specific refinery was that associated with rapeseed oil from the ITH refinery in Seville, and the epidemic began shortly after this oil was delivered to RAELCA for retail sale. On the basis of these findings, we conclude that oil refined by ITH and distributed by RAELCA was the principal, and probably the only, oil responsible for the TOS epidemic. Information about the history and treatment of this oil may yield important clues towards identifying the aetiologic agent of TOS.

Topics: Anilides; Brassica; Cholestadienols; Disease Outbreaks; Fatty Acids, Monounsaturated; Food Contamination; Food Packaging; Humans; Oleic Acids; Phytosterols; Plant Oils; Rapeseed Oil; Spain; Syndrome

The sterol composition of Tuber melanosporum was examined by medium-pressure liquid and high-performance liquid chromatography, nuclear magnetic resonance spectroscopy, and mass spectrometry. Ergosterol (ergosta-5,7,22-trienol) and brassicasterol (ergosta-5,22-dienol) were identified as the major components (90%). Their quantities and their relative concentrations, probably good indicators for differentiating mature truffles from immature colored ones, were determined during the maturation of the fungus. The results show that the total quantity of sterols decreased with the age of the fungus, but that the ergosterol/brassicasterol ratio remained relatively constant.

Topics: Ascomycota; Cholestadienols; Chromatography, High Pressure Liquid; Ergosterol; Magnetic Resonance Spectroscopy; Mass Spectrometry; Phytosterols; Sterols

A new sensitive and specific method for the evaluation of 3 beta-hydroxysteroid delta 7-reductase activity, the defective enzyme in the Smith-Lemli-Opitz (SLO) syndrome, is described. The assay is based on the use of gas chromatography-mass spectrometry with selected-ion monitoring to measure the mass of brassicasterol (ergosta-5,22-dien-3 beta-ol) produced by the incubation of ergosterol (ergosta-5,7,22-trien-3 beta-ol) with cultured human skin fibroblasts. Although the conversion of ergosterol to brassicasterol was slower than the transformation of [3H]7-dehydrocholesterol to [3H] cholesterol, cells from control subjects produced brassicasterol efficiently. In contrast, cells form SLO patients produced very little brassicasterol (P < 0.0001, patients vs. parents or vs. controls). These results indicate that the reduction of ergosterol can be used as an assay for 3 beta-hydroxysteroid delta 7-reductase in human skin fibroblasts, which avoids the many problems caused by the instability and lack of availability of radiolabeled 7-dehydrocholesterol. The present method made it possible to diagnose the SLO syndrome with high sensitivity and reliability using a commercially available compound.

Topics: Cholestadienols; Ergosterol; Fibroblasts; Gas Chromatography-Mass Spectrometry; Humans; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Phytosterols; Skin; Smith-Lemli-Opitz Syndrome; Stigmasterol

To investigate the metabolism and possible deleterious effects of 4-methyl and 4,4-dimethyl steroids in Manduca sexta, the 4,4-dimethyl sterols lanosterol and cycloartenol, the 4-methyl sterol obtusifoliol and the 4,4-dimethyl pentacyclic triterpenoid alpha-amyrin were fed in an artificial agar-based diet at various concentrations. Utilization and metabolism of these four compounds were compared with sitosterol, stigmasterol, brassicasterol, ergosterol and 24-methylenecholesterol, 24-alkyl sterols that are readily dealkylated and converted to cholesterol in Manduca and in most phytophagous insects. None of the 4-methylated compounds significantly inhibited development except at very high dietary concentrations. The delta 24-bonds of lanosterol and cycloartenol were effectively reduced by the Manduca delta 24-sterol reductase enzyme, as is the delta 24-bond of desmosterol which, in most phytophagous insects, is an intermediate in the conversion of sitosterol, stigmasterol and other C28 and C29 phytosterols to cholesterol. On the other hand, the 24-methylene substituent of obtusifoliol was not dealkylated. Each of the 4-desmethyl C28 and C29 sterols was readily converted to cholesterol, and a significant amount of 7-dehydrocholesterol was derived from ergosterol metabolism. The reason for the differences in substrate specificity of these sterols is not clear, but the information may be useful in the development of new, specific, mechanism-based inhibitors of sterol metabolism.

Topics: Animals; Cholestadienols; Cholesterol; Ergosterol; Manduca; Phytosterols; Sitosterols; Sterols; Stigmasterol

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.

Topics: Animals; Cholestadienols; Cholesterol; Desmosterol; Phytosterols; Polychaeta; Sex Attractants; Sexual Behavior, Animal; Steroids

Species of the genera Taphrina Fr. and Protomyces Unger were screened for the presence of carotenoid pigments and the sterols ergosterol and brassicasterol. All strains produced carotenoids in variable amounts: Taphrina: 0.3--39 micrograms/g dry weight; protomyces: 65--99 micrograms/g dry weight. It was concluded that the two genera cannot be separated on the basis of presence or absence of carotenoids. Thirty strains (24 species) of Taphrina produced brassicasterol as the principal sterol; twenty-one strains (17 species) did not form ergosterol. Only four isolates (4 species) produced ergosterol without formation of brassicasterol. Brassicasterol was the major sterol in 3 species of Protomyces, whereas ergosterol was absent. Brassicasterol is a rather unique sterol within the fungal kingdom and has hitherto not been found in the red yeasts. Therefore, this sterol is of taxonomic significance in contrast with ergosterol, which is widespread among fungi.

Topics: Ascomycota; Carotenoids; Cholestadienols; Ergosterol; Phytosterols; Species Specificity

Topics: Amphibian Venoms; Animals; Bufonidae; Cholestadienols; Cholesterol; Chromatography, Gas; Mass Spectrometry; Phytosterols; Sitosterols; Sterols; Stigmasterol; Vietnam

Topics: Chemical Phenomena; Chemistry; Cholestadienols; Cholesterol; Ergocalciferols; Ergosterol; Phytosterols; Research; Sterols