Free Sterol Fraction Research Articles

Sample preparation of free sterols from vegetable oils by countercurrent chromatography in co-current mode

Countercurrent chromatography (CCC) is a preparative instrumental method where both the mobile and stationary phases are liquids and which are predominantly used for the isolation of natural products. In this study, we widened the scope of CCC by using it as an instrumental method for the direct enrichment of the free sterol fraction from plant oils to which they contribute with ~ 1%. For the enrichment of sterols in a narrow band, we employed the so-called co-current CCC (ccCCC) mode in which both liquid phases of the solvent system (here: n-hexane/ethanol/methanol/water (34:11:12:2, v/v/v/v)) are moved at different flow rates in the same direction. Different from previous applications of ccCCC, the lower and predominant “stationary” phase (LPs) was pumped twice as fast as the mobile upper phase (UPm). This novel reversed ccCCC mode improved the performance but also required a higher demand of LPs compared to UPm. Therefore, the exact phase composition of UPm and LPs was determined by gas chromatography and Karl Fischer titration. This step enabled the direct preparation of LPs which considerably reduced the waste of solvents. Internal standards (phenyl-substituted fatty acid alkyl esters) were synthesised and utilised to frame the free sterol fraction. This approach allowed a fractionation of free sterols based on the UV signal and compensated run-to-run variations. The reversed ccCCC method was then applied to the sample preparation of five vegetable oils. In addition to free sterols, free tocochromanols (tocopherols, vitamin E) were also eluted in the same fraction as free sterols.Graphical

Free Sterol Composition of Deep-Sea Holothurian Orphnurgus cf. glaber

The free sterol fraction from the deep-sea holothurian Orphnurgus cf glaber Walsh, 1891, has been isolated and studied by GC–MS. Sterols containing Δ7, Δ0, Δ5, polyunsaturated and 5,8-epidioxy modified cores were found. A high percentage of compounds with polyunsaturated cores distinguish this composition from those of previously studied holothurians. Almost all identified compounds had saturated side chains, which is also a specific feature of this fraction. Peculiarities of biosynthesis and metabolism of the found sterols are discussed.

Method Development for the Determination of Free and Esterified Sterols in Button Mushrooms (Agaricus bisporus).

Ergosterol is the major sterol in button mushrooms (Agaricus bisporus) and can occur as free alcohol or esterified with fatty acids (ergosteryl esters). In this study, gas chromatography with mass spectrometry in the selected ion monitoring mode (GC/MS-SIM) was used to determine ergosterol and ergosteryl esters as well as other sterols and steryl esters in button mushrooms. Different quality control measures were established and sample preparation procedures were compared to prevent the formation of artifacts and the degradation of ergosteryl esters. The final method was then used for the determination of ergosterol (443 ± 44 mg/100 g dry matter (d.m.)) and esterified ergosterol (12 ± 6 mg/100 g d.m.) in button mushroom samples (n = 4). While the free sterol fraction was vastly dominated by ergosterol (∼90% of five sterols in total), the steryl ester fraction was more diversified (nine sterols in total, ergosterol ∼55%) and consisted primarily of linoleic acid esters.

Lipid composition of tropical and subtropical copepod species of the genus Rhincalanus (Copepoda: Eucalanidae): a novel fatty acid and alcohol signature

Rhincalanus nasutus, R. rostrifrons, and R. cornutus are primarily found in subtropi- cal and tropical waters and accumulate large lipid stores, an uncommon feature in low-latitude copepods. While R. nasutus fatty acid and alcohol profiles have been examined previously, little is known about lipids in R. rostrifrons or R. cornutus. Lipid profiles for wax ester, triacylglycerol, phospholipid, free fatty acid, sterol, and free fatty alcohol fractions were determined for these 3 species collected from the eastern tropical north Pacific, Gulf of California, and Gulf of Mexico. Storage lipids, primarily wax esters, were the dominant component (>88%) of total lipid in all 3 Rhincalanus species. R. nasutus, however, had distinctly different storage lipid fatty acid and alcohol profiles, with primarily 16:1(n-7)/18:1(n-9) fatty acids and 14:0/16:0 fatty alcohols, while R. cornutus and R. rostrifrons accumulated 14:0/16:0 fatty acids and 16:1/18:1 fatty alcohols. Species differences also were observed in sterol profiles, with R. rostrifrons having a cholesterol content of 75 to 76% versus 85 to 90% in R. nasutus and R. cornutus. The remainder of the sterol fraction was largely composed of 22-dehydrocholesterol in all species. In all 3 species, phospholipid fractions were dominated by 22:6(n-3), 16:0, 18:0, 20:5(n-3), and 18:1(n-9) fatty acids. The results of the present study suggest that genetic predisposition significantly governs lipid profiles, particularly storage lipids, within this genus. The storage lipid profiles observed in R. cornutus and R. rostrifrons were unique from their congeners and have not been reported previously for other copepod species.

Both the Peroxisome Proliferator‐Activated Receptor δ Agonist, GW0742, and Ezetimibe Promote Reverse Cholesterol Transport in Mice by Reducing Intestinal Reabsorption of HDL‐Derived Cholesterol

Peroxisome proliferator-activated receptor delta (PPARdelta) agonism increases HDL cholesterol and has therefore the potential to stimulate macrophage-to-feces reverse cholesterol transport (RCT). To test whether PPARdelta activation promotes RCT in mice, in vivo macrophage RCT was assessed using cholesterol-loaded/3H-cholesterol-labeled macrophages injected intraperitoneally. PPARdelta agonist GW0742 (10 mg/kg per day) did not change 3H-tracer plasma appearance, but increased fecal 3H-free sterols excretion by 103% ( p < 0.005) over 48 hours. Total free cholesterol efflux from macrophages to serum (collected from both control and GW0742 groups) was not different, although ABCA1-mediated efflux was significantly higher with GW0742. The metabolic fate of HDL labeled with 3H- cholesteryl ether or 3H-cholesteryl oleate was also measured. While 3H-cholesteryl ether tissue uptake was unchanged, the 3H-tracer recovered in fecal free sterol fraction after 3H-cholesteryl oleate injection increased by 88% with GW0742 ( p < 0.0005). This was associated with a lower Niemann-Pick C1 like 1 (NPC1L1) mRNA expression in the small intestine ( p < 0.05). The same experiments in mice treated with ezetimibe, which blocks NPC1L1, showed a similar 2-fold increase in fecal free sterol excretion after labeled macrophages orHDL injection. In conclusion, PPARdelta activation enhances excretion of macrophage or HDL-derived cholesterol in feces through reduced NPC1L1 expression in mice, comparable to the effect of ezetimibe.

Characterization of Total, Free and Esterified Phytosterols in Tetraploid and Hexaploid Wheats

Dietary plant sterols have received increasing attention in recent years due to their favorable health benefits. The present research focused on quantification of phytosterols as free, esterified and total forms in different tetraploid (5 cultivars of Triticum durum Desf., 9 cultivars of Triticum dicoccon Schrank) and hexaploid (5 cultivars of T. aestivum L., 12 cultivars of Triticum spelta L.) wheats. Tetraploid wheats showed the highest content of total sterol (79.4 and 79.5 mg of sterols /100 g dry weight for T. durum and T. dicoccon, respectively). Hexaploid cultivars were the best source of esterified sterols (40.7% and 37.3% of total sterols for Triticum aestivum and T. spelta, respectively). Significant amounts of free sterols (65.5% and 60.7% of total sterols for T. durum and T. dicoccon, respectively) were found in the tetraploid cultivars. The most abundant phytosterol in all wheat samples was sitosterol accounting for 45.1-59.1, 46.6-57.4 and 38.6-59.5% of total, free and esterified sterol fraction, respectively. These results demonstrate that although the sterol profile present in tetraploid and hexaploid wheat species is the same, differences in their relative amounts and distribution allow statistical differentiation between hexaploids and tetraploids, and between soft and durum wheats.

Cholesterol Substitution Increases the Structural Heterogeneity of Caveolae

Caveolin-1 binds cholesterol and caveola formation involves caveolin-1 oligomerization and cholesterol association. The role of cholesterol in caveolae has so far been addressed by methods that compromise membrane integrity and abolish caveolar invaginations. To study the importance of sterol specificity for the structure and function of caveolae, we replaced cholesterol in mammalian cells with its immediate precursor desmosterol by inhibiting 24-dehydrocholesterol reductase. Desmosterol could substitute for cholesterol in maintaining cell growth, membrane integrity, and preserving caveolar invaginations. However, in desmosterol cells the affinity of caveolin-1 for sterol and the stability of caveolin oligomers were decreased. Moreover, caveolar invaginations became more heterogeneous in dimensions and in the number of caveolin-1 molecules per caveola. Despite the altered caveolar structure, caveolar ligand uptake was only moderately inhibited. We found that in desmosterol cells, Src kinase phosphorylated Cav1 at Tyr(14) more avidly than in cholesterol cells. Taken the role of Cav1 Tyr(14) phosphorylation in caveolar endocytosis, this may help to preserve caveolar uptake in desmosterol cells. We conclude that a sterol C24 double bond interferes with caveolin-sterol interaction and perturbs caveolar morphology but facilitates Cav1 Src phosphorylation and allows caveolar endocytosis. More generally, substitution of cholesterol by a structurally closely related sterol provides a method to selectively modify membrane protein-sterol affinity, structure and function of cholesterol-dependent domains without compromising membrane integrity.

High Performance Thin Layer Chromatographic Analysis of Neutral Lipids in the Feces of BALB/c Mice Infected with Echinostoma caproni

High performance thin layer chromatography was used to determine neutral lipid profiles in the feces of BALB/c mice infected with adults of Echinostoma caproni (Trematoda). An approximate 25 worm burden per host induced changes in the neutral lipid profile of the mice at 1 to 5 weeks post infection (PI) relative to the uninfected controls. Infection caused a significant decrease (Student's t‐test, P<0.05) in the triacylglycerol fraction at 3 weeks PI and a significant increase (Student's t‐test, P<0.05) in the free sterol fraction at 2 weeks PI relative to the uninfected controls. Neutral lipid profiles of feces may serve as an indicator of infection by intestinal trematodes in animals and humans.

Sterols and related metabolites from five species of sponges

Free sterol fractions were isolated from the marine sponges Phyllospongia madagascarensis, Scalarispongia sp., Oceanapia sp., Monanchora clathrata and studied by GLC, GLC–MS, and spectroscopy NMR. P. madagascarensis and Scalarispongia sp. contained common Δ 5-sterols; cholesterol was shown to be a main sterol of both the sponges. Oceanapia sp. contained stanols and minor Δ 5-sterols with 24R-24,25-methylene-5α-cholestan-3β-ol as a main constituent. Many free sterols from M. clathrata were Δ 7-series compounds, and latosterol was a main sterol. Δ 4-3-Ketosteroids and Δ 5-sterol esters were found in the Antarctic sponge Haliclona sp., but free sterols were practically absent except for trace amount of cholesterol. A chemotaxonomic application of sterols in relation to the genera Phyllospongia, Oceanapia and the family Crambeidae is provided. The known cases of the absence of sterols in sponges and probable reasons of the phenomenon are discussed.

Fatty Acids, Triacylglycerols, and Sterols in Neem Oil (Azadirachta Indica A. Juss) as Determined by a Combination of Chromatographic and Spectral Techniques

Quantitative thin‐layer chromatography in silver ion and reversed phase modes, gas chromatography, gas chromatography–mass spectrometry, and 13C nuclear magnetic resonance spectrometry were employed to determine the lipid composition in neem (Azadirachta indica A. Juss) kernel oil. Thirteen fatty acids and 25 triacylglycerols species were identified and quantified. The seven main triacylglycerols species were found in almost equal amounts in the range 7–10%. Saturated fatty moieties occupied positions 1‐ and 3‐ in the triacylglycerol molecule, oleic acid was almost equally distributed, and linoleic acid predominantly occupied position 2. Sitosterol, stigmasterol, campesterol, and fucosterol were the main components in free sterol and sterol ester fractions.

Changes in the Composition of Phospholipid Fatty Acids and Sterols of Maize Root in Response to Monoterpenes

Terpenes are thought to be important in plant plant interactions because of their phytotoxic action on seed germination and growth. Herein, the effects of five volatile monoterpenes on root sterols and phospholipid fatty acid (PLFA) composition have been studied during maize seedling germination. The investigated monoterpenes (camphor, 1-8 cineole, geraniol, menthol, and thymol) were applied at their respective IC80 (concentration causing 80% inhibition). They quantitatively affected free sterols and PLFA composition, thus producing an increase in the percentage of unsaturated PLFAs, stigmasterol of the free sterol fraction, and saturated steryl ester fatty acids. Alcoholic and nonalcoholic monoterpenes appeared to have different modes of action. The former affected unsaturated fatty acid and stigmasterol to a greater extent, and accordingly they could interfere in seedling growth by changes in the membrane lipids.

Sterols of the heterotrophic dinoflagellate, pfiesteria piscicida (dinophyceae): is there a lipid biomarker?1

Within U.S. waters, blooms of the dinoflagellate, Pfiesteria piscicida, have been recorded on an almost regular basis in the Chesapeake Bay and surrounding mid‐Atlantic regions for the last two decades. Despite the apparent significance of such blooms to the environment and human health and the attendant economic consequences, little work has addressed the physiology and biochemistry, particularly that of sterol composition, of P. piscicida. GC‐MS characterization of trimethylsilyl ether derivatives of sterols from free sterol and sterol ester fractions was performed in an effort to determine whether P. piscicida produces unique sterols that may serve as potential biomarkers. This characterization revealed that like most dinoflagellates, the majority of sterols was present as free sterols. Furthermore, the profile of free sterols was found to resemble those of photosynthetic dinoflagellates, with the dominant compound being the previously reported dinoflagellate sterol, dinosterol. A number of other 4α‐methyl‐substituted sterols and steroidal ketones common to other dinoflagellates were also identified. No strong candidate(s) for a unique sterol biomarker was present.

Sterols from six marine sponges

The free sterol fractions from marine sponges Darwinella australiensis, Haliclona sp ., Agelas mauritiana, Clathria major, Didiscus aceratus and Teichaxinella labirintica from Western Australia were isolated and studied by HPLC, GLC, GLC-MS, and NMR methods. D. australiensis contained Δ 7-, Δ 5-, Δ 5,7-, Δ 5,7,9(11)-sterols, and cholest-7-en-3β-ol was shown to be a main sterol. The free sterols from A. mauritiana proved to be stanols and Δ 7-series compounds, chondrillasterol was identified as a predominant constituent. Haliclona sp . contained Δ 5-sterols with cholesterol as a main constituent. C. major and D. aceratus contained Δ 5-sterols, and clionasterol was shown to be a main sterol. T. labirintica was shown to contain 3β-hydroxymethyl-A-nor-sterols. Absolute configurations at C-24 of major sterols from C. major, D. aceratus and A. mauritiana were established by NMR method. Distribution of different sterols in the studied species was discussed to provide additional viewpoint on the probable application of these natural products as chemotaxonomic markers and to understand biological roles of unusual sterols in sponges using an idea of so-called biochemical coordination.

Solid-phase extraction–thin-layer chromatography–gas chromatography method for the detection of hazelnut oil in olive oils by determination of esterified sterols

The sterol composition of extra virgin olive oil is very characteristic and, thus, has become a helpful tool to detect adulterations with other vegetable oils. Special attention has been addressed to the separate determination of the free and esterified sterol fractions, since both have different compositions and can thus provide more precise information about the actual origin of the olive oil. In the case of admixtures with small amounts of hazelnut oil, this approach can be extremely useful, because the similarity between the fatty acid compositions of both oils hampers the detection of the fraud. A hyphenated chromatographic method was developed for a sensitive and precise determination of esterified sterols in olive oils. The oil was subjected to silica solid-phase extraction (SPE) fractionation, cold saponification of the collected fraction and purification on silica TLC. The sterol band was then injected into an SPB-5 (30 m×0.25 mm I.D., 0.25 μm film thickness) and the ratio [% campesterol×(% 7-stigmastenol) 2]/(% 7-avenasterol) was calculated. The method was tested on extra virgin olive oil; good sterol recoveries and repeatability were obtained. The results were compared with another method, which has a different sample preparation sequence (silica column chromatography, hot saponification and silica TLC). Similar results were achieved with both methods; however, the SPE–cold saponification–TLC–capillary GC was faster, required less solvent and prevented sterol decomposition. The SPE-method was applied to an admixture with 10% of hazelnut oil and to a screening of 11 oils (husk oil, virgin and refined olive oils) from different Mediterranean countries.

Influence of the vegetable oil refining process on free and esterified sterols

AbstractThe influence of the refining process on the distribution of free and esterified phytosterols in corn, palm, and soybean oil was studied. Water degumming did not affect the phytosterol content or its composition. A slight increase in the content of free sterols was observed during acid degumming and bleaching due to acid‐catalyzed hydrolysis of steryl esters. A significant reduction in the content of total sterols during neutralization was observed, which was attributed to a reduction in the free sterol fraction. Free sterols probably form micelles with soaps and are transferred into the soapstock. The steryl ester content remained constant during all neutralization experiments, indicating that hydrolysis of steryl esters did not take place during neutralization. During deodorization, free sterols are distilled from the oil, resulting in a gradual reduction in the total sterol content as a function of the deodorization temperature (220–260°C). A considerable increase in the steryl ester fraction was found during physical refining, probably owing to a heat‐promoted esterification reaction between free sterols and FA.

DETERMINATION OF NEUTRAL LIPIDS AND PHOSPHOLIPIDS IN THE CERCARIAE OF SCHISTOSOMA MANSONI BY HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY

ABSTRACT High performance thin layer chromatography (HPTLC) was used to analyze neutral lipids and phospholipids in the cercariae of a Puerto Rican strain of Schistosoma mansoni. The cercariae were obtained following isolation of Biomphalaria glabrata snails (NMRI strain) experimentally infected with S. mansoni miracidia. Cercariae were pelleted to contain from 1000 to 10,000 organisms per 1.5 mL of artificial spring water and then extracted in chloroform–methanol (2 : 1). The lipid extract was analyzed by previously published silica gel HPTLC methods, using a petroleum ether–diethyl ether–acetic acid (80 : 20 : 1) mobile phase and detection with phosphomolybdic acid spray reagent for neutral lipids. Visual observations of the chromatograms showed that the most abundant neutral lipids in the cercariae were free fatty acids and free sterols along with two additional faster moving zones that did not match the methyl oleate and cholesteryl oleate standard zones. Quantification of the free fatty acid and free sterol fractions by scanning densitometry revealed mean weights±standard deviation of 4.62±1.9 ng and 3.51±0.70 ng per cercaria, respectively. Phospholipids were separated on silica gel using a mobile phase of chloroform–methanol–water (65 : 25 : 4), and the plates were sprayed with a detection reagent of 10% cupric sulfate in 8% phosphoric acid. Visual observations of the chromatograms showed that the most abundant phospholipids were phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Quantification of PC and PE by scanning densitometry revealed mean weights±standard deviation of 67.0±18 ng and 38.7±8.8 ng per cercaria, respectively. This is the first study to provide quantitative data on neutral lipids and phospholipids on a per cercaria basis for S. mansoni. The possible functions of lipids and phospholipids in cercariae are discussed.

Analysis of free and esterified sterols in fats and oils by flash chromatography, gas chromatography and electrospray tandem mass spectrometry

A method for the analysis of free and esterified sterols has been developed. Fat or oil samples were separated on solid-phase extraction silica gel columns into a sterol ester fraction, a fraction of triacylglycerols, and a free sterol fraction containing partial acylglycerols and residual triacylglycerols. Sterol esters and acylglycerols of the free sterol fraction were transesterified to methyl esters. The fatty acid methyl esters from sterol ester fraction and the free sterols from sterol ester fraction and free sterol fraction were determined by GLC. Precursor ion electrospray MS–MS of sterol fragment ions of sterol ester fractions were recorded and used for determination of sterol ester proportions in butterfat and vegetable oil samples.

Free sterols from the holothurians Synapta maculata, Cladolabes bifurcatus and Cucumaria sp.

Free sterol fractions from the holothurians (sea cucumbers) Synapta maculata, Cladolabes bifurcatus and Cucumaria sp. have been isolated and studied by HPLC, GLC, GLC-MS and NMR methods. Forty seven sterols were identified, including several rare ones. In contrast with previously studied holothurians, the presence of a minor amount of Δ 7 sterols was indicated in the sterols of S. maculata. This animal contains predominantly Δ 9(11)sterols as well as an abnormally high concentration of Δ 5 sterols. In C. bifurcatus and Cucumaria sp., 14α-methyl and 4α,14α-dimethyl-Δ 9(11)sterols were found to be the main sterol constituents. Peculiarities of sterol distribution and the relationship between sterol compositions and taxonomic positions, ecology and toxicity of the corresponding sea cucumbers were discussed.

The distribution of free sterols, polyhydroxysteroids and steroid glycosides in various body components of the starfish Patiria (=Asterina) pectinifera

The distribution of free sterols, polyhydroxysteroids and steroid glycosides in different body components of the Far-eastern starfish Patiria (=Asterina) pectinifera has been studied. It was shown that free sterol fractions from aboral and oral body walls, gonads, stomach and pyloric ceca contained Δ 7 sterols with a preponderance of 5α-cholest-7-en-3β-ol. All these body components had also toxic steroid oligoglycosides. However, polyhydroxysteroids and related low molecular weight steroid glycosides were found in stomach and pyloric ceca only. In pyloric ceca, the sulfated monoside ‘asterosaponin’ P 1 was identified as a main polar steroid, whereas 6-sodium sulfate of cholestane-3β,4β,6α,7α,8,15β,16β,26-octaol predominated in the stomach. Probable biological functions of polar steroids and free sterols in this starfish were discussed. It was suggested that some polyhydroxysteroids and related monoglycosides play the same biological role as bile alcohols and bile acids do in vertebrates.

Canopy Position and Heat Treatments Influence Gamma-irradiation-induced Changes in Phenylpropanoid Metabolism in Grapefruit

Irradiation is being evaluated as a quarantine treatment of grapefruit (Citrus paradisi Macf. `Marsh'), but it can cause damage to the fruit. Research was conducted to determine if preirradiation heat treatments would improve fruit tolerance to irradiation as they improve tolerance to low temperature injury and to determine if canopy position influenced fruit tolerance to irradiation. Initially, grapefruit were irradiated at 0 or 2.0 kGy at a dose rate of 0.14 kGy·min-1 and selected biochemical changes were monitored over time. There was a marked increase in phenylalanine ammonia-lyase (PAL) activity following irradiation. Maximum activity (≈18-fold increase) was attained 24 hours after irradiation. Subsequently, grapefruit were harvested from interior and exterior canopy positions and irradiated at 0 or 1.0 kGy at a dose rate of 0.15 kGy·min-1 before storage for 4 weeks at 10 °C. Following storage, pitting of flavedo was the most evident condition defect noted as a result of irradiation. Pitting was observed on 27% and 15% of irradiated exterior and interior canopy fruit, respectively, whereas there was no pitting on nonirradiated fruit. Heat treatment before irradiation decreased susceptibility of fruit to damage. Pitting was 26%, 19%, and 17% when fruit were held 2 hours at 20 (ambient), 38 or 42 °C, respectively. Irradiation-induced PAL activity was reduced by temperature conditioning at 38 or 42 °C. Exterior canopy fruit flavedo contained higher levels of total phenols, including flavanols and coumarins compared with interior canopy fruit. Deposition of lignin was not related to canopy position. Neither irradiation nor heat treatment had an effect on total phenols or lignin deposition. Generally, cholesterol, campesterol, stigmasterol, β-sitosterol, and isofucosterol were found to be higher in four steryl lipid fractions in exterior canopy fruit compared with interior canopy fruit. Irradiation increased campesterol in the free sterol and steryl glycoside fractions and decreased isofucosterol in the free sterol fraction. Heat treatments had no effect on individual sterol levels. It seems that irradiation causes a stress condition in the fruit, which leads to pitting of peel tissue. Heat treatment before irradiation reduced damaging effects of irradiation.