Hepatic cholesterol synthesis from mevalonate and squalene in rats: Effect of feeding cholesterol supplemented diet during weaning and following starvation

The conversion of squalene to digitonin precipitable sterols by microsomes and soluble fractions from liver and the incorporation of mevalonate into nonsaponifiable lipids and digitonin precipitable sterols by 5000 g liver supernatant was studied in suckling rats and rats weaned on diet (Purina chow) supplemented with varying levels of cholesterol. The results indicate that the conversion of squalene to digitonin precipitable sterols was low in suckling rats and high in rats weaned on Purina chow diet. Weaning rats on 1% cholesterol supplemented diet effectively eliminated the post-weaning increase in mevalonate incorporation into nonsaponifiable and digitonin precipitable sterols, and in the conversion of squalene to digitonin precipitable sterols. The incorporation of mevalonate into nonsaponifiable lipids and digitonin precipitable sterols, and of squalene into digitonin precipitable sterols also was studied in liver preparations from adult rats which were starved and refed control or cholesterol supplemented diet. It was observed that the conversion of squalene to digitonin precipitable sterols by liver fractions from fasted rats was low, while that by liver fractions from rats refed control diet was higher. Furthermore, the post-fasting increase in the conversion of mevalonate to nonsaponifiable lipids and digitonin precipitable sterols and in squalene to digitonin precipitable sterols conversion essentially was eliminated by refeeding a 1% cholesterol supplemented diet. The low conversion of squalene to digitonin precipitable sterols in suckling rats, rats weaned on cholesterol supplemented diet, and adult rats that were starved, or starved and refed cholesterol supplemented diet, was due to the reduced activity of microsomal enzymes. It is concluded from this study that dietary cholesterol prevents the increase in cholesterol synthesis observed in developing and regenerating liver by suppressing the activities of one or more enzymes between mevalonate and squalene and between squalene and cholesterol.     

Characterization of Lipid Components in Two Microalgae for Biofuel Application

A full characterization of lipid components is critical for selecting the most suitable microalgae and for downstream processing for biofuel production. This study demonstrates extraction, quantification, and diversity of lipid components from two microalgae of different types. For total lipid quantification, three extraction methods were compared and the method of pre-drying, dry ice-assisted grinding, and sequential solvent extraction gave the highest total lipid recovery. For lipid class composition determination, the photosynthetic Nannochloropsis contained 37.0% polar lipids, 41.1% triacylglycerols, and 12.5% unsaponifiable matter, whereas, the heterotrophic Schizochytrium had much less polar lipids and unsaponifiable matter. Further separation and quantification showed that Nannochloropsis contained more glycolipids (37.1% of total polar lipids) than that in Schizochytrium (14.3%), while Schizochytrium contained much more phospholipids (85.7%) than that in Nannochloropsis (44.7%). The major components in unsaponifiable matter of Nannochloropsis were hydrocarbon and cholesterol (55.8 and 37.7% of the total GC quantifiable matter), which only accounted for 29.3% of total unsaponifiable matter. For Schizochytrium, 15.4% squalene, 28.9% cholesterol, and 43.2% stigmasta-4,7,22-trien-3-ol were found in its GC quantifiable matter, and the total quantified hydrocarbon and sterols accounted for 50.5% of unsaponifiable matter. The lipid compositions of the two types of microalgae are very different, therefore, processing performance, such as lipid extraction and conversion to biodiesel may be different. Similar lipid characterization for other biofuel types of microalgae needs to be made to ensure optimal biofuel processing and fuel quality.     

表面活性剂强化提取黄姜中薯蓣皂苷的研究

对7种表面活性剂强化提取薯蓣皂苷的效果进行了比较,筛选出对该提取过程具有较好强化作用的表面活性剂——十二烷基硫酸钠(SDS)。通过正交实验、单因素实验考察了提取温度、提取时间、w(SDS)、提取溶剂用量对薯蓣皂苷提取率的影响,获得了优化的提取条件:黄姜粉末5 g,提取温度70℃,提取溶剂用量12 mL/g黄姜,提取时间2 h,w(SDS)=3%,获得皂素92.59 mg。实验结果表明,SDS对水提取黄姜中薯蓣皂苷(7.74mg)有明显强化作用,提取效率高于传统的甲醇/水溶液(75.08 mg),接近索氏提取(95.67 mg)。     

A rapid method for extraction of total lipids from whey protein concentrates and separation of lipid classes with solid phase extraction

A modified procedure for extraction of total lipids from whey protein concentrates was developed such that stable emulsion with extracting solvents was avoided and the solvent system remained monophasic. Nonlipid contaminants from the extract were removed using gel filtration instead of traditional aqueous washing to prevent any loss of polar lipids. The extraction of total lipids by the modified procedure was complete and comparable with a reference procedure. Traditional thin-layer chromatography is tedious and more qualitative than quantitative for lipid class separation. Total lipids were further separated into free fatty acids, phospholipids, cholesterol ester, triacylglycerol, cholesterol, diacylglycerol, and monoacylglycerol, using modified solid phase extraction procedure. Columns with 2 g amino propyl packing allowed separation of up to 80 mg of total lipids into lipid classes gravimetrically. The values for anhydrous milk fat for all lipid classes agreed with those in the literature. Separation of total lipids into lipid classes with solid phase extraction is easy, quantitative, and can also be performed on a preparative scale.     

Effect of the extraction solvent polarity on the sesame seeds oil composition

This study highlights the effect of solvent polarity on the yield (Y%) and properties of oil extracted from Algerian sesame seeds. Extractions were carried out under Soxhlet conditions with the following solvents: hexane (Hx), ethanol (Eth), acetone (Ac), dichloromethane (Di), isopropanol (Iso), hexane:isopropanol (Hx:Iso), and chloroform:methanol (Chf:Me). The sesame oil yield obtained using different solvents ranged from 28.86 to 52.83%. Fatty acids and sterols analyses were performed by GC on capillary column. γ‐Tocopherol was the major tocochromanol compound detected by HPLC‐fluorescence. Fourteen fatty acids were identified, with the predominance of oleic and linoleic acids. The main sterol in sesame oil was β‐sitosterol, followed by stigmasterol, campesterol, and Δ⁵‐avenasterol which were present in lower concentrations. High correlations were found between arachidic, gadoleic, behenic, and lignoceric acids concentrations; these results were explained by the metabolic biosynthesis pathway of the biologically active long‐chain PUFA by successive elongation and desaturation. Principal component analysis (PCA) of the data obtained from sesame oil composition enabled an easy comparison of the different extraction solvents, and correlated their properties with the most characteristic components of the extracted oils with a view to understand solvent–oil interaction, and to establish the effects of extracting solvent on such oil composition. Practical applications: This study showed that the choice of solvent depends largely on the desired fraction to be extracted. Sesame oil was better extracted with less‐polar solvents but membrane‐associated lipids are more polar and require polar solvents capable of breaking hydrogen bonds or electrostatic forces. Owing to the differences in solvent capacity, the fatty acids, sterols, and tocopherols extracted along with the oil vary, leading to differences in the quality of the extracted oil. The results obtained in this study could be applied in industrial extraction to encourage the use of alternative extraction solvents.     

Hypercholesterolemia in rats fed cholesterol in agar gel diets

Female (Exp. I) or male (Exp. II) weanling rats were fed diets containing either 2% Solka-Floc or 2% agar for 28-day periods. Some groups received 1% cholesterol, either added in crystalline form or first dispersed in the oil portion of the diet, and some agar groups received their diet in a gelled form. Feces were collected for a 3-day period after 2 weeks (Exp. II) or during the fourth week (Exp. I) of experimentation. Serum and liver cholesterol, total liver lipids, fecal lipids, and fecal sterols were determined. The results indicated that cholesterol feeding increased serum cholesterol, total liver, and fecal lipids, liver cholesterol, and fecal sterols. Substitution of agar for Solka-Floc in dry (nongelled) diets further increased total liver lipids (Exp. I), but had no significant effect upon any other measured parameter. Gelling of 1% cholesterol agar diets, in contrast to the 1% cholesterol dry agar diet, resulted in reduced liver cholesterol in both experiments. Gelling significantly increased fecal sterols after 2 weeks feeding (Exp. II), but no significant differences were observed after 4 weeks feeding (Exp. I) when compared to 1% cholesterol-fed groups. Small, nonsignificant increases of liver cholesterol and total liver lipids with similar reduction of fecal sterols resulted from dispersing the cholesterol in the oil portion of the diet prior to mixing. The results indicate that (a) inclusion of 2% agar in rat diets and (b) dispersing cholesterol in oil had little effect upon serum, liver, or fecal lipids in cholesterol-fed rats. However, gelling the agar diets reduced liver cholesterol, possibly by initial reduction of dietary cholesterol absorption.     

The analysis of lipids via HPLC with a charged aerosol detector

Because most lipid extracts are a mixture of saturated and unsaturated molecules, the most successful strategies for the quantitative analysis of lipids have involved the use of so-called “mass” or universal detectors such as flame ionization detectors and evaporative light scattering detectors. Recently a new type of HPLC “mass” detector, a charge aerosol detector (CAD), was developed and is now commercially available. This detection method involves nebulizing the HPLC column effluent, evaporating the solvents charging the aerosol particles, and measuring the current from the charged aerosol flux. In the present study, the CAD was evaluated with several normal phase and reverse phase HPLC methods commonly used for the quantitative analysis of lipid classes and lipid molecular species. The CAD detected common lipids such as triacylglycerols, diacylglycerols, glycolipids, phospholipids, and sterols. Lower molecular weight lipids such as free FA had smaller peak areas (50–80% lower). FAME were not detected by the CAD, probably because they were completely evaporated and did not form aerosol particles. The minimum limits of detection of the CAD with lipids varied with different mobile phase solvents. Using solvent systems that were predominantly hexane, the minimum limits of detection of triacylglycerols, cholesterol esters, and free sterols were about 1 ng per injection and the mass-to-peak area ratio was nearly linear from the range of about 1 ng to about 20 mg per injection. Three other solvents commonly used for HPLC lipid analysis (methanol, isopropanol, and acetonitrile) caused higher levels of background noise and higher minimum limits of detection. These experiments indicate that the CAD has the potential to become a valuable tool for the quantitative HPLC analysis of lipids. Long-term studies are needed to evaluate full instrument performance.     

HPLC analysis of desmosterol, 7-dehydrocholesterol,and cholesterol

A simple and sensitive method to analyzed mixtures of desmosterol, 7-dehydrocholesterol and cholesterol is described. The method involves the oxidative conversion of the sterols with cholesterol oxidase, followed by high performance liquid chromatographic (HPLC) analysis. A C₁₈ reversed phase column (3 μm, 75×4.6 mm) and a mixture of methanol and acetonitrile (1∶1, v/v) at a rate of 1 ml/min are used to separate the sterols. The eluted sterols are quantified by measuring UV absoprtion at 240 nm. As little as 10 pmoles of sterol can be quantified under these conditions.     

Effects of a diet high in plant sterols,vegetable proteins,and viscous fibers (dietary portfolio) on circulating sterol levels and red cell fragility in hypercholesterolemic subjects

Plant sterols, soy proteins, viscous fibers, and nuts are advised for cholesterol reduction, but their combined effect on plant sterol absorption has never been tested. We assessed their combined action on serum sterols in hyperlipidemic subjects who were following low-saturated fat diets before starting the study and who returned to these diets post-test. The 1-mon test (combination) diet was high in plant sterols (1 g/1,000 kcal), soy protein (23 g/1,000 kcal), viscous fiber (9 g/1,000 kcal), and almonds (14 g/1000 kcal). Fasting blood was obtained for serum lipids and sterols, and erythrocytes were obtained for fragility prior to and at 2-wk intervals during the study. The combination diet raised serum campesterol concentrations by 50% and β-sitosterol by 27%, although these changes were not significant after Bonferroni correction; near-maximal rises were found by the end of the first week, but no change was found in red cell fragility despite a 29% reduction in the LDL cholesterol level. No significant associations were observed between changes in red cell fragility and blood lipids or sterols. We conclude that plant sterols had a minimal impact on serum sterol concentrations or red cell fragility in hyperlipidemic subjects on diets that greatly reduced their serum lipids.     

The Spectrum of Plant and Animal Sterols in Different Oil-Derived Intravenous Emulsions

Intravenous lipid constituents have different effects on various biological processes. Some of these effects are protective, while others are potentially adverse. Phytosterols, in particular, seem to be implicated with parenteral nutrition-associated cholestasis. The aim of this study is to determine the amount of plant and animal sterols present in lipid formulations derived from different oil sources. To this end, animal (cholesterol) and plant (β-sitosterol, campesterol, and stigmasterol) sterols in seven different commercially available intravenous lipid emulsions (ILEs) were quantified by capillary gas chromatography after performing a lipid extraction procedure. The two major constituents of the lipid emulsions were cholesterol (range 14–57% of total lipids) and β-sitosterol (range 24–55%), followed by campesterol (range 8–18%) and stigmasterol (range 5–16%). The fish oil-derived formulation was an exception, as it contained only cholesterol. The mean values of the different sterols were statistically different across ILEs (P = 0.0000). A large percentage of pairwise comparisons were also statistically significant (P = 0.000), most notably for cholesterol and stigmasterol (14 out of 21 for both), followed by campesterol (12 out 21) and β-sitosterol (11 out 21). In conclusion, most ILEs combined significant amounts of phytosterols and cholesterol. However, their phytosterols:cholesterol ratios were reversed compared to the normal human diet.     

A solvent extractor system for the rapid extraction of lipids and trace bioactive micronutrients in oilseeds

A low-cost laboratory extractor has been designed and constructed that selectively extracts polar and nonpolar components from oilseeds and other matrices. The extractor uses available high-performance liquid chromatography laboratory equipment for pumping the solvent into the extractor. Pressure, temperature, and valving arrangements are automatically controlled by commercially available components. Advantages of this system include low initial investment, reduced solvent consumption, shorter extraction times, quantitative lipid recovery, use of multiple extraction solvents, and reduction in cost per sample. The method has broader applications that include extraction of trace components from a variety of matrices, for example, the extraction of pesticides from foods and polychlorobiphenyls from soil. Class separation of components from different matrices can be achieved easily by selection of solvents with the appropriate polarity characteristics. Very small samples can be extracted simply by changing cell size or by adding an inert material to the cell to fill the void volume. Analyte collection can be accomplished by collecting in a test tube with an appropriate solvent, or on a solid-phase material. Optimization of extraction times, number of extractions, matrices, and solvent used is described. Neutral lipids were extracted from peanut meal in 70 min by the rapid extraction method compared to 1440 min required to extract the comparable amount of neutral lipids from a similar sample by the Soxhlet extraction method.     

Study of the neutral lipids of sunflower meal and isolates

Two types of sunflower protein isolates have been obtained from prepress and solvent extracted sunflower meal. The first was obtained by precipitation (at the isoelectric point) of the alkaline extract of the meal, and washing the curd with water. In the second, the alkaline extraction was carried out in the presence of sodium sulfite, and the curd was washed with water, ethanol and acetone. Both isolates were air-dried and then dried under vacuum at 50 C. From the total lipids, obtained with 86% ethanol, the neutral lipids were separated using a column of Florisil. The lipids studied were those of the two isolates mentioned above as well as those of the original meal. The following types of compounds were separated, identified and quantified: hydrocarbons, waxes, methyl esters, triglycerides, free fatty acids, diglycerides, free sterols, and hydroxy fatty acids.     

Integral lipids of human hair

It has long been recognized that hair is coated with nonpolar lipids originating in the sebaceous glands, and recently it has been shown that hair also contains cholesterol sulfate and small amounts of ceramides, similar to those found in the keratinized portion of the epidermis. In the present study, it is demonstrated that significant amounts of several additional lipids are tightly associated with hair in such a way as to be highly resistant to solvent extraction. These integral hair lipids included cholesterol sulfate (3.3 mg/g of extracted hair), cholesterol (0.6 mg/g), fatty alcohols (0.2 mg/g) and free fatty acids (4.3 mg/g). The principal fatty acid, comprising 40% of the total fatty acids, was identified as 18-methyl-eicosanoic acid by cochromatography with authentic standard on gas-liquid chromatography (GLC) and by mass spectrometry (MS).     

Bioorthogonal Probes for Imaging Sterols in Cells

Cholesterol is a fundamental lipid component of eukaryotic membranes and a precursor of potent signaling molecules, such as oxysterols and steroid hormones. Cholesterol and oxysterols are also essential for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Despite their importance, the use of imaging sterols in cells is currently very limited. We introduce a robust and versatile method for sterol microscopy based on C₁₉ alkyne cholesterol and oxysterol analogues. These sterol analogues are fully functional; they rescue growth of cholesterol auxotrophic cells and faithfully recapitulate the multiple roles that sterols play in Hedgehog signal transduction. Alkyne sterol analogues incorporate efficiently into cellular membranes and can be imaged with high resolution after copper(I)‐catalyzed azide–alkyne cycloaddition reaction with fluorescent azides. We demonstrate the use of alkyne sterol probes for visualizing the subcellular distribution of cholesterol and for two‐color imaging of sterols and choline phospholipids. Our imaging strategy should be broadly applicable to studying the role of sterols in normal physiology and disease.     

“Dark” Singlet Oxygenation of Hydrophobic Substrates in Environmentally Friendly Microemulsions

The molybdate‐catalyzed “dark” singlet oxygenation of hydrophobic compounds with hydrogen peroxide proceeds efficiently with low catalyst loadings (10 ^–3 mol %) in chlorine‐free w/o microemulsions. These micro‐heterogeneous systems are composed of sodium dodecyl sulfate (SDS)/n‐butanol/water/organic phase, the latter being either a ”green” solvent such as ethyl acetate or a liquid substrate, such as α‐terpinene or β‐citronellol. Very high reactor yields with improved product/SDS ratio can be obtained for the ”dark” singlet oxygenation of such liquid substrates.     

Extraction of α‐tocopherolquinone from vegetable oil deodorizer distillate waste

In industry, deodorizer distillate waste is one of the last products of refined edible oil after the removal of commercially important value components such as fatty acids, sterols, squalene, and vitamin E. The refinery process itself is the cause of a significant amount of loss in vitamin E due to distillation and thermal oxidation. The distillate waste has a very limited commercial value, therefore requires additional costs for a safe environmental disposal. One of the main vitamin E oxidation products found in large quantities in oil waste is tocopherolquinone (TQ). A literature search has revealed that in the past several techniques including a variety of solvent extractions, saponification, or column extraction have been used for TQ isolation with limited success. The present study is a new cost‐effective liquid–liquid extraction method developed to isolate α‐TQ from vegetable oil steam distillate or distillate waste. High recovery results ranging from 31 to 120% were obtained depending on the ratio between the sample and three different organic extraction solvents (acetonitrile, methanol, and hexane) combined.     

Occurrence of cholesterol as a major sterol component in leaf surface lipids

Cholesterol has been detected as one of the major sterols in the surface lipids of higher plant leaves. It was widely distributed among the plant leaves of various species as a common main sterol component with a few exceptions. The content of cholesterol amounted to 71.5% of the total sterols in the surface lipids of rape leaves. However, the proportion of cholesterol in the intracellular lipids of rape leaves was lower than that in the surface lipids, and the seed lipids contained only a trace amount of cholesterol, as reported in the literature. In the leaf surface lipids examined, a minor amount of cholestanol associated with cholesterol often was detected by capillary gas chromatography and gas chromatography-mass spectrometry. The related analysis for the surface lipids of fruits showed that cholesterol was one of the major component sterols also in those lipids of several species.     

<Superscript>31</Superscript>P NMR quantification and monophasic solvent purification of human and bovine lens phospholipids

Most lipid extraction procedures [Folch, J., Lees, M., and Sloane-Stanley, G.H., (1957) A Simple Method for the Isolation and Purification of Total Lipids from Animal Tissues, J. Biol. Chem. 226, 497–509; Bligh, E.G., and Dyer, W.J. (1959) A Rapid Method of Total Lipid Extraction and Purification, Can. J. Biochem. Physiol. 37, 911–917] employ biphasic solvent mixtures designed to dissolve the lipids in an organic phase and remove impurities in an aqueous phase. However, when applying these protocols to biological matrices such as that of the ocular lens, the formation of an emulsion layer between the organic and aqueous phases causes poor reproducibility in extraction yields and gives only a small amount of the lipid-containing chloroform phase. In this study, we quantified phospholipids at each step of the Folch et al. extraction protocol and compared the yield of human and bovine lens phospholipids obtained by the Folch-based approach and a novel monophasic methanol extraction method designed to circumvent the problems associated with biphasic extraction protocols. A monophasic methanol extraction coupled with ³¹P NMR spectroscopy was found to be the simplest, quickest, and most effective method for quantifying the phospholipid content of the lens.     

Sterol and steryl ester regulation of phospholipase A2 from the mosquito parasiteLagenidium giganteum

Lagenidium giganteum, a facultative parasite of mosquito larvae, cannot synthesize sterols, and requires an exogenous source of these lipids in order to enter its reproductive cycle. This parasite grows vegetatively in the absence of sterols, but requires cholesterol or structurally related compounds to produce motile zoospores, which are the only stage capable of infecting mosquitoes. Sterols structurally related to cholesterol and some steryl esters inhibited the activity ofL. giganteum phospholipase A₂ (PLA₂), an enzyme that hydrolyzes fatty acids from thesn-2 position of glycerophospholipids. Sterols that induce reproduction inhibitedL. giganteum PLA₂ activity, while sterols and steroids that do not support sporulation had minimal effect. Most steryl esters had no effect on enzyme activity, but cholesteryl arachidonate (CA) was a potent inhibitor of parasite PLA₂. Not all enzymes partly purified using a DEAE-Sephacel column were affected by these lipids, demonstrating selective inhibition of specific enzymes. Potency was enhanced by up to several orders of magnitude if epoxy fatty acids were esterified to the cholesterol nucleus. The steryl ester pool was dynamic during morphogenesis, and the fatty acid composition of the steryl esters did not mimic total cell or membrane (glycerophospholipid) fatty acid composition asL. giganteum proceeded through its growth cycle. Synthesis of CA and monoepoxy CA by the parasite was confirmed using electrospray mass spectrometry and collision-induced dissociation. Steryl derivatives selectively inhibited PLA₂ enzymes from bovine pancreas, snake venom, and human cytoplasmic 85-kDa PLA₂.     

Improved Methods for the Fatty Acid Analysis of Blood Lipid Classes

Two improved methods have been developed for preparation of fatty acid methyl esters (FAME) from major O-ester lipid classes in blood, i.e., cholesterol ester, triacylglycerol, and glycerophospholipids. The methods involve simple operations, and use neither harmful solvents such as chloroform or benzene nor highly reactive volatile reagents such as acetyl chloride. The FAME synthesis reaction proceeds under mild temperature conditions. The methods include (1) extraction of lipids from 0.2 ml of blood with 0.2 ml of tert-butyl methyl ether and 0.1 ml of methanol, (2) separation of the total lipids into lipid classes using a solid-phase extraction column or thin-layer chromatography, and (3) methanolysis of each lipid class at room temperature or at 45 °C. In all the operations, solvent concentration is performed only once prior to gas–liquid chromatography (GC). No noticeable differences in composition determined by GC have been found between FAME prepared by the present methods and those prepared by a conventional method involving lipid extraction with chloroform/methanol. The mild reaction and simplified procedures of the present methods enabled safe and reproducible analysis of the fatty acid compositions of the major ester-lipid classes in blood.