Sterols in hardening winter wheat

The main sterols in winter wheat crowns and roots were sitosterol and campesterol, with significant amounts of stigmasterol and traces of cholesterol. The main groups of sterol-containing lipids were free sterols, steryl glucosides, steryl esters and esterified steryl glucosides. Sterol analysis within each group showed little difference between them. Steryl esters were relatively rich in cholesterol and poor in stigmasterol. Free sterols were rich in stigmasterol. Low temperature caused an increase in sterol content but had little effect on sterol composition and sterol to lipid P ratio. There was some increase in steryl esters and some decrease in free sterols. Cholesterol and stigmasterol decreased in the steryl ester and free sterol fractions, respectively. There was little evidence for involvement of sterols in winter wheat frost hardening.     

Synthesis of fatty acid sterol esters using cholesterol esterase from Trichoderma sp. AS59

We recently reported the characterization of novel cholesterol esterase (EC. 3.1.1.13) from Trichoderma sp. and preliminary work on sterol ester synthesis. In the present study, we further examined the enzyme ability to synthesize cholesterol esters from cholesterol and free fatty acids of various chain lengths, and compared the fatty acid specificity in synthesis with that in hydrolysis. The enzyme catalyzed the synthesis of medium- and long-chain fatty acid cholesterol esters, but failed to synthesize short-chain fatty acid esters. The fatty acid specificities in the synthesis and hydrolysis of cholesterol esters were entirely different from each other. Unlike other lipolytic enzymes, the enzyme was largely independent of water content in the synthesis of cholesterol oleate, and it achieved near-complete esterification in the presence of an equimolar excess of oleic acid. Of additional interest is the finding that the addition of n-hexane markedly enhanced the esterification activities on all the medium- and long-chain saturated fatty acids used. Based on these findings, we attempted to synthesize stigmasterol stearate as a food additive to lower cholesterol levels in blood plasma, and found that the enzyme catalyzed effective synthesis of the ester without the need of dehydration during the reaction, indicating the potential utility of the enzyme in the food industry.     

Die quantitative verteilung der sterine und sterinderivate in organen von Solanum dulcamara

The following sterols were found in the roots, stems, leaves, unripe and ripe fruits of Solanum dulcamara: cholesterol, sitosterol, stigmasterol, campesterol, brassicasterol, isofucosterol and 24-methylenecholesterol. The most abundant components are cholesterol, sitosterol and stigmasterol (77–84%). In all parts of the plant the sterols are present in the free form and as esters, glycosides and acylated glycosides. The total sterol content and the content of combined forms were determined photometrically. In the leaves 58% of the sterols were found in the form of glycoside (26%), acylated glycoside (29%) and ester (2%). In the roots only 25% of the sterol were found in combined form. In the other organs the ratio of free and combined sterols was intermediate. In all cases, the ester fraction was the least.     

Effect of substrate composition and concentration on aortic cholesterol ester hydrolase activity.

Cholesterol ester hydrolase activity of pig aorta has been examined under optimum experimental conditions for hydrolysis of different cholesterol esters. The enzyme specific activity values were in the numerical order of substrates hydrolyzed: cholesteryl linoleate larger than or equal to linolenate greater than palmitate larger than or equal to stearate greater than oleate. The results are discussed in relation to the arterial accumulation of cholesterol esters.     

Genetic control of sterol esterification in developing wheat endosperm.

1. The action of gene Pln, previously characterized by the sterol ester patterns of mature whole wheat kernels, has been found to be restricted to the endosperm and not to affect the embryo, the pericarp or the seed coat. 2. The dominant allele Pln, which determines a sterol ester pattern with palmitate as the main ester, is also responsible for a low level of free sterol at maturity. A high level of free sterol is associated with the recessive allelet pln, which determines an ester pattern with linoleate as the main ester. 3. Divergence between the two phenotypes starts at about 21 days after anthesis, when cell proliferation has been completed, the aleurone layer has differentiated, and only cell enlargement is taking place. A marked increased in esterification, mainly by palmitate, which is controlled by the dominant allele, is concomitant with a sharp decrease in free sterol. 4. The increased net esterification is non-specific with respect to 4-demethyl sterols, because it affects the four main ones, namely sitosterol, stigmasterol, campesterol and cholesterol.     

Micellar distribution of cholesterol and phytosterols after duodenal plant stanol ester infusion

Properties of the intestinal digestion of the dietary phytosterols, cholesterol and cholestanol, and the mechanisms by which phytosterols inhibit the intestinal absorption of cholesterol in healthy human subjects are poorly known. We have studied the hydrolysis of dietary plant sterol and stanol esters and their subsequent micellar solubilization by determining their concentrations in micellar and oil phases of the jejunal contents. Two liquid formulas with low (formula 1) and high (formula 2) plant stanol concentrations were infused via a nasogastric tube to the descending duodenum of 8 healthy human subjects, and intestinal contents were sampled for gas-liquid chromatographic sterol analysis 60 cm more distally. During the duodenal transit, phytosterol esters were hydrolyzed. This was especially profound for sitostanol, as its esterified fraction per milligram of sitosterol decreased 80% (P < 0.001) in formula 1 and 61% (P < 0.001) in formula 2. Contrary to that, esterified fraction of cholesterol per milligram of sitosterol was increased fourfold (P < 0.001) in formula 1 and almost sixfold (P < 0.001) in formula 2, whereas that of cholestanol remained unchanged. Percentages of esterified sterols and stanols in total intestinal fluid samples were higher after the administration of formula 2 than of formula 1. Esterified cholesterol and stanols accumulated in the oil phase, and free stanols replaced cholesterol in the micellar phase. At high intestinal plant stanol concentrations, cholesterol looses its micellar solubility possibly by replacement of its free fraction in the micellar phase by hydrolyzed plant stanols, which leads to a decreased intestinal absorption of cholesterol.     

Effects of dietary sterols on development and reproduction of southwestern corn borer Diatraea grandiosella Dyar

Southwestern corn borer larvae, Diatraea grandiosella Dyar, were reared on artificial diets containing individual sterols (cholesterol, sitosterol, or stigmasterol) in concentrations ranging from 0.05 to 0.2%. Female larvae developed to pupae more rapidly as sitosterol and stigmasterol were increased in the diets. Increased cholesterol concentrations did not affect the larval period significantly, and development was not as rapid as with the phytosterols. Female larvae developed at significantly slower rates in all diets than did males, except at the highest concentrations of sitosterol and stigmasterol. Female pupae and adults were significantly heavier than the males, and pupal and adult weight increased as sterol concentrations increased. Number of eggs laid per fertilized female and egg hatchability were significantly increased as concentrations of the three sterols were increased in the larval diets. Sitosterol-reared females produced more eggs than did females reared on other sterols but egg hatchability was not significantly different among sterols.     

Changes in glycerolipids and their fatty acid composition during maturation of tobacco seeds

Triacylglycerol, which was one of the minor lipid components in immature seeds of tobacco, accumulated dramatically between 7 and 27 days after flowering and, in mature seeds at 37 days, the fatty acid methyl esters of the triacylglycerols comprised 96.3% of those of the total lipids. Diacylglycerols and sterol ester also increased significantly during seed development. Phosphatidylcholine and phosphatidylethanolamine, which were major components in immature seeds, decreased constantly with increasing maturation as well as the quantities of phosphatidylinositol and phosphatidylglycerol. Monogalactosyldiacylglycerols, digalactosyldiacylglycerols and sulfoquinovosyldiacylglycerols also decreased and disappeared in mature seeds. In the triacylglycerols the percentages of palmitate, stearate and linolenate fell with increasing seed age, while that of linoleate increased up to 75.3% in mature seeds. A similar trend was observed in the fatty acid composition in the diacylglycerols and sterol ester. Generally, in the phospholipids the proportions of linoleate and linolenate decreased with concomitant increases of stearate and oleate.     

Modulation of plant mitochondrial VDAC by phytosterols

We have investigated the effect of cholesterol and two abundant phytosterols (sitosterol and stigmasterol) on the voltage-dependent anion-selective channel (VDAC) purified from mitochondria of bean seeds (Phaseolus coccineus). These sterols differ by the degree of freedom of their lateral chain. We show that VDAC displays sensitivity to the lipid-sterol ratio and to the type of sterol found in the membrane. The main findings of this study are: 1), cholesterol and phytosterols modulate the selectivity but only stigmasterol alters the voltage-dependence of the plant VDAC in the range of sterol fraction found in the plant mitochondrial membrane; 2), VDAC unitary conductance is not affected by the addition of sterols; 3), the effect of sterols on the VDAC is reversible upon sterol depletion with 10 μM methyl-β-cyclodextrins; and 4), phytosterols are essential for the channel gating at salt concentration prevailing in vivo. A quantitative analysis of the voltage-dependence indicates that stigmasterol inhibits the transition of the VDAC in the lowest subconductance states.     

Fatty acid and sterol specificity in the biosynthesis of steryl esters by enzyme preparations from spinach leaves

Acetone powders prepared from the 20,000g participate fraction of spinach (Spinacia oleracea L.) leaves catalyzed the formation of steryl esters from free sterol and 1,2-diacylglycerol as the acyl donor. There was no sterol specificity when cholesterol, sitosterol, and campesterol were compared. When rates of sterol ester biosynthesis were compared using different 1,2-diacylglycerols it was found that the shorter chain fatty acids and the more unsaturated fatty acids were preferred. When the substrate concentration of diacylglycerol was varied, the maximal velocities obtained with the different substrates were dipalmitoleoyl- >dilinolenoyl- >dioleoyl- >dilinoleoyl-glycerol. It was demonstrated by silver nitrate thin-layer chromatography that the fatty acids of the supplied diacylglycerols were transferred to the sterol. When diacylglycerol mixtures were supplied, it was found that unsaturated diacylglycerols greatly stimulated conversion of saturated diacylglycerols to saturated steryl esters. For an equimolar mixture of dipalmitoyl-, dioleoyl-, dilinoleoyl-, and dilinolenoyl-glycerol, about equal amounts of the four steryl ester species were synthesized.     

Effect of Light on Mevalonic Acid Incorporation into the Phytosterols of Nicotiana tabacum L. Seedlings

Mevalonic acid-2-¹⁴C was readily incorporated into the free, esterified, and glycosidic sterol fractions of tobacco (Nicotiana tabacum L. var. Burley 21) seedlings. The time course of mevalonic acid-2-¹⁴C incorporation was different for the various individual sterols. Campesterol and sitosterol (group I) became radioactive as the free sterol and subsequently as the steryl ester. The reverse order was observed for cholesterol and stigmasterol (group II). Light stimulated the incorporation of mevalonic acid-2-¹⁴C into the group I free sterols and during the first 6 to 9 hours into the steryl esters of group II. The increase in specific radioactivity of the group II steryl esters was followed by a decline. Based on time course studies it is suggested that the group II steryl esters turn over rapidly and that light influences the rate of turnover.     

Shading Influence on the Sterol Balance of Nicotiana tabacum L

Tobacco plants (Nicotiana tabacum L.) were grown in the field and the apex was removed at the 42-day stage. Shading screens were set up which produced 0, 26, 67, and 90% shade. Plants were grown an additional 25 days before leaves from top, middle, and bottom stalk positions were harvested. Each leaf group was analyzed for free sterol, steryl ester, steryl glycoside, and acylsteryl glycoside. The free sterol content was lowest in top leaves and highest in bottom leaves; however, the top leaves had more steryl ester than the bottom leaves. Leaf position had no effect on steryl glycosides and acylsteryl glycosides. Shading did not influence the level of any sterol class; but in general, shading increased stigmasterol and decreased sitosterol. This trend was observed for all sterol classes, and the free sterols showed the largest and most consistent change. The younger top leaves showed a greater response than the older bottom leaves, but bottom leaves always had more stigmasterol than sitosterol even without shade.     

Characterization of Soybean Plasma Membrane during Development: FREE STEROL COMPOSITION AND CONCANAVALIN A BINDING STUDIES

Plasma membrane preparations from soybean root and hypocotyl contained the following free sterols: cholesterol, campesterol, stigmasterol, and sitosterol. The cholesterol level was relatively low in root plasma membrane (less than 0.5%) but was 1.4 to 2.4% in hypocotyl membrane. The relative levels of the three other sterols fluctuated with cellular development and tissue source. Campesterol level decreased with the development of both root and hypocotyl membrane. With development, stigmasterol increased greatly in root membrane but remained constant in hypocotyl membrane, and sitosterol, the major free sterol component of all membrane preparations, decreased in root membrane but increased slightly in hypocotyl membrane.     

Free sterols,steryl esters,glucosides, acylated glucosides and water-soluble complexes in Calendula officinalis

In 3- and 14-day-old seedlings and in the leaves of Calendula officinalis the following sterols were identified: cholestanol, campestanol, stigmastanol, cholest-7-en-3-β-ol, 24-methylcholest-7-en-3β-ol, stigmast-7-en-3β-ol, cholesterol, campesterol, sitosterol, 24-methylcholesta-5,22-dien-3β-ol, 24-methylenecholesterol, stigmasterol and clerosterol. Sitosterol was predominant in young and stigmasterol in old tissues. Young tissues contained relatively more campesterol but in old tissues a C₂₈Δ^5,22 diene was present suggesting transformation of campesterol to its Δ^5,22 analog, similar to that of sitosterol to stigmasterol. All the identified sterols were present as free compounds and also in the steryl esters, glucosides, acylated glucosides and water-soluble complexes. The variations in the amounts of these fractions in the embryo axes and cotyledons of 3- and 14-day-old seedlings and the distribution of individual sterols among the fractions are discussed.     

Optimisation of plant sterols incorporation in human keratinocyte plasma membrane and modulation of membrane fluidity.

The in vitro effects of plant sterols were investigated with regard to their uptake and membrane lipid fluidity in human keratinocytes. Among the different media tested to transport sterols (liposomes, micelles and organic solvents), the best results in terms of incorporation and viability were obtained by the use of the organic solvents dimethylsulfoxide and ethanol. After 48 h incubation exogenous sterol can account for about 30% of the total cell sterol content. The total sterol amount in plasma membranes increased 2-fold after incubation with cholesterol, whereas it was not altered when phytosterols were incorporated. The incorporation of cholesterol, sitosterol and stigmasterol led to an increase in the percent of unsaturated fatty acid C18:1 in the plasma membrane. The effect of this uptake on membrane fluidity was studied by means of fluorescence polarisation using DPH and TMA-DPH as fluorescent probes. Whereas cholesterol and sitosterol had no significant effect on the DPH fluorescence anisotropy (rs), the presence of stigmasterol induced a 12% decrease of rs reflecting an increase in membrane fluidity. We can conclude from this study that in the presence of sitosterol, the mean fluidity of the membrane is regulated whereas stigmasterol triggers a looseness of molecular packing of phospholipids acyl chains, in accordance with previous results obtained on purely lipid model membranes.     

Effects of substrate composition on the esterification and hydrolysis activity of lysosomal acid sterol ester hydrolase

Lipid microemulsions with various core and surface lipid compositions were prepared by co-sonication of cholesteryl esters, triolein (TO), egg phosphatidylcholine (egg PC), and cholesterol. The heterogeneous emulsion particle mixture was purified by gel filtration and particles with the size and general organization of low density lipoproteins were obtained. These lipid microemulsion particles were used for studies of the cellular metabolism of lipoprotein-derived cholesterol and cholesteryl esters as catalyzed by the enzyme acid sterol ester hydrolase (EC 3.1.1.13). The hydrolysis of cholesteryl oleate (CO) was more than twice and that of cholesteryl linoleate (CL) more than three times faster than the hydrolysis of cholesteryl stearate (CS) over the temperature range 25-39.6 degrees C. Both the synthesis and hydrolysis of cholesteryl esters were insensitive to the physical state of the microemulsion cores. The synthesis of cholesteryl esters by this enzyme was also insensitive to the ratios of cholesterol and egg PC in the microemulsion surface layers. Incorporation of triolein into the microemulsion cholesteryl ester core slightly increased the rate of cholesteryl ester synthesis. A decreasing fatty acyl chain length (C18:0 to C14:0) and an increasing degree of unsaturation (C18:0 to C18:2) enhanced the synthesis rate. It is suggested that the hydrolysis and synthesis of cholesteryl esters in microemulsions (and lipoproteins) take place only in the particle surface layer and that the rate of catalysis is directly dependent on the amount of substrate in this surface layer.     

Foliar sterols in soybeans exposed to chronic levels of ozone

Soybeans (Glycine max) exposed to chronic levels of ozone showed a linear decrease in biomass with increasing concentration. The foliar free sterols increased while the steryl ester, and the steryl glycosides, a minor component, decreased with increasing pollutant concentration. Of the free sterols, stigmasterol showed the largest increase, followed by sitosterol; campesterol, however, decreased. All steryl esters decreased; sitosterol showed the largest decrease and campesterol the least.     

The sterol esters of maize seedlings

1. The composition of the sterol ester fraction of the shoot, root, scutellum and endosperm of 10-day-old maize seedlings was investigated. 2. The scutellum and endosperm together contain 80% of the sterol ester of the seedling. 3. beta-Sitosteryl linoleate is the major sterol ester of the scutellum and endosperm. 4. beta-Sitosteryl and stigmasteryl palmitate, palmitoleate, oleate and linoleate are the major sterol esters of the root. 5. In the shoot phytosterol linoleate is less abundant than phytosterol myristate, palmitate, palmitoleate and oleate. 6. There is a greater proportion of cholesteryl ester in the shoot than in the other tissues of the seedling.     

Sterol Changes during Germination of Nicotiana tabacum Seeds

The identity, composition, and concentration of the total, free, esterified, and glycosidic sterol fractions were determined during germination of tobacco seeds. The total, free, and esterified sterols increased, with stigmasterol and campesterol accounting for most of the increase. Steryl glycosides decreased during germination, and stigmasteryl and sitosteryl glycosides showed the largest decrease. During germination, sitosterol was the major sterol in all fractions but stigmasterol and campesterol showed the greatest changes. The fatty acid composition of the steryl esters and acylated steryl glycosides most closely resembled the di- and triglycerides.     

Purification and characterization of a novel cholesterol esterase from Pseudomonas aeruginosa,with its application to cleaning lipid-stained contact lenses

With the aim of developing a new cholesterol esterase for eliminating lipids on used contact lenses, microorganisms were screened for the enzyme activity. A Pseudomonas aeruginosa isolated from soil was found to produce a desirable enzyme. The enzyme had an isoelectric point of 3.2, and molecular mass of 58 kDa. The optimal temperature was around 53 degrees C at pH 7.0, and the optimal pH was from 5.5 to 9.5. The enzyme was stable between pH 5 and 10 for 19 h at 25 degrees C, and retained its activity up to 53 degrees C on 30 min of incubation at pH 7.0. The rates of hydrolysis of cholesteryl esters of different fatty acids were in the following order: linoleate > oleate > stearate > palmitate > caprylate > myristate > laurate, caprate > caproate > butyrate, acetate. Addition of (tauro)cholate to a final concentration of 100 mM markedly promoted the hydrolysis of triglycerides of short-, medium-, and long-chain fatty acids. When used with taurocholate, the enzyme acted as an effective cleaner for contact lenses stained with lipids consisting of cholesteryl oleate, tripalmitin, and stearyl stearate.