Esterified Sterols Research Articles

The biosynthesis of the free sterols and sterol esters of Neurospora crassa

The composition of the free and esterified sterols in Neurospora crassa was examined as a function of incubation time in starvation medium containing [2-14C]mevalonic acid. The 14C incorporation was monitored in nuclear methylated and 4,14-desmethyl sterol fractions. After 7 h incubation, sterol esterification had increased from an initial 5% in the log phase culture to 48% of the total sterol pool, with a concomitant decrease in free sterols. The relationship of the free and esterified sterol components in ergosterol biosynthesis is discussed.

Effect of altered phospholipid head groups on the distribution of cholesterol in the housefly ( Musca domestica) larvae

The phospholipid composition of larvae of the housefly, Musca domestica, was altered by rearing them on defined diets containing adequate choline, no added choline, and choline plus 2-aminobutan-1-ol (2-AB-1-ol). The distribution of sterols in various tissues of insects with altered phospholipid head groups was examined and the sterol content related to the phospholipid and protein content. The results indicate that addition of 2-AB-1-ol to the control diets did not produce any significant changes in the sterol content (nmole/tissue) of various larval tissues. However, choline deficiency affected the growth of larvae. The composite gut fraction (gut, salivary glands and Malpighian tubules) of choline deficient insects contained a lower ratio of free sterol to protein or phospholipid than was found in the other two groups. The addition of graded amounts of choline to the deficient larval diets caused the molar ratio of sterol to phospholipid of the composite gut fraction to increase while that of the haemolymph fell in a complementary manner until approx. 1.40 μmole choline/g of casein had been added to the diets. The dietary regimes studied did not affect significantly the distribution of total sterols between polar, esterified and free sterols in the insect. The observed results following modification of phospholipid head groups in the tissues of insects are discussed.

Fatty acid profiles of esterified sterol glucosides from juice vesicles of citrus fruits

Vesicular lipids of 24 citrus cultivars were extracted and purified, and the esterified sterol glucosides (ESG) separated from other vesicular lipids by chromatography. Fatty acids of the ESG were analysed by gas-liquid chromatography as their methyl esters. Statistical analysis showed that five out of six citrus species or biotypes could be differentiated from each other on the basis of profiles of the five major ESG fatty acids. Differences between midand late-season sweet orange cultivers were detected. Small differences were observed between white- and red-fleshed grapefruit cultivars. Profiles of the minor acids showed higher relative percentages of iso-branched fatty acids in lemons and limes, and higher relative percentages of 12: 0 and 14: 0 fatty acids in mandarin and mandarin hybrids than in other cultivars.

Dietary restriction and the development of atherosclerosis

1. White Carneau pigeons develop genetic atherosclerosis. The aorta shows intimal cushions at birth, cholesterol accumulation at 9 months of age and definite atherosclerotic lesions by 3 years of age. To determine whether these age-related arterial changes can be prevented by dietary restriction started early in life, 3-month-old pigeons were subjected to dietary restriction (40% of ad lib. diet). 2. Plasma cholesterol levels, cholesterol excretion and aortic cholesterol concentrations were compared with those of pigeons on the ad lib. diet. The pigeons on dietary restriction showed a decrease of 30% in weight. The plasma cholesterol levels of these pigeons decreased 20% when compared with those of ad lib. group. In the dietary-restricted group, both the faecal excretion of bile acids (P less than 0.01) and the aortic cholesterol level (P less than 0.02) were significantly reduced, with changes in the free and in the esterified sterol fraction. 3. This study shows, for the first time, that age-related accumulation of aortic cholesterol can be prevented or at least delayed by dietary restriction started early in life.

Annual Variation in the Effect of Red Light on Sterol Biosynthesis in Digitalis purpurea L.

The effect of varying sequences of red and far red light on sterol biosynthesis in etiolated seedlings of Digitalis purpurea L. was examined. Red light caused a marked increase in the amounts of free and glycosidic sterols and a small decrease in esterified sterols during the first 4 hours after illumination. Far red light elicited the same response but to a lesser degree. Exposure to red followed by far red light or the reverse caused little or no increase in the amounts of free and glycosidic sterols. The magnitude of the increase in the amounts of sterols varied, depending upon the season in which the experiments were performed. The largest increments were obtained during the summer and fall, whereas the smallest were observed during the winter and spring. Correlation of these data with previous observations of an annual cycle in the sterol content of Digitalis seedlings showed that the maximum stimulation in sterol biosynthesis occurs when the endogenous level of sterols is minimal.Sterol monoglycosides, acylmonoglycosides, and an unidentified sterol conjugate from the lipid extracts were quantitated. Changes in conjugated sterol content were related to the particular light conditions of each experiment. The results are discussed in terms of physiological cycles and the possible influence of hormones upon the control of sterol biosynthesis in Digitalis.

Polar lipids of sun flower (Helianthus annuus L.) seeds

Qualitative and quantitative analysis of polar lipids in sun flower (Helianthus annuus L.) seed is reported in the present study. Ten classes of polar lipids i.e. phosphatidic acid, phosphatidyl inositol, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, diphosphatidyl glycerol, digalactosyl diglyceride, monogalactosyl diglyceride, esterified sterol glycoside and free fatty acids have been identified. Phosphatidyl choline and phosphatidyl inositol formed the major phosphatide fractions. Only four fatty acids i.e. palmitic, stearic, oleic and linoleic acids were present in measurable amounts in the total polar lipids. There was a great variation in the fatty acid composition in different classes of polar lipids. Arachidic acid was present in all the individual phosphatide fractions. Phosphatidyl glycerol was highly unsaturated followed by esterified sterolglycoside, phosphatidic acid, phosphatidyl ethanolamine + phosphatidyl glycerol, diphosphatidyl glycerol and monogalactosyl diglyceride.

Metabolic interconversion of free sterols and steryl esters in Saccharomyces cerevisiae

The interconversion of free and esterified sterols was followed radioisotopically with [U-14C]acetate and [methyl-14C]methionine. In pulse-chase experiments, radioactivity first appeared mainly in unesterified sterols in exponential-phase cells. Within one generation time, the label equilibrated between the free and esterified sterol pools and subsequently accumulated in steryl esters in stationary-phase cells. When the sterol pools were prelabeled by growing cells aerobically to the stationary phase and the cells were diluted into unlabeled medium, the prelabeled steryl esters returned to the free sterol form under several conditions. (i) During aerobic growth, the prelabeled sterols decreased from 80% to 45% esters in the early exponential phase and then returned to 80% esters as the culture reached the stationary phase. (ii) Under anaerobic conditions, the percentage of prelabeled steryl esters declined continuously. When growth stopped, only 15% of the sterols remained esterified. (iii) In the presence of an inhibitor of sterol biosynthesis, which causes accumulation of a precursor to ergosterol, prelabeled sterols decreased to 40% steryl esters while the precursor was found preferentially in the esterified form. These results indicate that the bulk of the free sterol and steryl ester pools are freely interconvertible, with the steryl esters serving as a supply of free sterols. Furthermore, there is an active cellular control over what types of sterol are found in the free and esterified sterol pools.

The content and composition of sterols and sterol esters in low erucic acid rapeseed (Brassica napus).

The low temperature crystallization technique for the enrichment of "minor" components, such as sterols and sterol esters, from vegetable oils was applied to low erucic acid rapeseed oils. The recovery of free sterols and sterol esters was estimated by use of(14)C-cholesterol and(14)C-cholesterol oleate. 80% of the free sterols and 45% of the sterol esters were recovered in the liquid fraction, while in two studies total recoveries were 95% and 99%, respectively. This technique showed some selectivity toward the sterol bound fatty acids when compared to direct preparative thin layer chromatography (TLC) of the crude oil. Gas liquid chromatography (GLC) analysis of the free and esterified sterols as TMS-derivatives showed very little selectivity in the enrichment procedure. The fatty acid patterns of the sterol esters demonstrated, however, a preference in the liquid fraction for those sterol esters which have a high linoleic and linolenic acid content. The content of free sterols was 0.3-0.4% and that of sterol esters 0.7-1.2% of the rapeseed oils in both winter and summer types of low erucic acid rapeseed (Brassica napus) when the lipid classes were isolated by direct preparative TLC of the oils. The free sterols in the seven cultivars or breeding lines analyzed were composed of 44-55% sitosterol, 27-36% campesterol, 17-21% brassicasterol, and a trace of cholesterol. The esterified sterols were 47-57% sitosterol, 36-44% campesterol, 6-9% brassicasterol, and traces of cholesterol and Δ5-avenasterol. The fatty acid patterns of these esters were characterized by ca. 30% oleic acid and ca. 50% linoleic acid, whereas these acids constitute 60% and 20%, respectively, of the total fatty acids in the oil. Little or no variation in sterol and sterol ester patterns with locality within Sweden was observed for the one cultivar of summer rapeseed investigated by the low temperature crystallization technique.

Sterol lipids in finger millet (Eleusine coracana).

AbstractNeutral lipids, glycolipids, and phospholipids (1.3%, 0.25%, and 0.10% of seed weight) were isolated from the total lipids (chloroform‐methanol) of finger millet seeds(Eleusine coracana), and four sterol‐containing lipids further isolated from neutral and glycolipids by preparative column and thin layer chromatography. On seed weight, these comprised: free sterols (S) 0.091%, sterol esters (SE) 0.013%, sterol glycosides (SG) 0.025%, acyl sterol glycosides (ASG) 0.020%, and total 0.149%. The major fatty acids, totaling 85–90%, were the same in both esterified sterols, but the proportions varied: 16:0, 18:1, and 18:2 comprising 24, 49, and 17% in SE (calculated iodine value 75) and 43, 36, and 7% in ASG (calculated iodine value 46). All four sterol lipids contained 80–84% of β‐sitosterol, the remainder being stigmasterol. The only sugar in SG and ASG was D‐glucose. It is deduced that the major representative species are: SE, β‐sitosterol oleate/palmitate; SG, β‐D‐glucopyranosyl‐(l → 3)‐β‐sitosterol; and ASG, 6–0‐palmitoyl‐β‐D‐glucopyranosyl‐(l → 3)‐β‐sitosterol.

Human endothelial cells in primary culture incorporation of acetate and mevalonate into lipids

Incorporation of [2- 14C]mevalonate and [2- 14C]acetate into lipids was measured in primary cultures of human endothelial cells isolated from the intima of umbilical veins. (1) In culture medium supplemented with 20% (v/v) serum, the removal of lipid or lipoproteins stimulated the conversion of acetate into sterols, while incorporation into the fatty acid and esterified sterol fractions decreased. (2) Mevalonate incorporation into sterols also increased significantly, indicating feedback inhibition at postmevalonate steps. (3) A labeled fatty acid fraction obtained after incubation with labeled mevalonate confirms that mevalonate also can serve as a substrate for fatty acids. (4) When incorporation into sterols was stimulated by lipoprotein removal, labeled sterols accumulated mainly intracellularly when either acetate or mevalonate was used as precursor. Thus endothelial cells are sensitive to the same negative feedback control mechanisms previously demonstrated in skin fibroblasts and smooth muscle cells.

Quantitative aspects of free and esterified sterols in Saccharomyces cerevisiae under various conditions

For extraction of free and esterified sterols from yeast cells, a method was devised in which both forms of sterols were extracted with light petroleum after the treatment of the cells with acetone, and then with dimethylsulfoxide. The content of sterol esters in the cells under aerobic conditions markedly increased with time, amounting to 95% of the total sterols under some conditions. However, the formed sterol esters were decreased, accompanied with an increase of free sterols, when the cells were put under anaerobic conditions. Variations of radioactivities of both sterols which had been labeled in the side chain by incubation of the cells with [Me- 14C]methionine were examined on the cells grown under various conditions. No variation was observed on the cells under aerobic conditions. On the other hand, the labeled esters were hydrolyzed to yield free sterols in the cells under anaerobic conditions. In the cells under aerobic conditions, the free sterols were found to consist mainly of ergosterol, whereas the esterified sterols contained considerable amounts of zymosterol, lanosterol, and other intermediate sterols besides ergosterol.

Biochemical and morphological characterization of mycobacteriophage R1

Large-scale propagation of mycobacteriophage R1 in broth culture has allowed the isolation of quantities of virus sufficient for characterization of its nucleic acid and lipid components as well as investigation of its ultrastructural attributes. Analysis of R1 DNA indicates that it is double stranded and possesses a molecular weight of 2.5 X 10(7) and a guanine-plus-cytosine content of 65.7 +/- 0.5%. The lipid fraction of R1 accounts for 14% of the total dry weight of the virus, 20% of which was identified as free or esterified sterols. A rapid loss of viral titer occurred after seconds of exposure to organic solvents. This result suggests that the lipid fractions of R1 is essential for its infectivity. Electron microscopic investigation of solvent-extracted R1 showed extensive deterioration of its normal morphology, including nucleocapsid disintegration and base plate separation. Routine phosphotungstate preparations demonstrated a particle with an oval head and a noncontractile tail. Altering the pH of the phosphotungstate negative stain from neutrality damage the viral particles. Uranyl formate-contrasted specimens displayed an elongated hexagonal nucleocapsid with a neck region; the cross-striated tail possessed a starlike base plate.

Lipid composition of the yeast

The lipids of two strains of Lipomyces kononenkoae, grown in batch culture, were extracted and analysed. The major lipids present were phospholipids, free sterols, esterified sterols, and triacylglycerols. Phospholipid analysis indicated that phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol were the major ones. The fatty-acyl residues were C12-C18 and contained 67–74% unsaturated residues. Polyunsaturated residues accounted for 15% and 30% in L. kononenkoae CBS 2514 and L. kononenkoae CBS 5608, respectively. Analysis of the fatty-acyl residues of a low-density vesicle fraction obtained from sphaeroplasts of L. kononenkoae CBS 2514 was carried out and the results are discussed in relation to plasma membrane synthesis. The suitability of L. kononenkoae for production of single-cell protein is also discussed.

24-methylenepollinastanol and 24-dehydropollinastanol—two sterols of pollen

The esterified and unesterified sterol fractions of bee-gathered mixed pollens were examined, and total sterol composition was determined. Two new sterols of pollens, 14α-methyl-9β,19-cyclo-5α-cholest-24-en-3β-ol (24-dehydropollinastanol) and 14α-methyl-5α-ergost-24(28)-en-3β-ol (24-methylenepollinastanol) were isolated and identified. Both sterols were found primarily in the esterified sterol fraction, and 24-methylenepollinastanol accounted for 43% of the sterols of this fraction. 24-Dehydropollinastanol and four other sterols which also contain a 9β,19-cyclopropane ring were found only in the esterified sterol fraction. 24-Methylenecholesterol was the major sterol of the unesterified sterol fraction.

Sterol composition ofNeurospora crassa.

The sterols extracted from freeze-dried, log-phase cultures ofNeurospora crassa were separated by thin layer and analyzed by capillary gas liquid chromatography and mass spectrometry. Ergosterol, episterol, and ergosta-7,22 dienol were the major sterols of the free sterol fraction. The major esterified sterols, which constituted 4.95% of the total sterol fraction, were ergosterol, episterol, and ergosta- 7,22,24(28)-trienol, with lesser quantities of 4α-methyl-ergosta-8,24(28)-dienol. With the exception of lanosterol, all sterols were alkylated at the C-24 position.

The effect of triparanol on the composition of free and esterified sterols of Saccharomyces cerevisiae

Triparanol altered the sterol composition of Saccharomyces cerevisiae and promoted an increase in the steryl ester and total sterol per organism. The accumulation of Δ 8 sterols, both free and esterified, in the presence of triparanol indicates that a major effect of the compound in yeast is the inhibition of the Δ 8→7 isomerase. Isolation of ergosta-5,8(9),22-trien-3 β-ol, hitherto detected only in ergosterol-deficient yeast mutants, further supports the concept that all of the other metabolic alterations required for the conversion of lanosterol to ergosterol can occur without the necessity of Δ 8→7 isomerization.

Isolation and Properties of Two Classes of Low-Density Vesicles from Saccharomyces cerevisiae

A mixture of small (0.43-mum diameter) and large (0.62-mum diameter) low-density vesicles from spheroplasts of Saccharomyces cerevisiae was fractionated by rate centrifugation in a gradient of 0 to 8% (wt/vol) Ficoll to yield fractions rich (90 to 95%) in small or large vesicles. The large, but not small, vesicles swelled when diluted into mannitol solutions containing less than 0.4 M mannitol. The pH-electrophoretic mobility curve of the large vesicles showed that they are probably enclosed in a phospholipid-protein membrane. The dyes neutral red and toluidine blue, accumulated into large vesicles by intact cells and spheroplasts, were largely lost from large vesicles when these were separated from stained spheroplasts. Sudan black III stained small and large vesicles, both classes of vesicle retaining the stain on separation. Fractions rich in large vesicles contained proportionately more phospholipid and less free sterols, diacylglycerols, and free fatty acids compared with those enriched in small vesicles. The two classes of vesicles contained about the same proportions of esterified sterols and triacylglycerols. The free fatty acids in both small and large vesicles were free from unsaturated fatty-acyl residues; diacylglycerols and triacylglycerols contained appreciable proportions of unsaturated fatty-acyl residues. Small vesicles were richer in lipase activity, whereas the larger vesicles contained greater beta-glucanase and alpha-mannosidase activities. Phospholipase activity could not be detected in any of the fractions.

Skin surface lipids of the dog.

The skin surface lipid of the dog has been reported to contain a high proportion of diol diesters having a lower mobility on thin layer chromatography than diesters from other species in spite of containing similar fatty acid and diol components. In the present study, dog skin surface lipid was separated by preparative thin layer chromatography into sterol esters (42%), wax diesters (32%), free sterols (9%), polar lipids (7%), and unidentified components (10%). The diesters contained 1,2-diols, each estrified with one long chain fatty acid and one isovaleric acid moiety. The diols were principally branched chain C21 and C22 compounds while the long chain fatty acids esterified with them were mainly C20 and C21 branched compounds. The fatty acids from the sterol esters were mostly saturated, branched chain C19 TO C23, together with 7% of straight chain monoenoic acids, principally C21 and C22. There were only trace amounts of free sterols other than cholesterol, while the esterified sterols contained 96% cholesterol and 4% lathosterol.

Effect of Plant Sterol Esters on the Absorption of Dietary Cholesterol

Cholesterol absorption decreases when either free or esterified plant sterols are added to the dietary fat. The effectiveness of various plant sterols, which were added to the dietary fat at the same molar concentration, in causing this decrease was determined in thoracic duct-cannulated rats. The oleate esters of β-sitosterol, campesterol, and stigmasterol, individually or in a mixture, were all similar in their ability to decrease the absorption of cholesterol from a diet that contained otherwise sterol-free triolein as the only fat. Their effectiveness was equal to that of free β-sitosterol or stigmasterol. β-Sitosterol esters of short (acetate), medium (decanoate), or long (oleate) chain fatty acids did not differ in their ability to lower cholesterol absorption. When the dietary fat contained 1% cholesterol, the addition of twice this amount of plant sterol resulted in a 30% reduction in cholesterol absorption. Increasing the amount of dietary plant sterols resulted in further decreases in the absorption of dietary cholesterol; the response had a log dose relationship. It is proposed that in the intestine, the plant sterol esters are hydrolyzed, and the resulting free sterol decreases the solubility of cholesterol in the oil and micellar phases with a consequent decrease in cholesterol absorption. A similar decrease in the percent absorption of dietary cholesterol ensued, if the level of dietary cholesterol was increased. It is proposed that this was brought about by the same mechanism as that responsible for the effect of the plant sterols. The extensive solubility of the phytosterol esters in fat, in contrast to the limited solubility of their unesterified forms, provides a means for administering effective amounts of these hypocholesterolemic agents.

Environmentally-induced changes in the neutral lipids and intracellular vesicles of Saccharomyces cerevisiae and Kluyveromyces fragilis

Saccharomyces cerevisiae, grown aerobically or anaerobically under conditions which induce a requirement for a sterol and an unsaturated fatty acid, synthesized approximately the same amounts of neutral lipid and intracellular low-density vesicles, although the neutral lipids in aerobically-grown cells contained more esterified sterol and less triacylglycerol than those in anaerobically-grown cells. Kluyveromyces fragilis synthesized much less neutral lipid and a smaller quantity of low-density vesicles than S. cerevisiae whether grown at 30 degrees C (generation time 1.1 h) or 20 degrees C (generation time 2.1 h). Both yeasts synthesized highly saturated triacylglycerols, relatively unsaturated phospholipids, and esterified sterols with an intermediate degree of unsaturation irrespective of the conditions under which they were grown. Free sterols in the yeasts were rich in ergosterol and 22(24)-dehydroergosterol, while the esterified sterol fractions were richer in zymosterol.