Steryl esters in a cell suspension culture of celery (Apium graveolens)

Arange of analytical techniques was used to investigate the composition of the steryl fatty acyl esters in a cell suspension culture of celery (Apium graveolens). Gas chromatography (GC) and GC-mass spectrometry (GC-MS), using electron ionization (EI) and negative ion chemical ionization (NICI), were employed to characterize the intact steryl esters. Assignments were supported by analysis of the sterol and fatty acid moieties released from the intact molecular species by alkaline hydrolysis. A selectivity for sterol esterification was noted, with the major free sterol, stigmasterol, occurring only in a very small amount in the esterified form. Instead, the precursors to Δ⁵-phytosterols, particularly cycloartenol, predominated in the ester fraction. The pentacyclic triterpene, β-amyrin, was also found as the palmitate and linoleate esters. Changes in composition and abundance of the steryl esters during the different growth phases of a celery cell suspension culture were investigated. The total amount of esterified sterols exceeded that of free sterols throughout the growth cycle. The changes observed during growth highlighted differences between the esters of precursor sterols and those of the 4-desmethyl-sterols, and it is postulated that the various steryl esters perform different functions in cell metabolism.     

Linear, steroidal, and triterpene esters, and steryl glycosides from Festuca argentina

Ester waxes and steryl glycosides of the grass Festuca argentina were studied. Saponification of the waxes from the petroleum ether extract led to n-hexacosanol as the major single linear alcohol, along with pentacyclic triterpenols, such as β-amyrin, germanicol, isobaurenol, lupeol, hopenol-a and hopeol, and low amounts of sterols, such as cholesterol, campesterol, stigmasterol, sitosterol and dihydrositosterol, identified by gas chromatography/mass spectrometry (GC/MS). Fatty acids were identified as methyl esters as C_12∶0, C_14∶0, C_16∶0, C_18∶0, C_18∶2, and C_20∶0. The occurrence of a wide chainlength range of fatty acids and a single linear alcohol closely matched for other reports on the tribe Festuceae. On the contrary, pentacyclic triterpenols with a variety of skeletons, especially isobauerenol, are not usual as esters of fatty acids in the Gramineae. Low amounts of steryl glycosides were also obtained from the methylene chloride percolate of the methanol extract. Upon acetylation followed by hydrolysis, aglycones were identified by capillary gas-liquid chromatography (GLC) and GC/MS. As Δ⁷-cholesterol, campesterol, stigmasterol, sitosterol, dihydrositosterol, and the sugars as glucose, xylose, and arabinose by GLC of the respective alditol acetates. This is the first report on the linear, steryl, and triterpenyl esters of F. argentina. It is noteworthy that Δ⁷-steryl glycosides are rare, and steryl monoarabinosides have not been proviously reported on the family Gramineae.     

Esterified lipids of the freshwater dinoflagellatePeridinium lomnickii

Steryl esters, phytyl esters and triacylglycerols of a naturally occurring freshwater dinoflagellate,Peridinium lomnickii, were identified using capillary gas chromatography-mass spectrometry (GC-MS). Steryl esters differing in degree of unsaturation were separated, prior to analysis, by argentation thin layer chromatography. 5α(H)-Cholestanol was more dominant, relative to 4α-methylstanols, in steryl esters than in the free sterols, but the same sterol moieties occurred in both fractions. Monoenoic fatty acids were enriched in the steryl esters relative to the free fatty acids. Major acyl groups in steryl esters were 16∶0 or 20∶1, with smaller amounts of 14∶0 and 18∶1. In triacylglycerols the acyl moieties were 14∶0, 16∶0, 18∶1, 16∶1 and 12∶0, in order of decreasing abundance. Phytyl esters, previously inferred to occur in a marine dinoflagellate only by analysis of transesterified products, were identified by GC-MS comparison with authentic compounds. Direct analysis of these esterified lipids has not been reported for freshwater phytoplankton. The 4α-methylstanyl esters, 5α(H)-cholestan-3β-yl esters and phytyl esters occurring inP. lomnickii are further features in common with marine dinoflagellates, additional to the 4α-methylsterols reported previously.     

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.     

Analysis of free and esterified sterols in vegetable oils

In vegetable oils, phytosterols occur as free sterols or as steryl esters. Few analytical methods report the quantification of esterified and free sterols in vegetable oils. In this study, esterified and free sterols were separated by silica gel column chromatography upon elution with n-hexane/ethyl acetate (90∶10 vol/vol) followed by n-hexane/diethyl ether/ethanol (25∶25∶50 by vol). Both fractions were saponified separately and the phytosterol content was quantified by GC. The analytical method for the analysis of esterified and free sterols had a relative standard deviation of 1.16% and an accuracy of 93.6–94.1%, which was comparable to the reference method for the total sterol analysis. A large variation in the content and distribution of the sterol fraction between different vegetable oils can be observed. Corn and rapeseed oils were very rich in phytosterols, which mainly occurred as steryl esters (56–60%), whereas the majority of the other vegetable oils (soybean, sunflower, palm oil, etc.) contained a much lower esterified sterol content (25–40%). No difference in the relative proportion of the individual sterols among crude and refined vegetable oils was observed.     

Lipid Profiles of Tunisian Coriander (<Emphasis Type="Italic">Coriandrum sativum</Emphasis>) Seed

Coriander (Coriandrum sativum L.) seeds were harvested from the region of Korba (North-East Tunisia) in order to characterize their fatty acids, phytosterols, tocopherols and tocotrienols (tocols) profiles. Nine fatty acids, with petroselinic acid accounting for 76.6% of the total fatty acids, followed by linoleic, oleic and palmitic acids, accounting for 13.0, 5.4 and 3.4%, respectively, of the total fatty acids were identified. Neutral lipids (NLs) were mainly composed of triacylglycerols (98.4%). Polar lipids were mainly composed of phosphatidylcholine as the major phospholipid (PL) subclass, whereas digalactosyldiacylglycerol was the major galactolipid (GL). Total sterols content was estimated to be 36.93 mg/g oil. Stigmasterol accounted for 29.5% of the total sterols. Other representative sterols were β-sitosterol, Δ⁷-stigmasterol and Δ⁵, 24-stigmastadienol, which accounted for 24.8, 16.3 and 9.2%, respectively. Gamma-tocotrienol was the predominant tocol at 238.40 μg/g seed oil. This was equivalent to 72.8% of the total tocols followed by γ-tocopherol (8.06%) and α-tocopherol (7.6%).     

Comparative studies of metabolism of 4-desmethyl, 4-monomethyl and 4,4-dimethyl sterols inManduca sexta

To investigate the metabolism and possible deleterious effects of 4-methyl and 4,4-dimethyl steroids inManduca sexta, the 4,4-dimethyl sterols lanosterol and cycloartenol, the 4-methyl sterol obtusifoliol and the 4,4-dimethyl pentacyclic triterpenoid α-amyrin were fed in an artificial agar-based diet at various concentrations. Utilization and metabolism of these four compounds were compared with sitosterol, stigmasterol, brassicasterol, ergosterol and 24-methylenecholesterol, 24-alkyl sterols that are readily dealkylated and converted to cholesterol inManduca and in most phytophagous insects. None of the 4-methylated compounds significantly inhibited development except at very high dietary concentrations. The Δ²⁴-bonds of lanosterol and cycloartenol were effectively reduced by theManduca Δ²⁴-sterol reductase enzyme, as is the Δ²⁴-bond of desmosterol which, in most phytophagous insects, is an intermediate in the conversion of sitosterol, stigmasterol and other C₂₈ and C₂₉ phytosterols to cholesterol. On the other hand, the 24-methylene substituent of obtusifoliol was not dealkylated. Each of the 4-desmethyl C₂₈ and C₂₉ sterols was readily converted to cholesterol, and a significant amount of 7-dehydro-cholesterol was derived from ergosterol metabolism. The reason for the differences in substrate specificity of these sterols is not clear, but the information may be useful in the development of new, specific, mechanism-based inhibitors of sterol metabolism.     

Sterols and their biosynthesis in some freshwater bivalves

A number of free sterols and sterol esters of three freshwater mussels was separated and identified. A slow rate of biosynthesisde novo of sterols was demonstrated inAnodonta cygnea. Injected cholesterol was found to undergo esterification, oxidation, Δ²²-dehydrogenation and C-24 alkylation. Methyl-[¹⁴C]methionine was proved to be incorporated in C-24 alkylsterols. Abnormally large amounts of cholesterol injected inA. cygnea were metabolized toward restoration of the normal composition of sterols. This was achieved by intensified metabolism of cholesterol, mainly by conjugation, oxidation and Δ²²-dehydrogenation.     

A method for the separation of seed oil steryl esters and free sterols: Application to peanut and corn oils

A method for separating and quantitating seed oil steryl esters and free sterols was developed using a combination of preparative column, thin layer (TLC), and gas liquid chromatography (GLC). Cholesteryl heneicosanoate and cholesterol served as internal standards. The method was applied to corn-oil samples (Mazola, Kroger) obtained from the local market and peanut-oil samples prepared in the laboratory from commercial varieties of peanuts (Florunner, Starr). Concentration (mg/100 g oil; mean ± SD) of steryl esters and free sterols in the 4 oils were: Mazola, 1420±40 and 370±8; Kroger, 950±40 and 320±4; Florunner, 74±0.5 and 150±3; and Starr, 51±0.5 and 130±2. Sitosterol was the major sterol in both the free sterol and steryl ester fractions of all oils and together with campesterol, stigmasterol and Δ⁵-avenasterol made up 90–95% of all sterols. Steryl esters of peanut oil contained higher proportions of linoleic acid and long-chain acids (C₂₀–C₂₄) than did whole oil. Corn-oil steryl esters also contained a higher proportion of linoleic acid than did whole oil. Squalene was the major hydrocarbon of all oils with the remaining hydrocarbon fraction consisting of a mixture of compounds. Presented at the AOCS meeting, Toronto, May 1982.     

Fatty acid desaturase specificity in tetrahymena

The ciliate,Tetrahymena, was provided a supplement of the fatty acid [1-¹⁴C] 18∶2Δ6,9. After a period of growth the cells were claimed, the lipids extracted, the polar lipids recovered and the mild alkali-labile fatty acid methyl esters generated. The fatty acids were resolved by high pressure liquid chromatography (HPLC), the 18∶3Δ6,9,12 (γ-linolenic acid) was recovered and its identity verified by high pressure liquid chromatography (HPLC), gas liquid chromatography (GLC), hydrogenation and oxidation. Fifty-three percent of the cell-associated label was found in γ-linolenic acid; thus, a Δ12 fatty acid desaturase converts the 6,9 octadecadienoic acid to the 6,9,12 derivative. No carboxyl or methyl terminus restriction appears on Δ9 monoenoic or dienoic fatty acid desaturation in this cell as is found in higher plants and animals.     

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 of¹⁴C-cholesterol and¹⁴C-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.     

Recovery of sterols as fatty acid steryl esters from waste material after purification of tocopherols

Tocopherols are purified industrially from soybean oil deodorizer distillate by a process comprising distillation and ethanol fractionation. The waste material after ethanol fractionation (TC waste) contains 75% sterols, but a purification process has not yet been developed. We thus attempted to purify sterols by a process including a lipase-catalyzed reaction. Candida rugosa lipase efficiently esterified sterols in TC waste with oleic acid (OA). After studying several factors affecting esterification, the reaction conditions were determined as follows: ratio of TC waste/OA, 1∶2 (wt/wt); water content, 30%; amount of lipase, 120 U/g-reaction mixture; temperature, 40°C. Under these conditions, the degree of esterification reached 82.7% after 24 h. FA steryl esters (steryl esters) in the oil layer were purified successfully by short-path distillation (purity, 94.9%; recovery, 73.1%). When sterols in TC waste were esterified with FFA originating from olive, soybean, rapeseed, safflower, sunflower, and linseed oils, the FA compositions of the steryl esters differed somewhat from those of the original oils: The content of saturated FA was lower and that of unsaturated FA was higher. The m.p. of the steryl esters synthesized (21.7–36.5°C) were remarkably low compared with those of the steryl esters purified from high-b.p. soybean oil deodorizer distillate substances (56.5°C; JAOCS 80, 341–346, 2003). The low-m.p. steryl esters were soluble in rapeseed oil even at a final concentration of 10%.     

AY-9944 inhibition of sterol biosynthesis inChlorella emersonii

WhenChlorella emersonii, a green alga, was cultured in the presence of 20 ppm AY-9944, a number of sterols accumulated which appear to be intermediates of sterol biosynthesis in this organism. The sterols isolated include 14α-methyl-ergost-8-en-3β-ol, 14α-methyl 24S-stigmast-8-en-3β-ol, 14α-methyl ergosta-8,24(28)-dien-3β-ol and 4α, 14α-dimethyl 24S-stigmast-8-en-3β-ol. Smaller quantities of several other sterols were found in addition to the normally occurring Δ⁷, chondrillasterol and Δ⁷. Control cultures were found to contain, in addition to the normally occurring sterols, smaller quantities of most of the sterols isolated from AY-9944 inhibited cultures. AY-9944 is a specific inhibitor of Δ⁷ in cholesterol biosynthesis in animals. However, sinceC. emersonii terminates sterol biosynthesis one step prior to the Δ⁷ step, AY-9944 apparently inhibits sterol biosynthesis prior to this step in this organism. The accumulation of 14α-methyl sterols in treated cultures suggests that AY-9944 is an effective inhibitor of the 14α-methyl removal inC. emersonii. Scientific Article No. A1865, Contribution No. 4775 of the Maryland Agricultural Experiment Station.     

Fatty acid composition of individual plasma steryl esters in phytosterolemia and xanthomatosis

The bulk of the plasma plant sterol in phytosterolemia occurs in the esterified form and is carried mostly in the low and high density lipoproteins. We have determined the fatty acid composition of the individual plasma steryl esters from a newly discovered subject with phytosterolemia and xanthomatosis. For this purpose the intact steryl esters were subject to high temperature gas liquid chromatography (GLC) on a polar capillary column, which separated the major esters on the basis of molecular weight and degree of unsaturation of the fatty acids. The saturated and unsaturated sterols esterified to saturated, monoenoic, dienoic and tetraenoic fatty acids were identified by GLC analysis of the sterol moieties of the corresponding AgNO₃-TLC fractions of the steryl esters. The GLC results were confirmed by reversed phase high performance liquid chromatography combined with mass spectrometry via direct liquid inlet interface. It was found that, in general, each fatty acid was esterified to the same complement of sterols, and that the esterified sterols possessed a composition comparable to that of the free plasma sterols, which was comprised of about 75% cholesterol, 6% campesterol, 4% 22,23-dihydrobrassicasterol and 15% β-sitosterol. The fatty acid composition of the steryl esters differed from that of the 2-position of the plasma phosphatidylcholines, which contained significantly less palmitic and oleic and more linoleic acid. On the basis of these results and a review of the literature it is suggested that the plasma cholesteryl and plant steryl esters in phytosterolemia originate from both synthesis in plasma via the lecithin-cholesterol acyltransferase and synthesis in tissues via the acylCoA-cholesterol acyltransferase.     

Free,esterified and glucosidic sterols in cocoa butter

Sterol lipids of cocoa butter (cocoa beansLome Tongo) were fractionated into free sterols, steryl esters (SE), steryl glucosides and acylated steryl glucosides (ASG). 4-Desmethyl, 4-methyl and 4,4′-dimethyl sterols or triterpene alcohols, which were isolated as free sterols or which resulted from hydrolysis, were determined by thin layer chromatography-flame ionization detection and identified by gas chromatography and combined gas chromatography-mass spectroscopy. Free sterols comprise the main sterol fraction in cocoa butter. Esterified sterols amount to 11.5% of total sterols and glucosidic sterols to 16.3%. Fatty acids and D-glucose from hydrolysis of esters and glucosides were analyzed. The fatty acids of SE and ASG are richer in unsaturated fatty acids than cocoa butter total fatty acids.     

Effect of dietary fats on desaturase activities and the biosynthesis of fatty acids in rat-liver microsomes

Four groups of rats were fed diets containing 15% (w/w) high-oleic safflower oil (SFO, rich incis-18∶1 acids), a mixture of 80% partially hydrogenated soybean oil plus 20% corn oil (H+CO, rich intrans-18∶1 acids), lard (L, rich in saturated fatty acids) and corn oil (Co, rich in 18∶2ω6). Fatty acid composition of liver microsomes and activities of the Δ⁵, Δ⁶ and Δ⁹ desaturases were determined. Microsomal Δ⁶ desaturase activity and arachidonic acid were lower in the H+CO group compared with SFO of L. No difference was found in the Δ⁵ or Δ⁶ desaturase activity of CO and SFO groups. Thus, the oleic-acid level of the SFO diet had no effect on the metabolism of 18∶2ω6. Fluorescent polarization studies, usingtrans-parinaric acid as a probe, showed no differences between the physical states of phospholipid vesicles made from lipids isolated from each group. We concluded that thetrans-18∶1 acids in partially hydrogenated soybean oil have a more inhibitory effect than saturated acids on EFA metabolism, even in the presence of adequate amounts of essential fatty acid.     

The rare caribbean spongeLeucosolenia canariensis: Phospholipid fatty acids and sterols

The phospholipid fatty acid composition of the Calcarean spongeLeucosolenia canariensis was studied, and no Δ^5,9 fatty acids were detected. These results are in contrast to the phospholipids from sponges belonging to the class Demospongiae where Δ5,9 fatty acids are predominant. Odd branched-chain fatty acids between 17 and 19 carbons accounted for 26% of the total fatty acids ofL. canariensis, while straight-chain fatty acids between 16 and 22 carbons accounted for 61% of the total fatty acid composition. The sterol composition ofL. canariensis is also reported, and only Δ^5,7,22 sterols were observed.     

Isolation and characterization of the monounsaturated long chain fatty acids ofMycobacterium tuberculosis

The positions of double bond in the monounsaturated C₁₅−C₃₂ fatty acids ofMycobacterium tuberculosis H37Ra were established by gas chromatography/mass spectrometry of the ozonized esters and their pyrrolidide derivatives. The monounsaturated C₁₅−C₂₁ fatty acids had the double bond primarily at the Δ⁹ position while the monounsaturated longer chain fatty acids (C₂₂−C₃₂) had the double bond in several positions. Many of the latter acids, especially the odd-numbered series, were very complex isomeric mixtures. Quantitation showed the most abundant even-numbered long chain fatty acid isomers to be as follow: C₂₂, Δ⁴; C₂₄, Δ⁵; C₂₆, Δ⁷ and Δ⁹; C₂₈, Δ⁹; C₃₀, Δ¹¹ and Δ¹³; C₃₂, Δ¹³ and Δ¹⁵.     

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

The 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.     

Pathways for fatty acid elongation and desaturation in Neurospora crassa

Neurospora crassa incorporated exogenous deuterated palmitate (16∶0) and ¹⁴C-labeled oleate (18∶1^Δ9) into cell lipids. Of the exogenous 18∶1^Δ9 incorporated, 59% was desaturated to 18∶2^Δ9,12 and 18∶3^Δ9,12,15. Of the exogenous 16∶0 incorporated, 20% was elongated to 18∶0, while 37% was elongated and desaturated into 18∶1^Δ9, 18∶2^Δ9,12, and 18∶3^Δ9,12,15. The mass of unsaturated fatty acids in phospholipid and triacylglycerol is 12 times greater than the mass of 18∶0. Deuterium label incorporation in unsaturated fatty acids is only twofold greater than in 18∶0, indicating a sixfold preferential use of 16∶0 for saturated fatty acid synthesis. These results indicate that the release of 16∶0 from fatty acid synthase is a key control point that influences fatty acid composition in Neurospora.