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.     

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.     

Characterization and composition of sterols in the free and esterified sterol fractions ofAspergillus oryzae

Free and esterified sterol fractions were isolated fromAspergillus oryzae, and their components analyzed mainly by gas chromatography-mass spectrometry. Cholesterol, brassicasterol, episterol (5α-ergosta-7,24[28]-dien-3β-ol), 4α-methyl-5α-ergosta-8[9],24[28]-dien-3β-ol, lanosterol and 24-methylene-24-dihydrolanosterol were well characterized, whereas 5-dihydroergosterol, sitosterol and 24[28]-dehydroergosterol were tentatively characterized. The principal sterol of the free sterol fraction was brassicasterol, and that of the esterified sterol fraction was episterol. Ergosterol, which is reported to be widely distributed in the fungi kingdom, was not detected.     

Free and esterified sterols of cotton buds and anthers

Capillary gas liquid chromatography analyses were conducted on free and esterified sterol fractions of cotton (Gossypium hirsutum cv. Stoneville 213) floral buds and anthers. The free sterols of both cotton buds and anthers consist mainly of the common plant sterols sitosterol, stigmasterol and 24ζ-methylcholest-5-en-3β-ol. The composition of esterified sterols of cotton buds and anthers were similar, and consisted of pollinastanol, 31-norcycloartanol, cycloartenol, 31-norcycloartenol, 24-dehydropolinastanol and sitosterol. Desmosterol was also present in both the free and esterified sterols of anthers. The identities of the sterols were confirmed by gas chromatography-mass spectrometry analyses. Esterified sterols accounted for 46.7 and 88.7% of total sterols of cotton bud and anthers, respectively. The ratio of esterified sterol to free sterol per gram of tissue is about 8∶1 in anthers. The Δ⁵-sterols of the esterified sterols of cotton buds and anthers account for only 17 and 9.2% of the total sterols, respectively.     

Free and esterified sterols of the marine dinoflagellateGonyaulax polygramma

Free and esterified sterols of the common marine dinoflagellateGonyaulax polygramma were identified using capillary gas chromatography—mass spectrometry (GC-MS). Fractions containing free 4α-methyl and 4-desmethyl sterols were isolated by column chromatography and shown to consist of at least 20 components. Major sterols included 4α,23,24-trimethyl-5α-cholestan-3β-ol (dinostanol), 4α,23,24-trimethyl-5α-cholest-22E-en-3β-ol (dinosterol), cholest-5-en-3β-ol (cholesterol), 23,24-dimethyl-5α-cholest-22E-en-3β-ol and 23,24-dimethylcholesta-5,22E-dien-3β-ol. Although the same group of sterols was found in the free and esterified sterol fractions, the proportions of individual sterols were quite different. The complexity of the sterol distributions, together with the predominance of dinostanol, distinguishes the sterol composition of this alga from those of other members of theGonyaulacaceae.     

Determination of free and esterified sterols in potential new oil seed crops by coupled on-line liquid chromatography-gas-chromatography

In vegetable oil, free and esterified sterols are characteristic constituents of the unsaponifiable matter and provide rich information about the oil quality. The sterol pattern of plants discussed as potential new oil seed crops have been determined by using coupled on-line normal phase liquid chromatography-gaschromatography (LC-GC). The content and composition of free sterols as well as the concentration of sterol esters have been analyzed in seed oils of different varieties of crambe (Crambe abyssinica), meadowfoam (Limnanthes alba, L. douglasii(, cape marigold (Dimorphotheca sinuata);, lesquerella (Lesquerella fendleri);, iron weed (Vernonia galamensis, V. petitiana);, spurge (Euphorbia lagascae, E. lathyris);, and various species of cuphea (Cuphea lanceolata, C. lutea, C. paucipetala, C. viscosissima, C. wrightii);.     

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.     

Peroxidation of free and esterified fatty acids by horseradish peroxidase

Linoleic and arachidonic acids and unsaturated esterified fatty acids of soybean lecithin liposomes were oxidized by horseradish peroxidase in the presence of hydrogen peroxide. The major products formed in the presence of oxygen were fatty acid hydroperoxides. In the absence of oxygen, other unidentified products were formed. Diene conjugation was about 5 times faster in oxygen than in nitrogen. Malondialdehyde was formed only in the presence of oxygen. Superoxide, singlet oxygen and hydroxyl radical may have been involved in the free fatty acid oxidation system but not in the liposome system. Replacement of hydrogen peroxide with the hydrogen peroxide (and superoxide) generators xanthine oxidase or galactose oxidase caused a more efficient oxidation in the presence of peroxidase than in its absence, suggesting that the in vivo toxicity of hydrogen peroxide and superoxide may be greatly increased in the presence of peroxidase.     

Enzymatic conversion of steryl esters to free sterols

Conversion of oleic acid phytosteryl esters (OASE) to free phytosterols (referred to as sterols) by an enzymatic process was attempted. Enzymatic hydrolysis of OASE reached a steady state at 55–60% hydrolysis, but addition of methanol (MeOH) significantly accelerated the conversion of OASE to sterols. Screening of commercially available enzymes indicated that Pseudomonas aeruginosa lipase was most effective for the conversion. Based on the study of several factors affecting the reaction, the optimal conditions were determined as follows: ratio of OASE to MeOH, 1∶2 (mol/mol); water content, 10 wt%; lipase amount, 20 U/g by weight of reaction mixture; temperature, 30°C. When the reaction was conducted for 48 h with stirring, the conversion reached 98%. FAME accumulated in the reaction mixture, but FFA did not, indicating that the FAME was poorly recognized as a substrate in the reverse conversion of sterols to OASE but the FFA was easily recognized as a substrate. The high conversion of OASE to sterols was therefore due to elimination of FFA from the reaction system. After the enzymatic reaction, the oil layer was fractionated at −20°C with 5 vol parts of n-hexane. Sterols were efficiently purified in the resulting precipitate (92% recovery, 99% purity).     

Quantification of free and esterified steryl glucosides in vegetable oils and biodiesel

Steryl glucosides (SG) are minor components that dramatically modify the low temperature performance of fatty acid methyl esters (FAME) used as biodiesel. SG are naturally present in vegetable oils but they may also be the result of the transesterification of esterified steryl glucosides (ESG). These are present in vegetable oils at a level of a few hundred milligrams per kilogram, depending on the nature of the feedstock. We developed an analytical method to quantify SG and ESG in vegetable oils and in FAME. The purification of SG and ESG was performed by liquid chromatography on silica gel, and the analysis of the trimethylsilyl derivatives was achieved by gas chromatography and flame ionization detection. The filterability of biodiesel is affected when the SG content is higher than 20 mg/kg. Therefore, the sensitivity of this new method is adapted for this purpose since the quantification limit is 10 mg/kg of SG and ESG. The recoveries are acceptable, between 75% and 90% depending on the species and content, and the reproducibility relative standard deviation, evaluated at 10%, is comparable to other studies.     

Digestion and absorption of free and esterified fish oil fatty acids in rats

This study was designed to test the hypotheses that digestibility and post-absorption metabolism of fish oil are influenced by impaired lipolysis and by the stereospecific composition of its triacylglycerols. Male Wistar rats were fed nonpurified diets containing one of the following fat sources: 9% native fish oil (NFO), 9% autorandomized fish oil (RFO), 8.1% fish oil-derived free fatty acids (FO-FFA) plus 0.9% glycerol, or 9% soybean oil (SO) as a reference fat. In a 24-day balance study, apparent digestibility of total dietary fat averaged 93.1% in the SO, NFO and RFO groups, and 90.9% in the FO-FFA group. Randomization of fish oil had no effect on apparent digestibility of individual fatty acids. In rats fed FO-FFA, apparent absorption of saturated and monounsaturated fatty acids was lower when compared to the NFO and RFO groups. Feeding the FO-FFA diet tended to increase plasma triglyceride content. The hypocholesterolemic effect of polyunsaturated n−3 fatty acids was not influenced by the dietary source. Similar effects on fatty acid profiles of plasma and liver phospholipids were caused by the NFO, RFO and the FO-FFA diets. We conclude that once polyunsaturated n−3 fatty acids are absorbed, their effect on lipid metabolism is not determined by the dietary source.     

Distribution of free and conjugated sterols in orange and tangor juice sacs

Comparative studies of the sterol composition of four sterol fractions, vis., free sterols, sterol esters, sterol glucosides and esterified sterol glucosides, were conducted on the juice sacs of six varieties of oranges and two tangor varieties. The sterols quantified in each fraction were β-sitosterol, campesterol, stigmasterol, cholesterol, 24-ethylidene cholesterol, brassicasterol and 24-methylene cholesterol. Each variety showed its own intrinsic composition for these sterols in the four sterol fractions. So. Market. Nutr. Res. Div., ARS, USDA.     

Plasma kinetics of free and esterified cholesterol in familial hypercholesterolemia: Effects of simvastatin

The objective of this study was to evaluate the kinetics of both free and esterified forms of cholesterol contained in a emulsion that binds to LDL receptors (LDE) in subjects with heterozygous familial hypercholesterolemia (FH), and the same subjects under the effects of high-dose simvastatin treatment, as compared with a control normolipidemic group (NL). Twentyone FH patients (44.0±13.0 yr, 12 females, LDL cholesterol levels 6.93±1.60 mmol/L) and 22 normolipidemic patients (44.0±15.0, 10 females, LDL cholesterol levels 3.15±0.62 mmol/L) were injected intravenously with ¹⁴C-cholesteryl ester and ³H-cholesterol. FH patients were also evaluated after 2 mon of 40 or 80 mg/d simvastatin treatment, and plasma samples were collected over 24 h to determine the residence time (RT, in h) of both LDE labels, expressed as the median (25%; 75%). The RT of both ¹⁴C-cholesteryl ester and ³H-cholesterol were greater in FH than in NL [FH: 36.0 (20.5; 1191.0), NL: 17.0 (12.0–62.5), P=0.015; and FH: 52.0 (30.0; 1515.0); NL 20.5 (14.0–30.0) P<0.0001]. Treatment reduced LDL cholesterol by 36% (P<0.0001), RT of ¹⁴C-cholesteryl ester by 49% (P=0.0029 vs. baseline), and ³H-cholesterol RT by 44% (P=0.019 vs. baseline). After treatment, the RT values of ¹⁴C-cholesteryl ester in the FH group approached the NL values (P=0.58), but the RT of ³H-cholesterol was still greater than those for the NL group (P=0.01). The removal of LDE cholesteryl esters and free cholesterol was delayed in FH patients. Treatment with a high dose of simvastatin normalized the removal of cholesterol esters but not the removal of free cholesterol.     

Soybean lipoxygenase-promoted oxidation of free and esterified linoleic acid in the presence of deoxycholate

Lipoxygenase (EC 1.13.11.12) catalyzes the reaction between oxygen and polyunsaturated fatty acids to give fatty acid hydroperoxides. Recent work showed that soybean lipoxygenase 1 can oxidize diacylglycerols when deoxycholate is present in the reaction medium. Conditions were sought to maximize 1,3-dilinolein oxidation with a commercial soybean lipoxygenase preparation. It was found that dilinolein was oxidized most rapidly in a multicomponent buffer medium that contained 10 mM deoxycholate between pH 8 and 9. When dilinolein oxidation was conducted in the individual components of the multicomponent buffer, the oxidation rate decreased two- to threefold. Addition of 0.2 M NaCl to one of the components, Tricine buffer, caused a twofold increase in the oxidation rate, demonstrating that high ionic strength is a major factor promoting rapid oxidation in the multicomponent buffer. In the deoxycholate multicomponent buffer, the order of reactivity toward oxidation was monolinolein>methyl linoleate≈ linoleic acid>dilinolein. Competition experiments in which mixtures of the substrates were presented simultaneously to lipoxygenase in the presence of deoxycholate showed that linoleic acid was the most reactive substrate. When no surfactant was present or when the surfactant was Tween 20, linoleic acid was the most rapidly oxidized substrate. Overall, the results demonstrate that monolinolein and methyl linoleate are just as reactive, or more so, as linoleic acid to oxidation by lipoxygenase under specified reaction conditions. In competition experiments, linoleic acid oxidation predominates, probably because its free carboxyl functionality allows it to be preferentially bound to the active site of lipoxygenase.     

Rapid separation of free sterols by reversed-phase high performance liquid chromatography

The separation of eight common, structurally closely related sterols on C8 and C18 reversed-phase columns with UV-detection at 206 nm is described. Good separation was obtained in less than 18 min on the C18 column using methanol-water as mobile phase and a column temperature of 30 C. Except for brassicasterol and cholesterol, the sterols also were readily separated on the C8 column. Applications of the method on sterols from natural sources are described.     

Detection of free chloride in concrete by NMR

Laboratory experiments to detect chloride in a cement matrix using pulse nuclear magnetic resonance (NMR) were conducted. The coils were in the centimeter scale and the magnetic field was 2.35 T. NMR signals were obtained from both aqueous chloride solution and samples of both regular and white Portland cement (WPC). A concrete sample from a sidewalk that had been in the field for 20 years was also tested. The experiments demonstrated that the signal-to-noise ratio (SNR) for a centimeter-scale cement sample volume is so small, even after averaging, that sample volumes much lower than that are unlikely to produce measurable signals at fields of 1 T or below. The consequence is that the potential for realizing an embedded NMR-based sensor including the magnet is low. Parametric studies identify feasible alternative coil diameters and magnetic field strengths for detecting chloride ion concentrations in hardened concrete.     

The effect of a fat free diet on esterified monoenoic fatty acid isomers in rat tissues

Rats were maintained 120–140 days on a normal diet (group 1) or one deficient in fatty acids (group 2). Isomer composition was determined of monoenoic fatty acids (16∶1, 18∶1) isolated from total lipids of heart, kidney, lung, brain and lumbar fat, and from separated neutral lipids and phospholipids of heart and kidney. Group 1: The number of major isomers of C_16∶1 and C_18∶1 was similar in all tissues but their proportions varied in different tissues and types of lipid. Group 2: The proportions of 16∶1(n−7) increased and of other 16∶1 isomers decreased in all tissues; 18∶1(n−9) was increased at the expense of (n−7) in heart, to a lesser extent in kidney, and was little changed in lung, lumbar fat, or brain. The decrease in proportion of 18∶1(n−7) was greatest in heart-muscle phospholipids. C_20∶3 comprised 95% (n−9) and 5% (n−7) in heart and kidney lipids. The changes in group 2 probably represent the body’s attempts to maintain lipids with the physical and chemical properties necessary to normal biological function. Nomenclature of fatty acids as in IUPAC-IUB Commission on Biochemical Nomenclature, “The nomenclature of lipids,” J. Biol. Chem. 242:4845–49 (1967).     

酯化淀粉的制备

本文以马铃薯淀粉为原料,醋酸乙烯酯为酯化剂,制备低取代度醋酸酯淀粉,研究了各反应因素对产品取代度的影响,并以取代度为指标,用单因素试验,确定了其最佳工艺条件,即反应体系pH值10.25,反应温度26℃,反应时间1h,酯化剂用量1g。