Separation of sterol acetates by column and thin-layer argentation chromatography

Column and thin-layer chromatographic systems employing silver nitrate-impregnated adsorbents are described for the separation of sterol acetates which differ in the number of double bonds in the steroid nucleus or side chain.     

A high-performance liquid chromatographic procedure for the determination of disaturated phosphatidylcholine in human plasma

Plasma disaturated phosphatidylcholine (DSPC) concentration has been implicated as a risk factor for atherosclerosis. However, suitable methods for the estimation of these compounds in plasma are not available. In this paper, a method for the estimation of DSPC using argentation thin-layer chromatography and high-performance liquid chromatography is described. It is quantitative for the measurement of individual and total DSPC species and is not dependent on fatty acid chain length. The method employs hydrolysis of total plasma phosphatidyl choline by phospholipase C, followed by benzoylation of the diacylglycerols. The benzoates are then fractionated on silver nitrate-impregnated silica gel thin-layer chromatography plates, and the disaturated species separated and quantitated by high-performance liquid chromatography. The method is sensitive and reproducible and allows many samples to be done at once. With this method, the amounts of DSPC were found to be significantly higher in a group of normolipidemic diabetic subjects, compared to age-matched controls.     

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.     

Combined silver-ion and reversed-phase high-performance liquid chromatography for the separation and identification of C20 metabolites of conjugated linoleic acid isomers in rat liver lipids

Reversed-phase high-performance liquid chromatography (RP-HPLC) and silver-ion high-performance liquid chromatography (Ag-HPLC) were successively combined for the separation of the longer-chain metabolites of conjugated linoleic acids (CLAs). Commercial silver nitrate-impregnated columns were used with an eluting solvent composed of a mixture of hexane–acetonitrile. Fatty acid methyl esters (FAMEs) from liver lipids of rats fed CLA were analysed. This method allowed separation both of the non-conjugated FAME, as C16:1, C18:2, C18:3, C20:4 and C22:5, but also the conjugated fatty acids like CLA, 8,12,14-20:3, 5,8,12,14-20:4 and 5,8,11,13-20:4. The presence of 8,11,13-20:3 is reported for the first time. This method is of interest for the isolation and identification of the C₂₀ conjugated metabolites that cannot be resolved by gas chromatography. Furthermore, it allows the isolation of FAME for further characterisation by GC–mass spectrometry (MS).     

Isolation and characterization of a glycolipid from Dictyostelium discoideum

A glycolipid was isolated from a lipid extract of the cellular slime mold Dictyostelium discoideum and characterized. From the results of analyses by thin-layer chromatography and infrared spectrometry, it was identified as a steryl glycoside. The steryl glycoside was further analyzed by gas-liquid chromatography/mass spectrometry as a trimethylsilyl ether derivative, and its quantitative and qualitative changes during the development of D. discoideum were examined. Δ²²-Stigmastenyl-d-glucoside was the major constituent of the steryl glycoside and comprised more than 90% of the total steryl glycoside fraction in cells at all stages of development. The content of the steryl glycoside was higher in vegetative-stage cells, late aggregation-stage cells, and 1-day sorocarps than in cells of other stages. The glycolipid fraction was often contaminated by a lipid which was also isolated and identified as a ceramide containing 2-hydroxy fatty acids and 4D-hydroxysphinganine.     

Thin-layer chromatography of ceramides

Ceramides with mono-, di-, and trihydroxy long-chain bases, and normal (saturated and unsaturated), branched-chain, and 2-hydroxy fatty acids have been analyzed by thin-layer chromatography. In most cases the compounds were also run as acetates. Borate, arsenite, and silver ions were used as complexing agents, and the effects of number, position, and stereochemistry of hydroxy groups, and of unsaturation, were studied. The results are discussed in view of analysis of natural ceramide species.     

Hydrolysis of steryl esters by a lipase (Lip 3) from Candida rugosa

A well-known lipase, Lip 3 of Candida rugosa, was purified to homogeneity from a commercial lipase preparation, using hydrophobic interaction and anion exchange chromatography. Lip 3, which has been reported to act on cholesteryl esters, was also found to be active on plant-derived steryl esters. Lip 3 had optimal activity at pH 5-7 and below 55 degrees C. It was able to hydrolyse steryl esters totally in a clear micellar aqueous solution. However, the action on a dispersed colloidal steryl ester solution was limited and only about half of the steryl esters were degraded. The degree of hydrolysis was not improved by addition of fresh enzyme. The composition of released fatty acids and sterols was, however, almost identical to that obtained by alkaline hydrolysis, showing that all the different steryl esters were hydrolysed equally and that none of the individual components were responsible for incomplete hydrolysis. Thus, it appeared that the physical state of the colloidal steryl ester dispersion limited the action of Lip 3. Wood resins contain both triglycerides and steryl esters among the hydrophobic components, which create problems in papermaking. The simultaneous enzymatic hydrolysis of triglycerides and steryl ester is therefore of considerable interest and Lip 3 is the first enzyme reported to act on both triglycerides and steryl esters.     

Steryl glycosides and acyl steryl glycosides from Musa paradisiaca

From peeled fruits of Musa paradisiaca (banana, vegetable variety), two new acyl steryl glycosides, sitoindoside-III and sitoindoside-IV, and two new steryl glycosides, sitosterol gentiobioside and sitosterol myo-inosityl-β-D-glucoside, have been isolated by gradient solvent extraction and extensive chromatography (CC, prep. TLC, GC and HPLC). The compounds have been characterized by comprehensive spectroscopic analyses (IR, ¹H NMR, GC, mass spectra, [α]_D) and crucial chemical transformation. Additionally, seasonal variations of the total sterols, free sterols, steryl esters, steryl glycosides and acyl steryl glycosides in the active samples of banana have been analysed. The results provide a basis for the observed fluctuations in the anti-ulcerogenic activity of the extracts, in different seasons, and the importance of appropriate formulation of the pure principles to optimize the activity.     

The fractionation of phosphatidylinositol into molecular species by thin-layer chromatography on silver nitrate-impregnated silica gel

1. Two methods for the fractionation of phosphatidylinositol into molecular species were developed. In addition to preserving the fatty acid moiety of the molecule, the first method preserves the phosphorus, and the second preserves both the phosphorus and inositol ring. 2. In the first method, phosphatidylinositol was oxidized with periodate and the products reacted with diazomethane. I.r. examination showed that the main product was identical with dimethylphosphatidic acid. Fractionation to molecular species was carried out on thin layers impregnated with silver nitrate. The fatty acid composition of the species was determined by gas-liquid chromatography, and their distribution in lamb liver phosphatidylinositol was studied by a method using [(3)H]methanol. 3. In the second method, phosphatidylinositol was acetylated under mild reaction conditions. The major product was the triacetylated derivative of this phospholipid. This was reacted with diazomethane and the methylated-triacetylated phosphatidylinositol was fractionated into molecular species on silver nitrate-impregnated thin layers. Solvent mixtures containing acetone and distilled chloroform were found most suitable for this purpose. The fatty acid composition of the molecular species was determined by g.l.c., and their distribution in lamb liver phosphatidylinositol was studied by a method using [1-(14)C]acetic anhydride during the acetylation reaction. 4. Results from both methods agree fairly well. The most predominant species of lamb liver phosphatidylinositol is the monoenoic (60%) followed by the tetraenoic (17%). The di- and tri-enoic species existed as minor components.     

Separation of Soybean Sterols by Florisil Chromatography and Characterization of Acylated Steryl Glucoside

Florisil column chromatography was demonstrated to be effective in differentiation between different forms of sterols. Sterols of ground soybeans are in four forms, free, ester, and free and acylated glucosides, as analyzed on acetone extracts. In soybean oil foots, steryl ester is present in negligibly small amount. The acylated steryl glucosides were isolated from oil foots in a crystalline state. A chemical structure, steryl 6-acyl d-glucoside, was assigned to the compound, and its probable identity with the glucosides reported by Lepage is discussed. The acylated glucoside preparation was shown to be heterogeneous in composition, carrying palmitic, stearic, oleic, linoleic and linolenic acids as the main acyl moieties and campesterol, stigmasterol and β-sitosterol as steryl moieties. The presence of the three sterols is common to three other forms of sterols.     

Sterols in Germinating Seeds and Developing seedlings of Longleaf Pine, Pinus palustris

Sterols in germinating embryos and young seedlings of longleaf pine (Pinus palustris Mill.) were identified and quantities determined for different periods after germination. Sterol analyses were performed by gas-liquid chromatography (GLC) and verified by combination of GLC-mass spectrometry. Campesterol and β-sitosterol were two major sterols which accounted for most of the sterol composition while stigmasterol was present in very small amounts. No cholesterol was revealed by GLC-mass spectrometry although there was a minor peak appearing on the sterol gas-liquid chromatograms with a retention time close to that of authentic cholesterol. By fractionation, three different forms of sterols were obtained: steryl esters, steryl glycosides, and free sterols. The sterols were mainly found in the esterified fraction, while steryl glycosides and free sterols only made up a small portion of the total sterol value. The total sterol content in general increased during seedling development, and this increase reflected mainly a change in steryl esters. The low levels of both free and glycosidic sterols remained nearly unchanged throughout the experimental germination period.     

南中国海野生海水鱼类外寄生车轮虫新记录

研究对采自南中国海的7种海水鱼类进行了调查研究, 并分离获得4种隶属于车轮虫属的种类,分别为浦氏车轮虫Trichodina puytoraci Lom, 1962; 日本车轮虫Trichodina japonica Imai, et al., 1991; 直钩车轮虫Trichodina rectuncinata Raabe, 1958 与鲀车轮虫Trichodina fugu Imai, et al., 1997。研究发现鲀车轮虫Trichodina fugu Imai, et al., 1997为寄主鱼虫纹东方鲀Takifugu vermicularis的病原, 且能在野生条件下将其致死。上述4种车轮虫的形态分类学数据均基于干银法标本获得。研究是对我国南中国海车轮虫的首次报道。       

Cholesteryl glucoside in Candida bogoriensis

Extraction of lipids with CHCl3/CH3OH/1 M HCl from fresh and frozen cells of Candida bogoriensis showed the presence of a steryl glucoside. The product was purified and identified as a beta-cholesteryl glucoside. It hydrolyzes in methanolic HCl and therefore it is a steryl glucoside. The quantitation of the extracted cholesteryl glucoside was pursued with high-pressure liquid chromatography. The chemical ionization mass spectra of the isolated and the synthesized beta-cholesteryl glucosides were compared and found identical. The cell wall of Candida bogoriensis was weakened with enzyme (glusulase) and after homogenisation and sucrose density gradient centrifugation, five layers separated. Only the pellets of one layer showed the presence of cholesteryl glucoside in thin-layer chromatography after extraction with solvents. In the same layer, the activity of sterol glucosyl transferase was found.     

Authentic standards for the reductive-cleavage method. The positional isomers of partially methylated and acetylated or benzoylated 1,5-anhydroribitol

The eight positional isomers of methylated and benzoylated 1,5-anhydroribitol were generated simultaneously from 1,5-anhydroribitol by sequential partial methylation and benzoylation, and the four meso isomers and two enantiomeric pairs of isomers so-formed were isolated in chemically pure form by high-performance liquid chromatography. The corresponding acetates were obtained by debenzoylation of the pure isomers and acetylation. Reported herein are the 1H NMR spectra of the benzoates and the electron-ionization mass spectra of the acetates and the tri-O-methyl derivative. Also reported for the acetates and the tri-O-methyl derivative are their linear temperature-programmed gas-liquid chromatography retention indices on three different capillary columns.     

Acylation of steryl glucosides by phospholipids. Solubilization and properties of the acyl transferase.

Particulate enzyme preparations of cotton fibers catalyze the acylation of exogenous steryl glucoside to form acylated steryl glucoside. The acyl transferase involved in this reaction was solubilized by treatment of the membrane fractions with Triton X-100 and was partially purified by chromatography on DEAE-cellulose and gel filtration. This solubilized enzyme had an absolute requirement for Triton X-100 and phospholipid in order to catalyze the acylation of the steryl glucoside. The best phospholipid substrate was phosphatidylethanolamine but egg and soybean phosphatidylcholine were also active. The phospholipid was shown to function as an acyl donor by demonstrating that [¹⁴C]fatty acid from ¹⁴C-labeled phospholipid could be transferred to steryl-[³H]glucoside to form [¹⁴C,³H]acylated steryl glucoside. Saponification of this compound yielded [¹⁴C]fatty acid and steryl-[⁷H]glucoside.     

Authentic standards for the reductive-cleavage method: the positional isomers of partially methylated and acetylated or benzoylated 1,5-anhydro-L-arabinitol

Described herein is the synthesis of the eight positional isomers of methylated and acetylated or benzoylated 1,5-anhydro-L-arabinitol. The compounds were generated simultaneously from 1,5-anhydro-L-arabinitol by sequential partial methylation and benzoylation and isolated in pure from by high-performance liquid chromatography. The desired acetates were obtained by debenzoylation and acetylation of the pure isomers. Reported herein are the 1H NMR spectra of the benzoates and the electron-ionization mass spectra of the acetates and the tri-O-methyl derivative. Also reported for the acetates and the tri-O-methyl derivative are their linear temperature-programmed gas-liquid chromatography retention indices on three different capillary columns.     

Expression of cholesteryl glucoside by heat shock in human fibroblasts

We investigated the heat-induced alteration of glycolipids in human cultured cells, TIG-3 fibroblasts, to show the expression of steryl glucoside by heat shock. A glycolipid band was detected on a thin-layer chromatography plate in lipid extracts from TIG-3 cells exposed to high temperature (42 degrees C) for 15 and 30 minutes, while it was hardly detectable without heat shock. Both cholesterol and glucose were almost exclusively detected by gas liquid chromatography as degradation products of the lipid. The structure of the lipid molecule was elucidated by electrospray mass spectrometry to be a cholesteryl glucoside. This is the first report to show the occurrence of a steryl glucoside in mammalian cells, and this substance is considered to have a significant role in heat shock responses in mammalian cells.     

Authentic standards for the reductive-cleavage method. The positional isomers of partially methylated and acetylated or benzoylated methyl 2-(acetylmethylamino)-2-deoxy-beta-D-glucopyranoside

Described herein is the synthesis of eight positional isomers of methylated and acetylated or benzoylated methyl 2-(acetylmethylamino)-2-deoxy-beta-D-glucopyranoside. The compounds were generated simultaneously from methyl 2-(acetylmethylamino)-2-deoxy-beta-D-glucopyranoside by sequential partial methylation and benzoylation and isolated in pure form by high-performance liquid chromatography (HPLC). The desired acetates were obtained by debenzoylation and acetylation of the pure isomers. Reported herein are the 1H NMR spectra of the benzoates and the electron-ionization mass spectra of the acetates and the tri-O-methyl derivative. Also reported for the acetates and the tri-O-methyl derivative are their linear temperature-programmed gas-liquid chromatography (GLC) retention indices on three different capillary columns.     

Structural analysis of novel bioactive acylated steryl glucosides in pre-germinated brown rice bran

Previous studies from our laboratory indicated that pre-germinated brown rice (PR) contained certain unknown bioactive lipids that activated two enzymes related to diabetes: Na+/K+ATPase and homocysteine-thiolactonase. In this paper, we report on the isolation and structural characterization of the activator lipids from PR bran as acylated steryl glucosides (ASGs). The activator lipid was isolated by silica gel column chromatography, and its chemical structure was determined by NMR, GC-MS, and tandem mass spectrometry. We demonstrated that the bioactive component consists of a mixture of acylated steryl beta-glucosides. Delta8-cholesterol and 2-hydroxyl stearic acid were identified as constituents of ASGs. The steryl glucosides (SGs) subsequent to alkaline hydrolysis lost this enzyme activator activity. Soybean-derived ASGs were not active. This activity may be quite peculiar to PR-derived ASGs. Our findings suggest that the molecular species of ASG may play an important contributing role in the anti-diabetic properties of a PR diet.     

Studies on furan fatty acids of salmon roe phospholipids

Mature salmon roe lipids were found to consist of triacylglycerols (63%), phospholipids (30%), sterols (4.2%), steryl esters (0.7%), and other minor components. In the steryl esters and phospholipids, furan fatty acids were detected instead of the triacylglycerols of the testes lipids in male fish. The representative 12,15-epoxy-13,14-dimethyleicosa-12,14-dienoic acid (F6) amounted to 3.8% and 0.6% of the total fatty acids in each fraction, respectively. However, the absolute amount of the acid in the phospholipid was much more than that contained in the steryl esters. The characteristic distribution of the furan acids found in the phospholipids was common to the steryl esters in the liver. Large amounts of furan acids were contained in phosphatidylcholine (PC) rather than in phosphatidylethanolamine. For positional analysis of furan fatty acids in PC, furan-containing species in the molecule were concentrated fourteenfold by using selective hydrogenation and repeated silica gel column chromatography. A series of furan fatty acids in PC was found to be exclusively linked to the sn-1 position. The amount of the acids in the roe exclusively linked to the sn-1 position. The amount of the acids in the roe phospholipids was comparable with that in the testes triacylglycerols. The physiological roles of furan fatty acids are discussed.