identification of the sterols of the yeasts Saccharomyces cerevisiae and Candida guilliermondii by the mass-spectrometric method

The sterol compositions of mutants of the yeasts ofSaccharomyces cerevisiae andCandida guilliermondii have been characterized by the methods of GLC, TLC, UV spectroscopy, and chromato-mass spectrometry. The possibility has been shown of identifying yeast sterols on the basis of a comparison of the intensities of the peaks of the fragmentary ions having a common nature for all sterols (m/z200). A change in the sterol composition in mutant yeasts as compared with the initial strain has been detected which is obviously connected with the blockage of the routes for the biosynthesis of ergosterol in the yeast cell.Lensovet Leningrad Technological Institute. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 196–201, March–April, 1986.     

Production of meiosis-activating sterols from metabolically engineered yeast

Meiosis-activating sterols (MAS), a class of potent regulators of reproductive processes, are difficult to obtain by chemical synthesis or isolation from natural sources. We demonstrate the development of metabolically engineered strains of Saccharomyces cerevisiae that accumulate MAS as the predominant sterol product. Homologous recombination was used to construct an erg24Delta erg25Delta hem1Delta mutant RXY4.3, which lacked sterol Delta14 reductase, C-4 oxidase, and delta-aminolevulinate synthase. The HEM1 deletion allowed sterol import and rendered RXY4.3 viable under aerobic conditions. This mutant accumulated 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (FF-MAS), and a similar erg25Delta hem1Delta mutant produced 4,4-dimethyl-5alpha-cholesta-8,24-dien-3beta-ol (T-MAS). Based on consistent yields of approximately 5 mug of FF-MAS per mL of culture, fermentation of genetically modified yeast compares favorably with other approaches to produce MAS.     

Cholesterol and phytosterols effect on sphingomyelin/phosphatidylcholine model membranes--thermodynamic analysis of the interactions in ternary monolayers

In this work thermodynamic analysis of the interactions between lipids in ternary sphingomyelin/DPPC/sterol Langmuir films were performed to compare the effect of cholesterol, beta-sitosterol and stigmasterol on a model membrane. The condensing effect of the respective sterols and the interactions between molecules in ternary mixtures were analyzed on the basis of the excess area per molecule and the excess free energy of mixing values. The stability of the mixed monolayers was verified with the free energy of mixing values. The conclusions on the ordering effect of sterols were drawn from the analysis of the compression modulus values. It was found that the stoichiometry of the mixed films of the highest thermodynamic stability and of the strongest interactions is the same for all the sterols investigated. The results obtained prove that the mammalian sterol induces the strongest contraction of the area and reveals the strongest stabilizing and ordering effect among the investigated sterol. Stigmasterol was found to condense a model membrane in a weaker extent as compared to beta-sitosterol, however, the differences in ordering properties of both phytosterols are less pronounced. The magnitude of the influence of the investigated sterols on a model membrane was thoroughly discussed from the point of view of the structure of their side chain, which determines the geometry of a sterol molecule.     

Analysis of free and esterified sterols in fats and oils by flash chromatography, gas chromatography and electrospray tandem mass spectrometry.

A method for the analysis of free and esterified sterols has been developed. Fat or oil samples were separated on solid-phase extraction silica gel columns into a sterol ester fraction, a fraction of triacylglycerols, and a free sterol fraction containing partial acylglycerols and residual triacylglycerols. Sterol esters and acylglycerols of the free sterol fraction were transesterified to methyl esters. The fatty acid methyl esters from sterol ester fraction and the free sterols from sterol ester fraction and free sterol fraction were determined by GLC. Precursor ion electrospray MS-MS of sterol fragment ions of sterol ester fractions were recorded and used for determination of sterol ester proportions in butterfat and vegetable oil samples.     

Sterols of scallop. I. Application of hydrophobic sephadex derivatives to the resolution of a complex mixture of marine sterols.

Column chromatography on a hydroxyalkoxypropyl derivative of Sephadex LH-20 and on Anasil B has been applied to the resolution of complex marine sterol mixture in combination with argentation thin-layer chromatography and gas chromatography. This approach permits isolation in quantity of individual sterols from a complex mixture and separation of sterol mixtures that were not resolved without the modified Sephadex step. Seventeen sterols were detected in the scallop Placopecten magellanicus. 24-Methyl-cholesterol, 24-ethyl-cholesterol, 24-methyl-22-dehydrocholesterol and 24-ethyl-22-dehydrocholesterol, i.e. sterols whose configuration at C-24 had not been definitively established, were isolated in sufficient quantities for further study by nuclear magnetic resonance spectroscopy.     

Sterol composition of a cultured marine dinoflagellate,Heterocapsa circularisquama

The composition of sterols was determined in a cultured marine dinoflagellate Heterocapsa circularisquama. Ten kinds of the sterol in H. circularisquama were identified by capillary gas chromatography-mass spectrometry. The major sterol was a 4-methyl sterol, 4alpha,23,24-trimethyl-5alpha-cholest-22E-en-3beta-ol (dinosterol) which is the common sterol in many dinoflagellates.     

Determination of plant sterol oxidation products in plant sterol enriched spreads, fat blends, and plant sterol concentrates

Plant sterols (PS) are very stable molecules but may undergo oxidation due to the presence of a double bond in the ring structure. In order to assess whether this occurs during heating and storage, an analytical procedure was developed for the determination of concentration levels and identity of PS oxidation products in functional food ingredients and products. The method is based on cold saponification, solvent extraction of unsaponifiables, isolation of sterol oxidation products by means of liquid chromatography, and final analysis by gas chromatography (GC) with flame ionization detection. Identification of the key PS oxidation products was performed by means of GC-mass spectrometry (GC-MS). Isotope dilution MS was used to verify the absence of the formation of potential artifacts by the method. The method described is applicable to spreads (containing 20-65% water), oils, sterol esters, pure sterols, and fat extracts from food. The between-day reproducibility of the total content of sterol oxidation products in control samples sample was 8%, and of individual sterol oxidation products, 6-15%. The recovery of sterol oxidation products was 91%. The limit of detection was 0.1 mg/kg.     

Application of comprehensive two-dimensional gas chromatography to the quantification of overlapping faecal sterols

Standard solutions containing a mixture of seven sterols and 5alpha-cholestane as internal standard, and sample mixtures that comprised varying ratios of sterol and stanols from green lip mussel tissue and dried cow faeces were analysed by using comprehensive two-dimensional gas chromatography (GC x GC). Quantitative results were compared with single-column GC analysis. The latter samples included sterols of interest, but which cannot be readily obtained elsewhere. It may also mimic potential environmental samples where dairy production and aquaculture (oyster, mussel cultivation) share the same catchment; environmental sterol signatures may exhibit characteristics of both sample types comprising this mixture. Whereas single-column GC-flame ionisation detection was unable to reliably quantitate target sterols, the GC x GC experiment permitted small amounts of sterols and stanols to be detected and separated. Likewise GC-MS analysis was unable to detect some of the minor sterols which coeluted on a single column. The GC x GC mode allows complete separation of several important sterols and stanols, such as 24-ethylcoprostanol, campesterol and 24-methylenecholesterol, demonstrating the enhanced resolving power of the GC x GC system. Separation of 24-ethyl-epi-coprostanol from several algal-derived interfering components was achieved, leading to higher degree of confidence in the quantitative analysis of faecal sterols. The effects of a number of operating variables--column length, carrier flow-rate and elution temperature--on component resolution and presentation of data in the two-column analysis are described.     

Biosynthetic studies of marine lipids 12.: Biosynthesis in marine sponges of sterois possessing the δ5,7-nucleus typical of fungi and the 24-alkyl side chain characteristic of plants

The biosynthesis of sterols with structural patterns common to plant and yeast sterols was studied by incorporation of eleven radiolabeled sterol precursors and mevalonate. The capability of sponges to convert Δ⁵- into Δ^5,7-sterols, which was as efficient as the conversion of Δ⁷- into Δ^5,7-sterols, was demonstrated by double labelling experiments. By examining epimeric pairs, stereospecific conversion of codisterol and clerosterol into the corresponding 7-dehydrosterols was established. 24β-24-Alkyl sterols with homo-conjugated 22,25-diene side chains are proposed as actual intermediates in the formation of Δ²²-24β-alkylsterols. An alternative pathway via oxidation at C-22 or C-23 with subsequent dehydration of an intermediate 22(R or S)- or C-23(R or S)-hydroxysterol is probably not operative due to lack of incorporation of any of the tritium labeled alcohols. These biosynthetic features, though unexpected for animals, nevertheless are attributed to the sponge because of the absence of symbiotic fungi and algae as shown by electron microscopy.     

N-(1-piperidinepropionyl)amphotericin B methyl ester (PAME)--a new derivative of the antifungal antibiotic amphotericin B: searching for the mechanism of its reduced toxicity

N-(1-piperidinepropionyl)amphotericin B methyl ester (in short, PAME), a low-toxicity amphotericin B derivative, has been investigated in Langmuir monolayers at the air/water interface alone and in mixtures with cellular membrane sterols (a mammalian sterol, cholesterol, and a fungal sterol, ergosterol) and a model phospholipid (DPPC). The analysis of the strength of interaction between PAME and both sterols as well as DPPC was based, on surface pressure measurements and analysis of the isothermal compressibility (C(s)(-1)), the mean area per molecule (A(12)), the excess free energy of mixing (DeltaG(Exc)) and the total free energy of mixing (DeltaG(M)). It has been found that the interactions between PAME and sterols are attractive; however, their strength is significantly weaker for mixtures of PAME with cholesterol than with ergosterol. This casts light on the improved selectivity of PAME toward fungal cells. The strongest interactions, found for PAME/DPPC mixtures, proved an important role of DPPC in the mechanism of reduced toxicity of PAME as compared to amphotericin B. Due to stable complex formation between PAME and DPPC the antibiotic is immobilized with DPPC molecules, which reduces the concentration of free antibiotic, which is capable of interacting with membrane sterols.     

Role of cholesterol 10-methyl group and effect of "extra" 14-methyl group on silkworm growth and development

In order to establish the functional importance of the 10-methyl group of cholesterol and the planarity of the steroid ring, silkworms (Bombyx mori) were reared on an artificial diet containing 19-norcholesterol (1), 14 alpha-methylcholesterol (3) or 19,19-difluorocholesterol (2). The former two sterols (1 and 3) only partially satisfied the silkworm sterol requirement; growth and development were seriously retarded. The fluorinated sterol (2) was much more deleterious and was totally inadequate in meeting the sterol requirement.     

Mechanism of azole antifungal activity as determined by liquid chromatographic/mass spectrometric monitoring of ergosterol biosynthesis

A liquid chromatography/mass spectrometry (LC/MS) method for separation and characterization of ergosterol biosynthetic precursors was developed to study the effect of Posaconazole on sterol biosynthesis in fungi. Ergosterol biosynthetic precursors were characterized from their electron ionization mass spectra acquired by a normal-phase chromatography, particle beam LC/MS method. Fragment ions resulting from cleavage across the D-ring and an abundant M - 15 fragment ion were diagnostic for methyl substitution at C-4 and C-14. Comparison of the sterol profile in control and treated Candida albicans incubations showed depletion of ergosterol and accumulation of C-4 and C-14 methyl-substituted sterols following treatment with Posaconazole. These C-4 and C-14 methyl sterols are known to be incapable of sustaining cell growth. The results demonstrate that Posaconazole exerts its antifungal activity by inhibition of ergosterol biosynthesis. Furthermore, Posaconazole appears to disrupt ergosterol biosynthesis by inhibition of lanosterol 14alpha-demethylase.     

The impact of sterol structure on the interactions with sphingomyelin in mixed langmuir monolayers

In this work, the Langmuir monolayer technique was applied to study the interactions between sphingomyelin and various sterols differing in the structure of the side chain (cholesterol, beta-sitosterol, stigmasterol). The mean area per molecule and the excess free energy of mixing values were analyzed in the context of sterol-induced condensing effect and interactions between molecules in the mixed monolayers. Moreover, the compression modulus values were calculated and widely discussed from the point of view of the ordering effect of sterols. It was found that all of the sterols investigated form the most stable monolayers with sphingomyelin at 2:1 sphingomyelin:sterol proportion and the strongest interactions exist between molecules in cholesterol-containing films. Moreover, cholesterol provokes the strongest area condensation and reveals the highest ordering properties, while plant sterols were found to differ only slightly with regards to their ordering properties. Additionally, the ordering effect of the sterols on dipalmitoylphosphatidylcholine (DPPC) films was analyzed and compared to that on sphingomyelin films.     

Analysis of olive and hazelnut oil mixtures by high-performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry of triacylglycerols and gas-liquid chromatography of non-saponifiable compounds (tocopherols and sterols)

We analysed the triacylglycerol, tocopherol and sterol composition of hazelnut oil, olive oil and their mixtures (90% olive oil with 10% hazelnut oil, 70% olive with 30% hazelnut oil and 50% olive oil with 50% hazelnut oil). The main triacylglycerols were 1,2,3-trioleylglycerol, 2,3-dioleyl-1-palmitoylglycerol, 2,3-dioleyl-1-linoleylglycerol and 2,3-dioleyl-1-stearoylglycerol. Non-saponfiable compounds (tocopherols and sterols) were derivatised as O-trimethylsilyl ethers. Alpha-tocopherol was the main vitamin E isomer in all samples; however, small amounts of beta-tocopherol and gamma-tocopherol were also found. Beta-sitosterol and delta5-avenasterol were the principal sterols in all samples; campesterol and stigmasterol were minor sterol compounds in all samples. Obtusifoliol, which was a major sterol in olive oil and oil mixtures, was not found in hazelnut oil. The discriminant analysis showed that hazelnut oil, olive oil and oil mixtures were clearly separated according to their triacylglycerol composition.     

Quantification of free and esterified sterols in Portuguese olive oils by solid-phase extraction and gas chromatography-mass spectrometry

A simple and accurate method based on solid-phase extraction (SPE), transesterification and gas chromatography-mass spectrometry (GC-MS) was developed for the quantitative analysis of free and esterified sterols of olive oil. In order to achieve better separation of esterified and free sterols, silica and alumina SPE adsorbents were tested. Separations by silica provided more reproducible results. The transesterification of both sterol fractions was found to be more user friendly than saponification as a method to liberate the sterols from the respective esters. The free sterols were then silylated with N,O-bis-trimethylsilyltrifluoroacetamide (BSTFA) with 1% of trimethylchlorosilane (TMCS). The most favourable conditions for exploitation of this reagent were established. The optimized methodology was suitable for evaluation of free and esterified sterols in Protected Designation of Origin (PDO) olive oils and monovarietal olive oils with different maturation indices. The prevailing phytosterols in all olive oils were beta-sitosterol and campesterol. The free sterols predominated, although they seemed to decrease with the maturation of the olive fruits.     

Separation of C27, C28 and C29 sterols by reversed-phase high-performance liquid chromatography on small particles.

The application of reversed-phase high-performance liquid chromatography to the analytical- and preparative-scale separation of sterols has been evaluated. The capacity factors, k', for a number of compounds chromatographed on a muBondapak C18 (LESS THAN 10 MUM) COLUMN ARE PRESENTED. C27, C28 and C29 sterols and also sterols differing in degree of unsaturation could be readily separated as their acetates in this system. The present reversed-phase chromatographic method is apparently not as selective as silver nitrate-silica gel thin-layer chromatography for the position of unsaturation in the sterol molecule.     

Study of penetration of amphotericin B into cholesterol or ergosterol containing dipalmitoyl phosphatidylcholine Langmuir monolayers

The interactions of amphotericin B (AmB) with sterols and phospholipids have been studied by adsorption of AmB from aqueous solutions into Langmuir monolayers from dipalmitoyl phosphatidylcholine (DPPC), ergosterol, cholesterol and their mixtures. The results show that AmB exhibits stronger interaction with cholesterol than ergosterol in one-component monolayers. However, for DPPC–sterol monolayers, the effectiveness of AmB penetration depends on the proportion of both film components in the mixed film as well as on the strength of interaction between DPPC and particular sterol.     

High resolution GC of unsaponifiable matter and sterol fraction in vegetable oils

Summary The analysis of both the total unsaponifiable matter and the sterol fractions in vegetable oils has been performed with a new polar column (TAP, Chrompack). The use of a polar column, which is characterized by high thermal stability, has led to the identification of a greater number of constituents than the use of a nonpolar column (i.e. SE 52, SE 54). The polar column enhances the separation of the main classes of unsaponifiable matter (sterols, 4-methyl sterols and 4,4-dimethyl sterols). When used for the unsaponifiable and sterol fraction analyses, it offers a powerful tool for characterizing the lipid source.     

Sterols of grapefruit,orange, and mandarin pulps

The composition and percentage distribution of the sterols in preparations of the free (I), glycosylated (II), and esterified (III) sterols in the pulp of citrus fruits have been studied. In the sterol preparations, cholesterol, campesterol, stigmasterol, and β-sitosterol have been identified and detected, and three other sterols have been detected but not identified. It has been established that the sequences of the relative amounts of sterols in preparations (I), (II), and (III) of the grapefruit and the orange are similar and differ from that of mandarin pulp.     

Further data on a histochemical reaction as applied to the thin-layer chromatography of sterols.

A method for the characterization of sterols on silica gel G layers is proposed. After the chromatograms have been sprayed with a permanganate-sulphuric acid oxidative reagent and the reaction has been terminated with sodium hydrogen sulphite, the plates are sprayed with the colour-developing reagent (an acid solution of alcian blue or toluidine blue). The plates are also viewed under UV radiation (254 and 366 nm). Only 3 of the 28 sterol samples assayed (spot content 8 mug) did not show positive reactions after oxidation, which suggests that this step can be used as a "universal" detection method for sterols. After staining the plate, several sterols are shown to be easily differentiated from one another. The exposure of the plates to UV radiation assists characterization. In general, the reaction exhibits satisfactory sensitivity for the qualitative and differentiating detection of sterols; it is also rapid and easy to carry out.