Improved separation of sterols by column chromatography

Some closely related sterols have been separated on columns of neutral alumina-Super-Cel impregnated with silver nitrate. Most notable are the separations of some Delta(8)-sterols from their Delta(7)-isomers, separations which have been difficult, if not impossible, to achieve with other published column chromatographic methods.     

Soybean sterol composition and utilization by Phytophthora sojae

The sterol fraction of Glycine max (soybean) was found to contain a mixture of 13 major sterols which differed dramatically in composition between seeds and shoots. Typical C4-desmethyl Delta(5)-sterols, including sitosterol, predominate the sterol mixture of shoots, whereas C4-methyl sterol intermediates, cycloartenol and 24(28)-methylene cycloartanol, accumulate in seeds. The significance of modified sterol profile of shoot compared to seed was relevant to the physiology of Phytophthora sojae, a phytopathogen of soybean shown to be auxotrophic for sterol. Sterols native to the host plant containing a C4-methyl group, such as cycloartenol, were not utilized by the fungus. Alternatively, all Delta(5)-sterols added to the culture media of P. sojae supported normal growth and promoted viable oospore production. The results demonstrate the importance of sterols in plant-fungal interactions and offer the possibility of bioengineering the phytosterol pathway for resistance to phytopathogens which scavenge specific sterols of the host plant to complete the life cycle.     

Insights into the role of specific lipids in the formation and delivery of lipid microdomains to the plasma membrane of plant cells

The existence of sphingolipid- and sterol-enriched microdomains, known as lipid rafts, in the plasma membrane (PM) of eukaryotic cells is well documented. To obtain more insight into the lipid molecular species required for the formation of microdomains in plants, we have isolated detergent (Triton X-100)-resistant membranes (DRMs) from the PM of Arabidopsis (Arabidopsis thaliana) and leek (Allium porrum) seedlings as well as from Arabidopsis cell cultures. Here, we show that all DRM preparations are enriched in sterols, sterylglucosides, and glucosylceramides (GluCer) and depleted in glycerophospholipids. The GluCer of DRMs from leek seedlings contain hydroxypalmitic acid. We investigated the role of sterols in DRM formation along the secretory pathway in leek seedlings. We present evidence for the presence of DRMs in both the PM and the Golgi apparatus but not in the endoplasmic reticulum. In leek seedlings treated with fenpropimorph, a sterol biosynthesis inhibitor, the usual Delta(5)-sterols are replaced by 9beta,19-cyclopropylsterols. In these plants, sterols and hydroxypalmitic acid-containing GluCer do not reach the PM, and most DRMs are recovered from the Golgi apparatus, indicating that Delta(5)-sterols and GluCer play a crucial role in lipid microdomain formation and delivery to the PM. In addition, DRM formation in Arabidopsis cells is shown to depend on the unsaturation degree of fatty acyl chains as evidenced by the dramatic decrease in the amount of DRMs prepared from the Arabidopsis mutants, fad2 and Fad3+, affected in their fatty acid desaturases.     

A new immobilized cell system with protection against toxic solvents

A new immobilized cell system providing protection against toxic solvents was investigated so that normal fermentations could be carried out in a medium containing toxic solvents. The system consists of immobilized growing cells in Ca-alginate gel beads to which vegetable oils, which are inexpensive absorbents of solvents, had been added. The ethanol fermentation of Saccharomyces cerevisiae ATCC 26603 was used as a model fermentation to study the protection afforded by the system against solvent toxicities. The fermentation was inhibited by solvents such as 2-octanol, benzene, toluene, and phenol. Ethanol production of one batch was not finished even after 35 h using immobilized growing yeast cells in conventional Ca-alginate gel beads in an ethanol production medium (5% glucose) containing 0.1% 2-octanol, which is used as a solvent for liquid-liquid extraction and is one of the most toxic solvents in our experiments. With the new immobilized growing cell system using vegetable oils, however, four repeated batch fermentations were completed in 35 h. Castor oil provided even more protection than soy bean, olive, and tung oils, and it was possible to complete six repeated batches in 35 h. The immobilized cell system with vegetable oils also provided protection against other toxic solvents such as benzene and toluene. A possible mechanism for the protective function of the new immobilized cell system is discussed.     

荧光光谱分析法在地沟油鉴别中的应用研究

由于地沟油的成分含量复杂性和不定量性,导致了现有的单一检测方法不能同时满足快速和准确的辨认。荧光光谱具有高灵敏度和分辨率的特性,由此提出了一种利用荧光光谱快速检测食用油中是否掺有地沟油的新方法。将花生油分成7组,每组油所含的地沟油的比例不同,用220 nm到800 nm的激发和发射光检测各组样品油,收集其荧光数据后做归一化处理进行分析。在荧光实验中,特别是在365 nm和720 nm激发波长波段和434 nm发射波长波段,样品油的荧光强度与所含地沟油的体积分数大小明显成反比,当地沟油的体积分数大于5%时,荧光强度的衰减更为明显。结果证明了荧光光谱法检测地沟油的可行性,而且步骤更为简单。利用荧光光谱的非接触和高灵敏度的优势,能够更为简便地检测到加入了5%以上地沟油的花生油。     

An Arabidopsis mutant deficient in sterol biosynthesis: heterologous complementation by ERG 3 encoding a Δ7-sterol-C-5-desaturase from yeast

The mutant STE 1 was isolated by screening an ethylmethane sulfonate (EMS)-mutagenized population of Arabidopsis thaliana which consisted of 22 000 M₂ plants divided into 1100 pools of 20 plants by gas chromatography of sterols extracted from small leaf samples. STE 1 was characterized by the accumulation of three Δ⁷-sterols concomitantly with the decrease of the three corresponding Δ⁵-sterols which are the end products of the sterol pathway in wild-type leaves. The structure of these Δ⁷-sterols was determined after two steps of purification on HPLC, by gas chromatography coupled with mass spectrometry (GC-MS) and proton nuclear magnetic resonance spectrometry (¹H-NMR). The accumulation of Δ⁷-sterols suggested that the mutant is deficient in the activity of the Δ⁷-sterol-C-5-desaturase. Genetic analysis showed that the accumulation of Δ⁷-sterols was due to a single recessive nuclear mutation. The mutant line STE 1 was backcrossed four times to the wild-type. The resulting STE 1 plants had wild-type morphology and set seeds normally, suggesting that the Δ⁷-sterols in STE 1 are good surrogates of physiologically active Δ⁵-sterols to sustain normal development. STE 1 roots were transformed with the Saccharomyces cerevisiae ERG 3 gene encoding the Δ⁷-sterol-C-5-desaturase under the control of the CaMV 35S promoter. Seven transgenic STE 1 root-derived calli showed an increase in Δ⁵-sterols and a concomitant decrease in Δ⁷-sterols in comparison with STE 1 untransformed root-derived calli. Northern blot analysis using the ERG 3 probe showed a strong expression of ERG 3 in three of the seven transgenic calli. These results suggest that the accumulation of Δ⁷-sterols in the STE 1 mutant is due to a deficiency of the Δ⁷-sterol-C-5-desaturation step in the plant sterol biosynthesis pathway.     

Isolation and chemical characterization of delta 5,7,24-cholestatrien-3 beta-ol from pig tissues

Although indirect evidence has implicated Delta(5,7,24)-cholestatrien-3-ol as a possible intermediate in cholesterol biosynthesis, this sterol has not previously been isolated from tissues. Administration of two inhibitors of cholesterol biosynthesis to pigs led to the accumulation of Delta(5,7,24)-cholestatrien-3-ol in the tissues, and this sterol was isolated from the lung. Proof of its chemical identity was based upon UV, IR, NMR, circular dichroism, and mass spectra, as well as comparison with synthetic Delta(5,7,24)-cholestatrien-3-ol. A fragment at m/e 143 is particularly prominent in the mass spectrum of Delta(5,7)-sterols, and this fact may prove useful for the detection of this functional group. It is proposed that Delta(5,7,24)-cholestatrien-3-ol may be an intermediate in sterol biosynthesis in both animals and plants.     

Generation of lipid peroxyl radicals from edible oils and their biological activities: a need for consideration for anti-radical components and purification processing

Lipid hydroperoxides (LOOH or oxidized oils) are known as unfavorable food components. Molecular details of the fate and mechanisms of LOOH to exert adverse effects in vivo are, however, little understood. In the present study, we demonstrated that LOOH generated alkylperoxyl radical (LOO*) after reaction with various heme compounds such as myoglobin, cytochrome c, hemin, hematin, etc., but little formation of other radical species was noticed such as L* or LO*. It was also shown that LOO* thus formed exhibits cytotoxicity and caused DNA damages including strand breakage and abasic site formation. This highly toxic LOO* is effectively scavenged by hot water extracts of vegetable (soup), flavonoids, polyphenols as well as tocopherols. Another important finding is that crude vegetable oils are rich in potent-LOO* scavenging activity, which exhibits potent anti-oxidant activity as well; whereas highly purified oils are scanty in such components and LOO* scavenging activity. These findings imply that a considerate processing in the refining of oils should be needed to retain such potent endogenous anti-oxidative radical scavenging-components.     

Co-occurrence of Δ5- and Δ7 -sterols in the fungus Dictyuchus monosporus

The desmethyl sterol composition of the oomycete Dictyuchus monosporus is unusual in that it is a mixture of 56.9 % Δ⁵-sterols and 42.6 % Δ⁷-sterols. The Δ⁵-sterols are cholesterol, 24 methylenecholesterol and fucosterol; the Δ⁷-sterols are cholest-7-enol, ergosta-7,24(28)-dienol and stigmasta-7,E-24(28)-dienol. Stigmasta-7,E-24(28)-dienol, is identified for the first time from natural sources. In addition, traces of lanosterol are present.     

Applied models to biodegradation kinetics of lubricant and vegetable oils in wastewater

Bioremediation technologies are used in order to remove pollutants from the environment in a safe, economical and harmless way during the treatment of waste, especially with the use of techniques such as biodegradation. A lubricant and vegetable oil contaminated water sample was studied in order to evaluate the biodegradability of different types of oils, considering the relevance of the obtained data in the bioremediation procedures. The objective of this paper is to use respirometry technique as a biodegradation process data source, and then apply to the obtained data the experimental design of mathematical models to characterize and determinate how the different types of oils are capable of affecting the parameters in biodegradation kinetics. The kinetics was then evaluated through selected models with a reasonable fit to experimental data. The Bartha and Pramer respirometer is used as a method to accurately measure the CO₂ formation in the organic compounds degradation by microorganisms. Therefore, the difference in biodegradation efficiency process is compared in the different groups of oils using mathematical models fitting the obtained data for the kinetics of biodegradation. The results demonstrated that used lubricant automotive oils are more susceptible to the biodegradation process, since their molecular structures had already been altered after use. In general, automotive lubricant oils shown better performance in biodegradation than vegetable oils. The models proposed for the obtained data in each of these assays demonstrated that vegetable oils biodegradation rate tends to decrease faster and end sooner than the automotive oils. Also, the modeling predicted that higher rates of biodegradation and total CO₂ production are to be expected in automotive lubricant oils rather than vegetable oils.     

Study on the effect of cultivation parameters and pretreatment on Rhizopus oryzae cell-catalyzed transesterification of vegetable oils for biodiesel production

Enzymatic methanolysis of vegetable oils for biodiesel production has become a hot point recently, in which study on whole cell as catalyst is an important field. In this paper, whole cell (Rhizopus oryzae IFO 4697) was adopted directly as biocatalyst for biodiesel production. Effects of carbon source on cell growth and whole cell-catalyzed methanolysis of vegetable oils for biodiesel production were studied. The results showed that different oils contained in the cultivation medium had varied effects on the whole cell-catalyzed methanolysis of oils; with some specified oil as the carbon source for cell cultivation, those cells expressed higher catalytic activity in catalyzing the transesterification of the same oil for biodiesel production. The initial reaction rate was increased notably (204%) with oil pretreatment on the cells before catalyzing the reaction, which was possibly due to the improved mass transferring of substrates. Under the optimized conditions, the maximum methyl ester yield could reach 86%.     

Study on the effect of cultivation parameters and pretreatment on Rhizopus oryzae cell-catalyzed transesterification of vegetable oils for biodiesel production

Enzymatic methanolysis of vegetable oils for biodiesel production has become a hot point recently, in which study on whole cell as catalyst is an important field. In this paper, whole cell (Rhizopus oryzae IFO 4697) was adopted directly as biocatalyst for biodiesel production. Effects of carbon source on cell growth and whole cell-catalyzed methanolysis of vegetable oils for biodiesel production were studied. The results showed that different oils contained in the cultivation medium had varied effects on the whole cell-catalyzed methanolysis of oils; with some specified oil as the carbon source for cell cultivation, those cells expressed higher catalytic activity in catalyzing the transesterification of the same oil for biodiesel production. The initial reaction rate was increased notably (204%) with oil pretreatment on the cells before catalyzing the reaction, which was possibly due to the improved mass transferring of substrates. Under the optimized conditions, the maximum methyl ester yield could reach 86%.     

Determination of tocopherols as lipid antioxidants in vegetable oils and animal fats

The content of vitamin E (tocopherols) in natural vegetable oils and fats has been determined by microcalorimetry. This technique belongs to kinetic methods for determining vitamin E, which are based on the capacity of tocopherol to inhibit liquid-phase radical oxidation reactions. It has been shown on a model reaction of initiated oxidation of cumene that fatty oils inhibit the radical reaction with an induction period proportional to the content of tocopherols in oil. From experimental curves, the content of tocopherols in oils obtained by different technological methods has been estimated. It has been shown that the amount of tocopherols is an indicator of the oil quality; therefore, the method proposed can be used to control the way of oil manufacture, oil quality, and the presence of synthetic antioxidants.     

Selective growth inhibition of a human malignant melanoma cell line by sesame oil in vitro.

Ayurveda, an ancient and comprehensive system of natural medicine, recommends regular topical application to the skin of sesame oil, above all other oils, as a health-promoting procedure. We examined the effect of sesame oil and several other vegetable oils and their major component fatty acids on the proliferation rate of human normal and malignant melanocytes growing at similar rates in serum-free media. We found that sesame and safflower oils, both of which contain large amounts of linoleate in triglyceride form, selectively inhibited malignant melanoma growth over normal melanocytes whereas coconut, olive and mineral oils, which contain little or no linoleate as triglyceride, did not. These oils were tested at a range of 10-300 micrograms/ml. We found that of the fatty acids tested, only linoleic acid was selectively inhibitory while palmitic and oleic were not. These fatty acids were tested in the range of 3-100 micrograms/ml. These results suggest that certain vegetable oils rich in linoleic acid, such as the sesame oil, recommended for topical use by Ayurveda, may contain selective antineoplastic properties which are similar to those demonstrated for essential polyunsaturated fatty acids and their metabolites. This suggests that whole vegetable oils may have potential clinical usefulness.     

Inhibition of the sterol pathway in leek seedlings impairs phosphatidylserine and glucosylceramide synthesis but triggers an accumulation of triacylglycerols

Like most higher plants, leek seedlings (Allium porrum L.) contain a mixture of Delta(5)-sterols in which sitosterol largely predominates. As previously reported (Plant Physiol., 117 (1998) 931), these compounds, which are synthesized at the endoplasmic reticulum level, were shown to be actively transported to the plasma membrane via a membrane-mediated process, together with phosphatidylserine (PS). In the present work, leek seedlings were allowed to germinate for 7 days in the presence of fenpropimorph, a sterol biosynthesis inhibitor. Such a treatment was found to trigger an almost complete replacement of the usual sterols by 9beta,19-cyclopropylsterols (mainly cycloeucalenol and 29-norcycloartenol). Extensive lipid analyses and labeling experiments with sodium [14C]acetate were performed to examine potential changes in the content and the rate of synthesis of the other lipid molecular species. The results indicate that the inhibition of the sterol pathway was accompanied by a severe decrease in PS and glucosylceramide synthesis as well as by a redirection of fatty acids toward the storage triacylglycerol pathway. Triacyglycerols are shown to accumulate concomitantly with a significant increase in intracellular lipid droplets in both aerial parts and roots of leek seedlings. Taken together, the present data emphasize that a coordinated regulation of the biosynthetic pathways of sterols and some specific lipid molecular species could take place during plant membrane biogenesis.     

Stereoselective method development and validation for determination of concentrations of amphetamine-type stimulants and metabolites in human urine using a simultaneous extraction-chiral derivatization approach

Amphetamine-type stimulants (ATS) are a group of chiral amine drugs which are commonly abused for their sympathomimetic and stimulant properties. ATS are extensively metabolised by hepatic cytochrome P450 enzymes. As metabolism of ATS has been shown to be highly stereospecific, stereoselective analytical methods are essential for the quantitative determination of ATS concentrations for both in vivo and in vitro studies of ATS metabolism. This paper describes a new stereoselective method for the simultaneous determination of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxymethamphetamine (HMMA), 4-hydroxy-3-methoxyamphetamine (HMA), 3,4-hydroxymethamphetamine (HHMA) and 3,4-hydroxyamphetamine (HHA) in human urine samples validated according to the United States Food and Drug Administration guidelines. In this method, analytes are simultaneously extracted and derivatized with R-(-)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (R-MTPCl) as the chiral derivatization reagent. Following this, the analytes were subjected to a second derivatization with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) which targets the hydroxyl groups present in HMMA, HMA, HHMA and HHA. The derivatized analytes were separated and quantified using gas chromatography-mass spectrometry (GC-MS). The method was evaluated according to the established guidelines for specificity, linearity, precision, accuracy, recovery and stability using a five-day protocol. Intra-day precision ranged from 0.89 to 11.23% RSD whereas inter-day precision was between 1.03 and 12.95% RSD. Accuracy values for the analytes ranged from -5.29% to 13.75%. Limits of quantitation were 10 μg/L for AM, MA, MDMA, HMA and HMMA and 2μg/L for MDA, HMA and HHA. Recoveries and stability values were also within accepted values. The method was applied to authentic ATS-positive samples.     

Role of highly conserved residues in the reaction catalyzed by recombinant Delta7-sterol-C5(6)-desaturase studied by site-directed mutagenesis

The role of 15 residues in the reaction catalyzed by Arabidopsis thaliana Delta7-sterol-C5(6)-desaturase (5-DES) was investigated using site-directed mutagenesis and expression of the mutated enzymes in an erg3 yeast strain defective in 5-DES. The mutated desaturases were assayed in vivo by sterol analysis and quantification of Delta5,7-sterols. In addition, the activities of the recombinant 5-DESs were examined directly in vitro in the corresponding yeast microsomal preparations. One group of mutants was affected in the eight evolutionarily conserved histidine residues from three histidine-rich motifs. Replacement of these residues by leucine or glutamic acid completely eliminated the desaturase activity both in vivo and in vitro, in contrast to mutations at seven other conserved residues. Thus, mutants H203L, H222L, H222E, P201A, G234A, and G234D had a 5-DES activity reduced to 2-20% of the wild-type enzyme, while mutants K115L, P175V, and P175A had a 5-DES activity and catalytical efficiency (V/K) that was similar to that of the wild-type. Therefore, these residues are not essential for the catalysis but contribute to the activity through conformational or other effects. One possible function for the histidine-rich motifs would be to provide the ligands for a presumed catalytic Fe center, as previously proposed for a number of integral membrane enzymes catalyzing desaturations and hydroxylations [Shanklin et al. (1994) Biochemistry 33, 12787-12794]. Another group of mutants was affected in residue 114 based on previous in vivo observations in A. thaliana indicating that mutant T114I was deficient in 5-DES activity. We show that the enzyme T114I has an 8-fold higher Km and 10-fold reduced catalytic efficiency. Conversely, the functionally conservative substituted mutant enzyme T114S displays a 28-fold higher Vmax value and an 8-fold higher Km value than the wild-type enzyme. Consequently, V/K for T114S was 38-fold higher than that for T114I. The data suggest that Thr 114 is involved in stabilization of the enzyme-substrate complex with a marked discrimination between the ground-state and the transition state of a rate-controlling step in the catalysis by the 5-DES.     

Fats and atheroma: an inquest.

All well-controlled trials of cholesterol-reducing diets and drugs have failed to reduce coronary (CHD) mortality and morbidity. Nevertheless, commercial, professional, and even government-sponsored propaganda continues. Experimentally some vegetable oils and hardened fats can be more damaging to arteries than butter. There are other hazards to heart muscle from vegetable oils. Israelis consume a high polyunsaturated fat diet equal to that recommended for prevention of CHD in USA but their CHD incidence is very high. Urban Bedouins are also affected. The primary clofibrate prevention trial underlines unacceptable risks which could apply also to diets. Official medical endorsement of these cholesterol reducing measures should be withdrawn.