Artifact formation during transmethylation of lipid peroxides.

By-product formation during base-catalyzed transesterification of lipid peroxides was observed in model experiments. A partially oxidized linoleic acid methyl ester as well as purified hydroxylinoleates served as test compounds. By-products formed during a simulated transmethylation of purified hydroxylinoleates were separated from the main components by means of thin-layer chromatography. The loss attributable to these by-products amounted to 10%. Oxygenated fatty acid derivatives were completely destroyed by acid-catalyzed transmethylation. Catalytic hydrogenation prior to base-catalyzed transmethylation proved to be a simple means to minimize side-product formation. By using this technique the yield of hydroxystearates from a partially autoxidized linoleic acid methyl ester preparation was improved significantly.     

花生△12脂肪酸脱氢酶基因高效表达载体构建与原核表达

花生是世界范围内广泛种植的重要油料作物之一,其种子中富含油酸和亚油酸。△^12脂肪酸脱氢酶（FAD2）是亚油酸合成的关键酶,催化油酸（18：1）在△^12位上脱氢生成亚油酸（18：2）,但由于△^12脂肪酸脱氢酶本身的特性,目前还没有有效的方法将其纯化并在蛋白水平作进一步的研究,尚需对其结构和功能之间以及表达调控进行更深入全面的研究。本文利用从花生中克隆的△^12脂肪酸脱氢酶基因（GenBank接受号为AY1006）构建高效表达载体,把花生△^12脂肪酸脱氢酶基因全长序列插入到大肠杆菌高效表达载体pRSETB中,构建了pRSET／HO-A融合表达载体,并转化到大肠杆菌表达菌BL21（DE3）pLysS中,在IPTG诱导下,pRSET／HO-A融合表达载体在BL21（DE3）pLysS菌株中高效表达了△^12脂肪酸脱氢酶。利用Clon-Tech蛋白纯化Kit进一步分离了目的蛋白,同时加入外源性底物油酸在20℃温育6h后,进行脂肪酸甲酯化处理,通过气相色谱（GC）和气相色谱,质谱（GC-MS）分析表明,所编码的酶具有△^12脂肪酸脱氢酶的活性,能将外源性的底物油酸转化为亚油酸,转化率为11．8％。花生△^12脂肪酸脱氢酶基因的原核表达目前国内外还未见报导,本实验为其进一步的大量纯化和结构功能分析奠定了基础。     

Stereospecificity of the hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids produced by cultured bovine endothelial cells

Characterization of the stereospecificity of the derivatives of arachidonic acid and linoleic acid produced by endothelial cells is needed to define the enzymatic origin of these compounds and their role in vascular physiology. In studies utilizing two bovine endothelial cell lines (CPAE and AG04762), both free 15-hydroxyeicosatetraenoic acid (15-HETE) and 11-hydroxyeicosatetraenoic acid (11-HETE) were generated during incubations with exogenous arachidonic acid and both free 9-hydroxyoctadecadienoic acid (9-HODE) and 13-hydroxyoctadecadienoic acid (13-HODE) were generated during incubations with exogenous linoleic acid. Esterification of 15-HETE, 9-HODE and 13-HODE during these incubations was demonstrated. The analyses included reversed-phase high performance liquid chromatography of the free acid and its methyl ester and chiral separation of the methyl ester on straight phase chiral columns. The ratio of 9-HODE/13-HODE averaged 2.7 in the chromatographic analyses of the extracts of the incubations with linoleic acid. The combined production of 13-HODE and 9-HODE from linoleic acid was four times greater than that of 15-HETE and 11-HETE from arachidonic acid. With regard to the products of the CPAE endothelial cell line, the S/R ratio of the stereoisomers averaged 1.5 for free 15-HETE, 5.7 for free 13-HODE and 0.2 for free 9-HODE. The 11-HETE had strict (R) stereospecificity. The products from the AG04762 endothelial cell line had similar stereochemistry. All these stereochemical findings point to the activity of a cyclooxygenase rather than that of a lipoxygenase.     

藏红花花瓣和雄蕊挥发油化学成分GC-MS分析及比较

采用水蒸气蒸馏法从藏红花花瓣和雄蕊中提取挥发油,用GC-MS技术结合计算机检索对其二者化学成分进行分离和鉴定,用色谱峰面积归一化法计算各组分的相对含量.花瓣中共鉴定出16种化合物,主要成分为正二十六烷(11.60％)、正十五烷(11.31％)、棕榈酸甲酯(10.82％)、油酸甲酯(10.35％)、2,4-二叔丁基苯酚(9.63％)、亚油酸甲酯(7.18％)、藏红花醛(5.66％);雄蕊中共鉴定出20种化合物,主要成分为油酸甲酯(30.83％)、亚油酸甲酯(24.12％)、环已醇(16.80％)、硬脂酸甲酯(12.88％)、棕榈酸甲酯(8.97％)、花生酸甲酯(1.18％)、苯并噻唑(1.01％).     

Evidence for participation of cytochrome b5 in microsomal delta-6 desaturation of fatty acids.

The delta-6 desaturation of linoleic acid to gamma-linolenic acid and oleic acid to 6,9-octadecadienoic acid by rat liver microsomes was investigated. Using a specific antibody prepared against purified rat liver cytochrome b5, we demonstrated that cytochrome b5 participated in delta-6 desaturation of both fatty acids. The reaction products were identified as their methyl ester derivatives by argentation thin-layer chromatography, gas-liquid chromatography, and reductive ozonolysis followed by gas-liquid chromatography.     

Free and esterified 13(R,S)-hydroxyoctadecadienoic acids: principal oxygenase products in psoriatic skin scales

Characterization of the chemical form and stereo-specificity of the fatty acid derivatives of arachidonic and linoleic acid in psoriatic epidermis is needed to define the enzymatic origin of these compounds and their possible role in pathogenesis. In an analysis of psoriatic skin scales, both free and esterified 13-hydroxyoctadecadienoic acids were the principal fatty acid derivatives, present in mean concentrations of 115 and 17 ng/mg scales, respectively. The analysis included reversed-phase high performance liquid chromatography of the free acid and of its methyl ester, gas chromatography, gas-liquid chromatography-mass spectrometry of the methyl ester derivatives, and chiral separation. The free and esterified 13-hydroxyoctadecadienoic acids isolated from the psoriatic scales contained a mixture of the S/R stereoisomers, averaging 1.9:1 for free 13- hydroxyoctadecadienoic acid. These findings are not compatible with the strict S-stereospecificity for oxygen insertion exhibited by mammalian lipoxygenase but rather could point to the action of a cyclooxygenase. The demonstration that a hydroxylated fatty acid derivative is esterified in vivo in psoriatic keratinocytes suggests that the physiology of these cells may be altered early in the process of keratinization.     

Murine 12(R)-lipoxygenase: functional expression, genomic structure and chromosomal localization

A cDNA, recently cloned (by Krieg et al. (1998)) from mouse skin, was shown to encode a 12(R)-lipoxygenase. When expressed in HEK cells, the recombinant protein converted methyl arachidonate into the corresponding 12-HETE ester which was shown to be the R-enantiomer by chiral phase chromatography. Neither arachidonic acid nor linoleic acid were substrates for the recombinant protein. The structure of the 12(R)-lipoxygenase gene is unique among all animal lipoxygenases in that it is divided into 15 exons and 14 introns spanning approximately 12.5 kb. By interspecific backcross analysis, the 12(R)-lipoxygenase gene was localized to the central region of mouse chromosome 11.     

Modification of cellular fatty acid composition of Hep-G2 cells: effect of antioxidants on cholesterol esterification and secretion

Modification of fatty acid composition of Hep-G2 cells was achieved by 7-9 days of supplementation of culture medium with palmitic, oleic or linoleic acid. Cholesterol release into serum-free culture medium during 24 h of incubation was significantly lower in cells supplemented with linoleic acid, when compared to those supplemented with palmitic, oleic or no additional fatty acid. In cells cultured in the presence of linoleic acid, less [3H]cholesterol was esterified to cholesteryl ester and the mass of cholesteryl ester was significantly lower than in cells cultured with palmitic acid or with no additional fatty acid. The reduction in [3H]cholesterol secretion and the impairment in cholesterol esterification in linoleic acid-treated cells was prevented by addition of butylated hydroxytoluene or probucol concurrently with the fatty acid. The antioxidants also increased esterification and [3H]cholesterol release in cells supplemented with the other fatty acids. It is suggested that cholesterol secretion and esterification are sensitive to peroxidation.     

Growth characteristics and polyene sensitivity of a fatty acid auxotroph of Candida albicans.

A fatty acid auxotroph of Candida albicans 6406, designated A' 44 and originally isolated as an oleic acid requiring strain, has been shown to be a delta9 desaturase mutant. Although lacking this step in fatty acid biosynthesis, it appears to retain the ability to desaturate monounsaturated fatty acids. The polyene sensitivity of the organism grown on different fatty acid supplements varied between 0-08 +/- 0-02 and 1-20 +/- 0-30 microgram amphotericin B methyl ester ml-1 for exponentially growing cells. In spite of this variation, the sterol composition remained fairly constant, the major differences lying in fatty acid composition. Stationary-phase cells were more resistant to amphotericin B methyl ester, although again this change was not associated with changes in sterol content. The organism was most resistant when grown in the presence of oleic or linoleic acid. Protoplasts derived from resistant organisms grown on these two fatty acids were also resistant, indicating that the structure of the cell wall was less important than that of the plasma membrane in determining polyene sensitivity under these conditions.     

Linoleic acid biosynthesis in the pea aphid,Acyrthosiphon pisum (Harris)

The pea aphid Acyrthosiphon pisum (Harris) incorporated [1-¹⁴C]acetate into a phospholipid dienoic fatty acid in a time-dependent manner. In 2-h incubations, the incorporation of radioactivity into the 18:2 fraction was minimal, whereas after 45 h 18:2 was the major fatty acid labeled. Ozonolysis of the isolated dienoic fatty acid methyl ester followed by radio-gas-liquid chromatography showed that radioactivity was associated with fragments containing carbons 1–9 and 13–18. These data established the location of the double bonds in the 9,12 positions and indicated that the entire molecule was labeled from [1-¹⁴C]acetate. Tetracycline-treated aphids synthesized linoleic acid in the same proportions as untreated controls. Scanning electron microscopy showed that over 50% of the treated insects had greatly reduced numbers of intracellular symbiotes or lacked them or most of the existing symbiotes had an abnormal appearance. Therefore, we conclude that intracellular symbiotes are not involved in the biosynthesis of linoleic acid in the pea aphid.     

二十二碳六烯酸甲酯标准样品的研制

本制备采用以深海鱼油为基础原料,在碱性条件下皂化水解为游离脂肪酸,经尿素－甲醇包合分离后,以硫酸－甲醇甲酯化,再经硝酸银－硅胶柱层析分离,从而得到高纯度的二十二碳六烯酸甲酯标准物质,并经定值测试,确认该标准物质的含量≥98％以上。     

Oxidative metabolism of linoleic acid by human leukocytes

Upon incubation with human leukocytes, [1-14C] linoleic acid is almost exclusively transformed into 13-hydroxy-9Z, 11E-octadecadienoic acid (13-HODE) if the linoleic acid concentration is lower than 50 microM. Identification of 13-HODE was done by GLC-MS at the level of its methyl ester, trimethylsilyl ether and by comparison with authentic 13-HODE in two different HPLC systems. Analysis of the products by chiral phase HPLC shows that 13(S)-hydroxy-9Z, 11E-octadecadienoic acid is by far the major metabolite formed by human leukocytes. Comparison of reactions performed with intact or lyzed cells suggests that the formation of 13(S)-HODE by human leukocytes occurs in two steps, a dioxygenation catalyzed by a 15-lipoxygenase and a reduction of intermediate 13-HPODE by a glutathione-dependent peroxidase.     

Biotransformation of linoleic acid by porcine leukocytes

Linoleic acid is an important essential fatty acids of leukocyte cell membrane phospholipids from some animals, e.g. from pigs and rabbits, and is a known substrate for lipoxygenase(s), especially in plant systems. Lipoxygenase activity has also been well documented in leukocytes using arachidonic acid as a substrate. These findings and our own interest in the fate of linoleic acid have prompted us to investigate the biotransformation of this essential fatty acids in leukocytes.Porcine leukocytes were isolated from whole blood by dextrane precipitation of the erythrocytes and by centrifugation. Broken cells were incubated with exogenous linoleic acid and four major biotransformation products, X₁, X₂, X₃ and X₄, were formed. Following isolation by silicagel column chromatography and thin layer chromatography, the products were derivatized and characterized by GC/MS. Derivatization included hydrogenation, methyl ester formation, n-butyl boronate formation and trimethylsilylation, and various types of derivatives were made in order to facilitate the structure elucidation. The major product X₁, which represented 60.5% of the total metabolites formed, was identified as 13-hydroxy-9,11-octadecadienoic acid. Product X₂ (16.2%) was shown to be 11-hydroxy-12,13-epoxy-9-octadecenoic acid. Products X₃ and X₄ (respectively 5.2 and 7.5%) resulted in identical thermore, each of the products X₃ and X₄ was shown to be a mixture of two positional isomers, i.e. of 9,12,13-trihydroxy-10-octadecenoic acid (70%) and 9,10,13-trihydroxy-12-octadecenoic acid (30%). With regard to the structure elucidation of the latter isomers, the mixed hydrogenated, n-butylboronate, methyl ester, TMS-ether derivatives were shown to be of particular value for the determination of the vicinal diol position.The metabolism of linoleic acid in porcine leukocytes is analogous to that by cereal lipoxygenases. A major difference however is that porcine leukocyte lipoxygenase predominantly yields products, which arise through 13-lipoxygenation, whereas, in cereals, transformation products of 9-hydroperoxy-10,12-octadecadienoic acid are formed to the same extent as metabolites of 13-hydroperoxy-9,11-octadecadienoic acid.     

Effects of different types of polyunsaturated fatty acids on cholesterol esterification in human fibroblasts.

We have enriched human fibroblasts with oleic acid, with linoleic acid and with eicosapentaenoic acid. The accumulation of cholesteryl esters in the cells and the rate of esterification of cholesterol by microsomal acyl-CoA:cholesterol acyltransferase (ACAT) were measured in these cells. Cholesteryl ester levels were lower in cells enriched with eicosapentaenoic acid compared with cells enriched with oleate or linoleate. We also observed significantly lower ACAT activities in the microsomes from fibroblasts enriched with the n-3 polyunsaturated fatty acids relative to cells enriched with oleic acid or linoleic acid. We suggest that the presence of n-3 polyunsaturated fatty acids might suppress cholesteryl ester accumulation and inhibit atherogenesis.     

Stimulation of hematopoiesis and bone marrow suppressor cells by the subcutaneous injection of linoleic acid

The effects of injection of linoleic acid into C57Bl/6 mice on hematopoietic and immunological parameters were examined. Administration of linoleic acid stimulated hematopoiesis as it increased spleen weight and cellularity, increased the number of bone marrow and splenic granulocytic-monocytic progenitor cells, and increased the colony stimulating factor activity in the serum of the treated mice. Associated with the hematopoietic stimulation in linoleic acid-treated mice was a decline in the spleen cell blastogenic responses and the appearance of bone marrow suppressor cells which were inhibitory to normal spleen cell blastogenesis. The linoleic acid-induced bone marrow suppressor cells resembled cells of the monocyte lineage in that they were sensitive to treatment with L-leucine methyl ester, partially sensitive to treatment with anti-Ia antibodies and complement, and their suppressor activity was minimized by indomethacin, a prostaglandin synthesis inhibitor. These results suggest that administration of linoleic acid results in hematopoietic stimulation and, concurrently, in the appearance of suppressor cells in the bone marrow. The bone marrow suppressor cells resemble immature cells of the monocyte lineage and appear to mediate their suppressive effects through the production of prostaglandins.     

Formation of lipoxin B by the pure reticulocyte lipoxygenase via sequential oxygenation of the substrate

The pure reticulocyte lipoxygenase converts 15LS-hydroxy-5,8,11,13(Z,Z,Z,E)-icosatetraenoic acid (15LS-HETE) methyl ester to a complex mixture of products containing 5DS,14LR,15LS-trihydro(pero)xy-6E,++ +8Z,10E,12E-icosatetraenoate methyl ester (lipoxin B methyl ester), 5DS,15LS-DiH(P)ETE methyl ester and four 8,15LS-DiH(P)ETE methyl ester isomers [DiH(P)ETE = dihydro(pero)xy-icosatetraenoic acid]. After a short incubation period (15 min) 5DS,15LS-DiH(P)ETE methyl ester was found to be the main product, whereas after a 3-h incubation lipoxin B methyl ester was the predominant product. The reaction shows a remarkable stereoselectivity since only small amounts of other trihydroxy tetraenes are formed. Anaerobiosis, heat inactivation of the enzyme, or incubation in the presence of lipoxygenase inhibitors (icosatetraynoic acid, nordihydroguaiaretic acid) completely abolished the reaction. The complete steric structure of the major tetraene product (lipoxin B methyl ester) was established by ultraviolet spectroscopy, HPLC on four different types of columns, gas chromatography/mass spectrometry, gas/liquid chromatography of the ozonolysis fragments of the menthoxycarbonyl derivatives, and by 400-MHz 1H-NMR. Atmospheric oxygen was incorporated at carbon-5 and carbon-14 into the major product. 5DS,15LS-DiH(P)ETE methyl ester was shown to be an intermediate in the synthesis. Lipoxin B was also formed during the oxygenation of arachidonic acid, 15LS-HETE and 5DS,15LS-DiHETE. The results presented here indicate that lipoxin B can be formed by pure lipoxygenases via a sequential oxygenation of arachidonic acid or its hydro(pero)xy derivatives.     

根瘤蚜对不同抗性葡萄的选择性及葡萄根系挥发性物质的鉴定

为了探讨不同抗性水平砧木及品种对根瘤蚜吸引及驱避效应以及鉴定挥发性物质组分,采用离体根段培养法测定葡萄根瘤蚜Phylloxera viticola Fitch对砧木5BB,140Ru及栽培品种巨峰的选择性,发现根瘤蚜对栽培品种巨峰的根段具有很强的选择性,至接种后3 d,67.13％的根瘤蚜优先选择巨峰;而对5BB和140Ru的选择性却很差,分别有15.29％和17.58％的根瘤蚜选择5BB及140Ru。根瘤蚜在不同品种根段上的生长发育和繁殖也存在明显差异。对砧木5BB和巨峰根系的挥发性组分进行测定,发现2个品种挥发物独有组分数量均较高,巨峰独有组分为23个,占总成分数的48.94％,5BB的独有组分为32个,占总成分数的57.14％。5BB和巨峰的共有组分为24个,主要成分均为亚油酸甲酯和反油酸甲酯,但含量有所不同,其中,5BB以倍半萜类的丁香醇及脂肪酸甲酯类的亚油酸甲酯,反油酸甲酯,棕榈油酸甲酯含量较高,高于巨峰中相应物质含量的4.23%~6.46％,而巨峰以邻苯二甲酸二丁酯及胆甾烷含量较高,分别高于5BB相应组分含量的6.81％和1.07％。结果说明葡萄根瘤蚜对不同抗性水平砧木及品种确实存在不同的选择性,并且不同抗性水平的砧木及品种的挥发性物质存在显著差异。     

Synthesis and biological activity of hydroxylated derivatives of linoleic acid and conjugated linoleic acids

Allylic hydroxylated derivatives of the C18 unsaturated fatty acids were prepared from linoleic acid (LA) and conjugated linoleic acids (CLAs). The reaction of LA methyl ester with selenium dioxide (SeO₂) gave mono-hydroxylated derivatives, 13-hydroxy-9Z,11E-octadecadienoic acid, 13-hydroxy-9E,11E-octadecadienoic acid, 9-hydroxy-10E,12Z-octadecadienoic acid and 9-hydroxy-10E,12E-octadecadienoic acid methyl esters. In contrast, the reaction of CLA methyl ester with SeO₂ gave di-hydroxylated derivatives as novel products including, erythro-12,13-dihydroxy-10E-octadecenoic acid, erythro-11,12-dihydroxy-9E-octadecenoic acid, erythro-10,11-dihydroxy-12E-octadecenoic acid and erythro-9,10-dihydroxy-11E-octadecenoic acid methyl esters. These products were purified by normal-phase short column vacuum chromatography followed by high-performance liquid chromatography (HPLC). Their chemical structures were characterized by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR). The allylic hydroxylated derivatives of LA and CLA exhibited moderate in vitro cytotoxicity against a panel of human cancer cell lines including chronic myelogenous leukemia K562, myeloma RPMI8226, hepatocellular carcinoma HepG2 and breast adenocarcinoma MCF-7 cells (IC₅₀ 10-75 μM). The allylic hydroxylated derivatives of LA and CLA also showed toxicity to brine shrimp with LD₅₀ values in the range of 2.30-13.8 μM. However these compounds showed insignificant toxicity to honeybee at doses up to 100 μg/bee.     

Evidence in the formation of conjugated linoleic acids from thermally induced 9t12t linoleic acid: a study by gas chromatography and infrared spectroscopy

Thermally induced isomerisation leading to the formation of conjugated linoleic acids (CLAs) has been observed for the first time during the thermal treatment of 9t12t fatty acid triacylglycerol, and methyl ester. Fifteen microlitre portions of the triacylglycerol sample containing 9t12t fatty acid (trilinoelaidin) were placed in micro glass ampoules and sealed under nitrogen, then subjected to thermal treatment at 250 °C. The glass ampoules were removed at regular time intervals, cut open, and the contents were analysed by infrared spectroscopy using a single reflectance attenuated total internal reflectance crystal accessory. The samples were then subjected to derivatisation into their methyl esters. The methyl esters of the isomerised fatty acids were analysed by gas chromatography. The same procedure was repeated with methyl ester samples containing 9t12t fatty acid (methyl linoelaidate). Each sample was subjected to infrared measurements and gas chromatographic analysis after appropriate dilution in heptane.The results show that the thermally induced isomerisation of 9t12t fatty acids from both triacylglycerol molecules and methyl esters give identical CLA profiles as those found for the thermally induced isomerisation of 9c12c fatty acids. The infrared spectrometry provides additional evidence confirming the formation of CLA acids during thermal treatment. A mechanism for the formation of the CLAs from 9t12t fatty acid molecules is also formulated for the first time. This mechanism complements the pathways of formation of CLAs from 9c12c fatty acids during thermal treatment.     

Biodiesel production from algal oil using cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz) as feedstock

As a potential source of biomass supplies, cassava (Manihot esculenta Crantz) has been studied for bioethanol production, but not for the production of biodiesel. In this study, we used cassava hydrolysate as an alternative carbon source for the growth of microalgae (Chlorella protothecoides) which accumulated oil in vivo, with high oil content up to 53% by dry mass under a 5-L scale fermentation condition. The oils were extracted and converted into biodiesel by transesterification. The biodiesel obtained consisted of mainly unsaturated fatty acids methyl ester (over 82%), cetane acid methyl ester, linoleic acid methyl ester, and oleic acid methyl ester. This work suggests the feasibility of an alternative choice for producing biodiesel from cassava by microalgae fermentation. We report herewith the optimized condition for the fermentation and for the hydrolysis of cassava as the carbon source.