Synthesis of hydroxy fatty acids from linoleic acid by human blood platelets

The metabolism of linoleic acid by washed human platelets was investigated. [1.14C] linoleic acid was converted to [1.14C] hydroxy octadecadienoic acids (HODEs) at about the same rate with which [1.14C] 12-HETE was produced from [1.14C] arachidonic acid. The total radioactivity in HODEs was distributed among two isomers: 13-HODE (85%) and 9-HODE (15%) as defined by CG-MS. The production of HODEs by intact washed platelets was inhibited by indomethacin (IC50:5 x 10(-7) M) which suggest that hydroxy fatty acids were produced by PGH-synthase. By contrast, the production of HODEs by platelet cytosolic fractions was not modified under indomethacin treatment but completely abolished by NDGA (10(-3) M) and inhibited by the platelet lipoxygenase inhibitors 15-HETE (2.10(-5) M) and baicalein (10(-5) M). Platelets thus contain two different active systems which may convert linoleic acid to hydroxy fatty acids. Since these compounds remained essentially associated with the platelets, their presence may significantly participate in the mechanisms of platelet activation.     

Triglyceride-rich lipoprotein lipolysis releases neutral and oxidized FFAs that induce endothelial cell inflammation

Triglyceride-rich lipoprotein (TGRL) lipolysis products provide a pro-inflammatory stimulus that can alter endothelial barrier function. To probe the mechanism of this lipolysis-induced event, we evaluated the pro-inflammatory potential of lipid classes derived from human postprandial TGRL by lipoprotein lipase (LpL). Incubation of TGRL with LpL for 30 min increased the saturated and unsaturated FFA content of the incubation solutions significantly. Furthermore, concentrations of the hydroxylated linoleates 9-hydroxy ocatadecadienoic acid (9-HODE) and 13-HODE were elevated by LpL lipolysis, more than other measured oxylipids. The FFA fractions elicited pro-inflammatory responses inducing TNFalpha and intracellular adhesion molecule expression and reactive oxygen species (ROS) production in human aortic endothelial cells (HAECs). The FFA-mediated increase in ROS was blocked by both the cytochrome P450 2C9 inhibitor sulfaphenazole and NADPH oxidase inhibitors. Compared with linoleate, 13-HODE was found to be a more potent inducer of ROS production in HAECs, an activity that was insensitive to both NADPH oxidase and cytochrome P450 inhibitors. Therefore, although the oxidative metabolism of FFA in endothelial cells can produce inflammatory responses, TGRL lipolysis can also release preformed mediators of oxidative stress (e.g., HODEs) that may influence endothelial cell function in vivo by stimulating intracellular ROS production.     

Preferential formation of 13-hydroxylinoleic acid by human peripheral blood eosinophils

Lipid mediators released by inflammatory and immune cells play an important role in inflammatory and immune processes. Most attention has been focussed on arachidonic-derived mediators, including prostaglandins, thromboxanes, leukotrienes, and lipoxins. Literature data, however, suggest that also metabolites of the unsaturated fatty acid linoleic acid may be important in this respect. We have studied the formation and release of 9-hydroxy- and 13-hydroxy-linoleic acid (9-HODE and 13-HODE) by enriched populations of human peripheral blood neutrophils, eosinophils, basophils, monocytes, and lymphocytes. We demonstrate that the eosinophil preferentially produces 13-HODE, whereas the other cell types produce equal amounts of 9-HODE and 13-NODE. The biological significance of these findings is discussed.     

Arachidonic and linoleic acid metabolism in mouse intestinal tissue: evidence for novel lipoxygenase activity.

Previous studies in our laboratory revealed a high expression of 15-lipoxygenase-1 in human colorectal carcinomas, suggesting the importance of lipoxygenase in colorectal tumor development. In this report, we have investigated the metabolism of arachidonic and linoleic acid by intestinal tissues of Min mice, an animal model for intestinal neoplasia. The polyp and normal tissues from Min mice intestine were homogenized, incubated with arachidonic or linoleic acid, and analyzed by reverse-, straight-, and chiral-phase HPLC. Arachidonic acid was converted to prostaglandins E2 and F2alpha. Little 12- or 15-hydroxyeicosatetraenoic acid was detected. Cyclooxygenase (COX)-2 was detected in polyps and the adjacent normal tissues by Western immunoblotting, but neither COX-1 nor leukocyte-type 12-lipoxygenase, the murine ortholog to human 15-lipoxygenase-1, was detected. These tissue homogenates converted linoleic acid to an equal mixture of 9(S)- and 13(S)-hydroxyoctadecadienoic acid (HODE). Inhibition of lipoxygenase activity with nordihydroguaiaretic acid blocked HODEs formation, but the COX inhibitor indomethacin did not. Degenerative-nested PCR analyses using primers encoded by highly conserved sequences in lipoxygenases detected 5-lipoxygenase, leukocyte-type 12-lipoxygenase, platelet-type 12-lipoxygenase, 8-lipoxygenase, and epidermis-type lipoxygenase-3 in mouse intestinal tissue. All of these PCR products represent known lipoxygenase that are not reported to utilize linoleic acid preferentially as substrate and do not metabolize linoleic acid to an equal mixture of 9(S)- and 13(S)-HODE. This somewhat unique profile of linoleate product formation in Min mice intestinal tissue suggests the presence of an uncharacterized and potentially novel lipoxygenase(s) that may play a role in intestinal epithelial cell differentiation and tumor development.     

Formation of monohydroxy derivatives of arachidonic acid, linoleic acid, and oleic acid during oxidation of low density lipoprotein by copper ions and endothelial cells.

An important event in the formation of atherosclerotic lesions is the uptake of modified low density lipoprotein (LDL) by macrophages via scavenger receptors. Modification of LDL, which results in its recognition by these receptors, can be initiated by peroxidation of LDL lipids. The first step in this process is the formation of monohydroperoxy derivatives of fatty acids, which are subsequently degraded to the corresponding monohydroxy compounds, or to a variety of secondary oxidation products. In order to understand this process more completely, we have developed a mass spectrometric procedure to measure the amounts of specific hydroperoxy/hydroxy fatty acids formed by oxidation of the major unsaturated fatty acids in human LDL, oleic acid, linoleic acid, and arachidonic acid. Oxidation of human LDL in the presence of a relatively strong stimulus (20 microM CuSO4) resulted in very large increases in the amounts of the major monohydroxy derivatives of linoleic acid (9- and 13-hydroxy derivatives) and arachidonic acid (5-, 8-, 9-, 11-, 12-, and 15-hydroxy derivatives) in LDL lipids in the early stages of the reaction. After 20 h, the amounts of these products declined due to substrate depletion, but large amounts of monohydroxy derivatives of oleic acid (8-, 10-, and 11-hydroxy derivatives) were detected. Although thiobarbituric acid-reactive substances clearly increased under these conditions, the changes were not nearly so dramatic as those observed for monohydroxy fatty acids. Oxidation of LDL in the presence of endothelial cells, a much milder stimulus, resulted in 2.5- to 3-fold increases in the amounts of monohydroxy derivatives of linoleic and arachidonic acids, as well as thiobarbituric acid-reactive substances, with more modest increases in the amounts of hydroxylated derivatives of oleic acid. There was little positional specificity in the oxidation of the above fatty acids in the presence of either stimulus, suggesting that the formation of these products proceeds primarily by lipid peroxidation, rather than by catalysis by lipoxygenases. However, an important role for lipoxygenases in the initiation of these reactions cannot be excluded. In conclusion, oxidation of LDL in the presence of copper ions or endothelial cells results in the formation of a large number of monohydroxy derivatives of oleic, linoleic, and arachidonic acids. The relative amounts of products formed from each of these fatty acids depends on the strength of the stimulus as well as the incubation time.     

Lipidic last breath of life in patients with alcoholic liver disease

Alcoholic liver disease (ALD) begins with the accumulation of lipid droplets in the liver. Lipids which accumulate in the liver can stimulate inflammation, and the fatty acid derivatives, hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecadienoic acids (HODEs), may play an important role in this process. We evaluated the concentrations of linoleic and arachidonic acid derivatives in the plasma of patients with ALD, non-alcoholic fatty liver disease (NAFLD) and healthy individuals. The groups consisted of 173 subjects: 63 patients with ALD, 90 with NAFLD and 20 healthy volunteers. Plasma 12-, 15-, and 5-HETE as well as 9- and 13-HODE were assessed using HPLC and isoprostane 8-epi-PGF 2α III was evaluated with an ELISA. In addition the mRNA expression of lipoxygenases (5-LOX, 15-LOX-1, 15-LOX-2) in the liver samples of patients with ALD cirrhosis was measured. A significant difference between the plasma concentrations of the analyzed derivatives was found when divided according to gender. The most significant differences were found between healthy individuals and ALD patients, as well as ALD and NAFLD individuals regardless of gender. The increased plasma HODEs and HETEs concentrations were in line with the increase in 5- and 15-LOX-1 and 15-LOX-2 mRNA in liver samples from ALD cirrhosis patients. LOXs expression and peroxidation of polyunsaturated fatty acids by free radical-propagated chemical oxidation may be contributing factors in liver necroinflammatory injury in ALD.     

Synthesis of hydroxy fatty acids from linoleic acid by human blood platelets

The metabolism of linoleic acid by washed human platelets was investigated. 1.¹⁴C linoleic acid was converted to 1.¹⁴C hydroxy octadecadienoic acids (HODES) at about the same rate with which 1.¹⁴C 12-HETE was produced from 1.¹⁴C arachidonic acid. The total radioactivity in HODEs was distributed among two isomers: 13-HODE (85%) and 9-HODE (15%) as defined by GC-MS. The production of HODES by intact washed platelets was inhibited by indomethacin (IC50:5×10⁻⁷M) which suggest that hydroxy fatty acids were produced by PGH-synthase. By contrast, the production of HODEs by platelet cytosolic fractions was not modified under indomethacin treatment but completely abolished by NDGA (10⁻³M) and inhibited by the platelet lipoxygenase inhibitors 15-HETE (2.10⁻⁵M) and baicalein (10⁻⁵M). Platelets thus contain two different active systems which may convert linoleic acid to hydroxy fatty acids. Since these compounds remained essentially associated with the platelets, their presence may significantly participate in the mechanisms of platelet activation.     

Stereochemistry in the formation of [9-hydroxy-10,12-octadecadienoic acid and 13-hydroxy-9,11-octadecadienoic acid from linoleic acid by fatty acid cyclooxygenase

9-Hydroxy-10,12-octadecadienoic acid and 13-hydroxy-9,11-octadeca-dienoic acid are formed from linoleic acid upon incubation with the microsomal fraction of homogenates of the sheep vesicular gland (Hamberg M. and Samuelsson B. (1967) J. Biol. Chem. bd242, 5344–5354. This communication is concerned with the stereochemical aspects of the conversion.The ratio between the 9- and 13-hydroxy isomers was 77:23. Steric analysis of the individual isomers showed that the hydroxyl group of both isomers had mainly the L configuration i.e. 9L:9D, 79:21 and 13L:13D, 78:22. Incubation of [11l-³H; 1-¹⁴C]linoleic acid led to the formation of 9- and 13-hydroxyoctadecadienoates which had largely lost the tritium label (6% and 7% retention of tritium relative to precursor, respectively) showing that the hydrogen which is removed from C-11 during the conversion has the l (pro-S) configuration.     

Profiling assay for lipoxygenase products of linoleic and arachidonic acid by gas chromatography-mass spectrometry.

A method for determination of the lipoxygenase products of linoleic acid (9- and 13-hydroxyoctadecadienoic acid; 9-HODE, 13-HODE) and of arachidonic acid (5-, 8-, 9-, 11-, 12-, and 15-hydroxyeicosatetraenoic acid; 5-, 8-, 9-, 11-, 12-, and 15-HETE) is described. The method combines solid-phase extraction, derivatization to the corresponding fully hydrogenated methylester/trimethylsilylether derivatives and capillary gas chromatography coupled with electron impact mass spectrometry. Each regioisomeric HODE and HETE shows a unique pair of mass spectrometric fragment ions originating from fission of the fatty acid carbon chain at the hydroxylated position. The carboxyl-terminal fragment is used for quantification relative to a carboxyl-18O2-labeled analogue added as internal standard and the methyl-terminal fragment is monitored for confirmation. The assay can be extended for quantification of the complete hydroxylation profile of linoleic and arachidonic acid. Applications of this assay are demonstrated for the quantification of HODEs and HETEs in normal, hyperplastic, and neoplastic mouse epidermis. In mouse epidermis papilloma, the tissue levels of 8- and 12-HETE were found to be increased by one to two orders of magnitude compared to levels in normal epidermis.     

Differentiation-inducing activity of retinoic acid isomers and their oxidized analogs on human promyelocytic leukemia HL-60 cells.

Retinoidal activity of retinoic acid isomers [all-trans-retinoic acid (ATRA), 9-cis-retinoic acid (9CRA) and 13-cis-retinoic acid (13CRA)] and their oxidized derivatives [19-hydroxy and 19-oxo derivatives of ATRA (19-hydroxy-ATRA and 19-oxo-ATRA), 19-oxo derivative of 9CRA (19-oxo-9CRA), and 19-hydroxy derivative of 13CRA (19-hydroxy-13CRA)] was evaluated by means of a human promyelocytic leukemia HL-60 cell differentiation induction assay. All the compounds examined showed this activity with ED50 values of 2-30 nM, which are in accordance with their binding activity to nuclear retinoic acid receptors (RARs).     

Identification and egg hatching activity of monohydroxy fatty acid eicosanoids in the barnacle Balanus balanoides.

Monohydroxy fatty acids (MHFAs) were isolated from homogenates of the barnacle Balanus balanoides and identified by gas chromatography-mass spectrometry (GC-MS) as 14- and 17-hydroxy docosahexaenoic acids, 8-, 11-, 12-, 15- and 18-hydroxy eicosapentaenoic acids, 13- and 16-hydroxyoctadecatrienoic acids and 9-, 13- and 15-hydroxyoctadecadienoic acids. Each monohydroxy fatty acid was tested for egg hatching activity in a bioassay using Elminius modestus egg masses, but 8-hydroxy-5, 9, 11, 14, 17-eicosapentaenoic acid (8-HEPE) was the only MHFA with barnacle egg hatching activity. Studies on the egg hatching activity of MHFAs prepared from the oxidation of polyunsaturated fatty acids showed that activity was confined to the 8-hydroxy isomer of eicosapentaenoic acid and arachidonic acid, and that unsaturation at C5 and C14, but not C17, was essential for activity. In addition, the 8(R) conformation is necessary for activity, as 8(R)-HEPE caused egg hatching at 10(-7) M whereas the enantiomer 8(S)-HEPE was inactive.     

9- and 13-hydroxy-linoleic acid possess chemotactic activity for bovine and human polymorphonuclear leukocytes

The presence of polymorphonuclear leukocytes (PMNs) within the airways is a characteristic feature of a variety of lung diseases. Pulmonary alveolar macrophages (PAMs) and epithelial cells release many different factors which contribute to the recruitment of inflammatory cells into infected airways. PAMs and tracheal epithelial cells are able to produce linoleic acid metabolites (9-HODE and 13-HODE) besides arachidonic acid metabolites. The objective of the present study was to determine whether 9-HODE and 13-HODE possess chemotactic activity for isolated PMNs. It was found that 9-HODE and 13-HODE induced a chemotactic response of both human and bovine PMNs in vitro. The HODEs evoked chemotaxis with a linear dose response from 10(-10) to 10(-6) M to the same extent as the arachidonic acid metabolite 15-HETE. At 10(-8) M, 9-HODE and 13-HODE were approximately half as potent in inducing chemotaxis as compared to LTB4.     

11, 15, 19-trihydroxy-9-ketoprost-13-enoic acid and 11, 15, 19-trihydroxy-9-ketoprosta-5, 13-dienoic acid in human seminal fluid.

Two novel prostaglandins (PG) have been found in human seminal fluid which had been frozen immediately after collection. They were characterized by combined gas-liquid chromatography-mass spectrometry of various derivatives as 19-hydroxy prostaglandin E1 (11, 15, 19-trihydroxy-9-ketoprost-13-enoic acid) and 19-hydroxy prostaglandin E2 (11, 15, 19-trihydroxy-9-ketoprosta-5, 13-dienoic acid). They were present in three to five times the quantity of prostaglandins E1 and E2. Incubation of seminal fluid for 3 hr at 25 degrees C reduced levels of 190H-PGEs2.5-fold and PGE22-fold, while increasing levels of PGAs and PGBs 2-fold. No 190H PGA or 190H PGB was detected in extracts of unincubated fluid. The PGAs, PGBs and their 19-hydroxy analogs are probably artifacts arising metabolically or as a result of classical isolation techniques.     

氧脂素对赭曲霉在大豆培养基质中合成赭曲霉毒素A的影响

【背景】赭曲霉毒素A (ochratoxin A,OTA)是曲霉属和青霉属等真菌的次级代谢产物,严重威胁农产品及食品安全,氧脂素羟基十八碳二烯酸(hydroxyoctadecaenoic acids,HODEs)被认为可能是曲霉属的群体感应信号分子,调节曲霉的生长发育和次级代谢物生成。【目的】主要研究HODEs对赭曲霉(Aspergillusochraceus)菌株AS3.4412产生OTA的影响,检测孢子密度、培养基类别以及内、外源HODEs作用下OTA产量的不同变化。【方法】分别在PDB、黄豆和黑豆培养基中进行赭曲霉的培养,采用高效液相色谱-荧光检测法测定OTA含量,采用高效液相色谱-质谱法测定氧脂素含量,根据变化规律寻找赭曲霉群体密度、氧脂素、OTA三者间的关系。【结果】低密度赭曲霉培养物(103 spores/mL)中9(S)-HODE/13(S)-HODE及OTA产量高于高密度赭曲霉(106 spores/mL);外源添加9(S)-HODE能促进OTA合成,13(S)-HODE可以抑制OTA合成;赭曲霉侵染抗氧化能力更高的黑豆产生更多的OTA。【结论】OTA的合成受到赭曲霉群体密度和氧脂素的影响,推测9(S)-HODE和13(S)-HODE是赭曲霉群体感应信号分子,并且二者在调节OTA合成中具有相反的作用。     

A novel unsaturated fatty acid hydratase toward C16 to C22 fatty acids from Lactobacillus acidophilus

Hydroxy FAs, one of the gut microbial metabolites of PUFAs, have attracted much attention because of their various bioactivities. The purpose of this study was to identify lactic acid bacteria with the ability to convert linoleic acid (LA) to hydroxy FAs. A screening process revealed that a gut bacterium, Lactobacillus acidophilus NTV001, converts LA mainly into 13-hydroxy-cis-9-octadecenoic acid and resulted in the identification of the hydratase responsible, fatty acid hydratase 1 (FA-HY1). Recombinant FA-HY1 was purified, and its enzymatic characteristics were investigated. FA-HY1 could convert not only C18 PUFAs but also C20 and C22 PUFAs. C18 PUFAs with a cis carbon-carbon double bond at the Δ12 position were converted into the corresponding 13-hydroxy FAs. Arachidonic acid and DHA were converted into the corresponding 15-hydroxy FA and 14-hydroxy FA, respectively. To the best of our knowledge, this is the first report of a bacterial FA hydratase that can convert C20 and C22 PUFAs into the corresponding hydroxy FAs. These novel hydroxy FAs produced by using FA-HY1 should contribute to elucidating the bioactivities of hydroxy FAs.     

Role of IL-13 in a model of Strongyloides venezuelensis infection in rats

IL-13 is a cytokine known to play a role in several pulmonary diseases, including asthma and fibrosis. The role of IL-13 in the context of pulmonary changes induced by helminth infection is unclear. Rats experimentally infected with Strongyloides venezuelensis and treated with anti-IL-13 neutralizing antibody were used to evaluate the role of IL-13 on functional and inflammatory changes of host lungs, and on parasite control. S. venezuelensis-induced airway hyperreactivity was IL-13-independent, but IL-13 played an essential role in driving airway mucus production and eosinophil infiltration. IL-13 was important for the control of egg production but not establishment in the intestine.     

Fatty acid structural requirements for activity of arachidonoyl-CoA synthetase

We have examined the fatty acid substrate specificity of arachidonoyl-CoA synthetase from human platelet membranes. A variety of positional isomers and chain-length analogs of arachidonic acid [20:4(5, 8, 11, 14)] were synthesized, and assayed for their ability to inhibit arachidonoyl-CoA formation or to serve as substrates for the synthetase. The chain-length specificity of the synthetase for delta 8,11,14 trienoic fatty acids was C19 greater than C18 = C20 much greater than C21 greater C22. Inhibition activity by positional isomers of arachidonate was 20:4(5, 8, 11, 14) approximately equal to 20:4(6, 9, 12, 15) = 20:4(7, 10, 13, 16) much greater than 20:4(4, 7, 10, 13), however, Vmax for arachidonate was greater than that for 20:4(6, 9, 12, 15). The enzyme apparently "counts" double bonds from the carboxyl terminus. As counted from the methyl terminus we found that several n-6,-9,-12 fatty acids were ineffective as inhibitors [18:3(6, 9, 12); 19:4)4, 7, 10, 13); 21:3(9, 12, 15)], whereas all methylene-interrupted tri- and tetraenoic fatty acids which contained delta 8 and delta 11 double bonds were potent inhibitors. The delta 11 double bond was best associated with optimal inhibition: 20:3(5, 11, 14) had a lower Ki than 20:3(5, 8, 14). 13-Methyl-20:3(8, 11, 14) did not inhibit the enzyme. Partially purified enzyme from calf brain, depleted of nonspecific long-chain acyl-CoA synthetase, exhibited the same fatty acid specificity as crude platelet enzyme.     

Increased levels of monohydroxy metabolites of arachidonic acid and linoleic acid in LDL and aorta from atherosclerotic rabbits.

Lipid peroxidation results in the formation of peroxy and hydroperoxy metabolites of polyunsaturated fatty acids which can directly or indirectly affect many cellular processes. Lipid hydroperoxides are rapidly metabolized to the corresponding monohydroxy products by various cellular peroxidases. We have measured the amounts of monohydroxy metabolites of linoleic acid (18:2) and arachidonic acid (20:4) in lipids derived from aorta and LDL from rabbits fed a diet enriched in cholesterol and peanut oil for either 8 or 15 weeks. Increased amounts of the 9-hydroxy, and, to a lesser extent, the 13-hydroxy metabolite of 18:2 were observed in aorta and LDL from cholesterol-fed rabbits at both 8 and 15 weeks. The amounts of esterified 11-, 12- and 15-hydroxy metabolites of 20:4 in aortae from cholesterol-fed rabbits were similar to controls after 8 weeks, but about 3-fold higher after 15 weeks. These monohydroxy metabolites of 20:4 were also detected in LDL lipids in cholesterol-fed rabbits. The greater amounts of hydroxy-18:2 in the cholesterol-fed group could be explained by an approx. 2-4-fold increase in 18:2 in aorta and LDL. In contrast, the amounts of 20:4 in aortic lipids were lower in cholesterol-fed rabbits than in controls. Thus, the percentage of esterified 20:4 which had been oxidized to its 11, 12, and 15-hydroxylated metabolites was about 5-times higher in the cholesterol-fed group. Our results would be consistent with the hypothesis that increased amounts of peroxidized 18:2 and 20:4 in lipids could be involved in the development of atherosclerotic lesions in cholesterol-fed rabbits.     

Studies on the Chemical Constituents of the Leaves of WTBX]Pseudotaxus chienii (Cheng) Cheng

Fourteen compounds were isolated from the leaves of Pseudotaxus chienii (Cheng) Cheng which is uniquely indigenous to China, and their structures were identified mainly by spectrum analyses. Among them, 13 known compounds were determined as: 2-guaiacylpropane-1, 3-diol (1), vanillic acid (2), 3-methoxy-4-hydroxy cinnamic acid (3). 3. 5-dimethoxy-phenol (4), taxicatin (5), 5-oxymaltol (6), quercetin-3-rhamnoside (7), (±)-catechin (8), ecdysterone (9), β-sitosterol (10), D-glucose (11), (±)-10-nonacosanol (12) and octacosanoic acid (13). All these compounds butβ-sitosterol were isolated from this plant for the first time. Compounds 1 and 6 have never been reported in plant kingdom. Another compound was tentatively established as a new compound, named pseudotaxlactone (14).