Arachidonic acid,protein kinase C activators and bud formation in Hydra vulgaris

1. The effect of the protein kinase C (PKC) activators verrucosin B (VB), 1,2-sn-dioctanoylglycerol (diC₈) and phorbol-12-myristate-13-acetate (PMA), of arachidonic acid (AA) and of substances interfering with its release, re-uptake and metabolism was studied in Hydra vulgaris.2. All PKC activators potently inhibited bud formation, VB and PMA being 10,000 × more potent than diC⁸. VB effect was maximal already after 10 min incubation with hydra and persisted at 24 hr incubations.3. AA and substances inhibiting its re-uptake from cell membrane or its metabolism also inhibited bud formation, whereas oleyl-oxyethyl-phosphorylcholine (OOPC), an inhibitor of phospholipase A₂, potently induced bud formation.4. The findings described herein suggest a role for both PKC activation and AA in the inhibition of bud formation in H. vulgaris.     

Lipoxygenase metabolites of arachidonic and linoleic acids modulate the adhesion of tumor cells to endothelium via regulation of protein kinase C.

12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE) and 13(S)-hydroxyoctadecadienoic acid (13[S]-HODE), lipoxygenase metabolites of arachidonic acid and linoleic acid, respectively, previously have been suggested to regulate tumor cell adhesion to endothelium during metastasis. Adhesion of rat Walker carcinosarcoma (W256) cells to a rat endothelial cell monolayer was enhanced after treatment with 12(S)-HETE and this 12(S)-HETE enhanced adhesion was blocked by 13(S)-HODE. Protein kinase inhibitors, staurosporine, calphostin C, and 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, inhibited the 12(S)-HETE enhanced W256 cell adhesion. Depleting W256 cells of protein kinase C (PKC) with phorbol 12-myristate-13-acetate abolished their ability to respond to 12(S)-HETE. Treatment of W256 cells with 12(S)-HETE induced a 100% increase in membrane-associated PKC activity whereas 13(S)-HODE inhibited the effect of 12(S)-HETE on PKC translocation. High-performance liquid chromatographic analysis revealed that in W256 cells 12-HETE and 13-HODE were two of the major lipoxygenase metabilites of arachidonic acid and linoleic acid, respectively. Therefore, these two metabolites may provide an alternative signaling pathway for the regulation of PKC. Further, these findings suggest that the regulation of tumor cell adhesion to endothelium by 12(S)-HETE and 13(S)-HODE may be a PKC-dependent process.     

Binding of 13-HODE and 5-, 12- and 15-HETE to endothelial cells and subsequent platelet, neutrophil and tumor cell adhesion

Some studies report that endothelial cells preferentially take up the lipoxygenase-derived arachidonic acid metabolite, 5-hydroxyeicosatetraenoic acid (5-HETE), released from stimulated leukocytes (polymorphonuclear leukocytes, PMNs), whereas others report that endothelial cells preferentially take up 12-HETE released from platelets. The biological relevance of these observations, however, is unknown. Recently, we and others have found that, under basal conditions, endothelial cells, PMNs and tumor cells metabolize linoleic acid via the lipoxygenase enzyme to 13-hydroxyoctadecadienoic acid (13-HODE). We propose that endogenous levels of these metabolites regulate blood-vessel wall cell adhesion. In this study, we have measured (1) the relative binding of 5-, 12- and 15-HETE, and 13-HODE to endothelial cell monolayers, and (2) their effects on endothelial cell adhesivity with platelets, PMNs and tumor cells. There was a dose-related and specific binding of 5-[3H]HETE to endothelial cells but no binding of 12- or 15-HETE or 13-HODE. Platelet or PMN adhesion to endothelial cells was unaffected by the 5-HETE binding, but tumor cell adhesion was blocked by 40% (P less than 0.01). Interestingly, preincubation of endothelial cells with 13-HODE, 12-HETE or 15-HETE decreased platelet adhesion to endothelial cells (P less than 0.05), even though these metabolites did not bind to the endothelial cells. We conclude that 5-HETE preferentially binds to endothelial cells and interferes with a specific receptor for tumor cells, whereas the other metabolites neither bind to cells nor affect cell adhesion.     

Glucuronidation of arachidonic and linoleic acid metabolites by human UDP-glucuronosyltransferases

Arachidonic acids (AA) and linoleic acids (LAs) are metabolized, in several tissues, to hydroxylated metabolites that are important mediators of many physiological and pathophysiological processes. The conjugation of leukotriene B4 (LTB4), 5-hydroxyeicosatetraenoic acid (HETE), 12-HETE, 15-HETE, and 13-hydroxyoctadecadienoic acid (HODE) by the human UDP-glucuronosyltransferase (UGT) enzymes was investigated. All substrates tested were efficiently conjugated by human liver microsomes to polar derivatives containing the glucuronyl moiety as assessed by mass spectrometry. The screening analyses with stably expressed UGT enzymes in HK293 showed that glucuronidation of LTB4 was observed with UGT1A1, UGT1A3, UGT1A8, and UGT2B7, whereas UGT1A1, UGT1A3, UGT1A4, and UGT1A9 also conjugated most of the HETEs and 13-HODE. LA and AA metabolites also appear to be good substrates for the UGT2B subfamily members, especially for UGT2B4 and UGT2B7 that conjugate all HETE and 13-HODE. Interestingly, UGT2B10 and UGT2B11, which are considered as orphan enzymes since no conjugation activity has so far been demonstrated with these enzymes, conjugated 12-HETE, 15-HETE, and 13-HODE. In summary, our data showed that several members of UGT1A and UGT2B families are capable of converting LA and AA metabolites into glucuronide derivatives, which is considered an irreversible step to inactivation and elimination of endogenous substances from the body.     

Incorporation of arachidonic and linoleic acid hydroperoxides into cultured human umbilical vein endothelial cells

The current study assessed the differential incorporation of 12-hydroperoxyeicosatetraenoic acid (12-HPETE), arachidonic acid (AA), 12-hydroxyeicosatetraenoic acid (12-HETE) and the linoleic acid (LA) oxidation products, 13-hydroxyoctadecadienoic acid (13-HODE) and 13-hydroperoxyoctadecadienoic acid (13-HPODE), into human umbilical vein endothelial cells (HUVEC). Approximately 80-90% of AA (10(-8)-10(-5)M) and 80% of LA (10(-8)-10(-5)M) were incorporated into HUVEC within 12h, while less than 50% of the hydroxy metabolites (12-HETE, 12-HPETE, 13-HODE, 13-HPODE) were incorporated into HUVEC over 48h. Further, treatment of HUVEC with either 12-HPETE or 13-HPODE (concentrations of 10(-5)M) had no effect on cell number at a 48h time point when compared with control. These results demonstrate that exogeneous hydroxy metabolites are incorporated into HUVEC to a lesser degree than were endogenous fatty acids. Further, we speculate that 12-HPETE and 13-HPODE are rapidly metabolized to substances without significant cytotoxic effects.     

Transformation of arachidonic acid to 19-hydroxy- and 20-hydroxy-eicosatetraenoic acids using Candida bombicola

Candida bombicola (ATCC 22214) and C. apicola (ATCC 96134), grown on glucose (100 g l^–1) and arachidonic acid (5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid; AA), 1.25 g l^–1, synthesized sophorolipid up to 0.93 g l^–1. Acid hydrolysis of sophorolipid yielded 19-hydroxy-5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid (19-HETE) and 20-hydroxy-5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid (20-HETE) which were identified by TLC and GC-MS; the ratio of synthesis was 73:27, respectively. Conversion of AA by immobilized Candida bombicola, suspended in beads of 2% (w/v) calcium alginate for 96 h, gave an 83% conversion of 1 g AA l^–1 to 19- and 20-HETE. There was no significant loss in the efficiency of the immobilized cells after ten uses.     

Effect of oxylipins on <Emphasis Type="Italic">Neurospora crassa</Emphasis> growth and differentiation

The effect of the natural oxylipins 3(R)-hydroxy-(5Z,8Z,11Z,14Z)-eicosatetraenoic acid (3-HETE) and 18-hydroxy-(9Z,12Z)-octadecadienoic acids (18-HODE) on the growth and hypha aggregation, as well as on some light-depending processes, such as carotenoid biosynthesis, protoperithecia formation (sexual cycle), and conidiation (asexual cycle), of the ascomycete Neurospora crassa was studied. Hypha aggregation and growth slowdown were induced by 3-HETE, 18-HODE, and linoleic acid. At concentrations from 5 to 50 μM, these compounds had no significant effect on the light-induced carotenogenesis. At the same time, these 3-HETE and 18-HODE concentrations, unlike linoleic acid, induced the formation of protoperithecia in the dark. At the concentration of 5 μM, an additive effect of oxylipins and light was revealed. The studied oxylipins had different effects on the asexual reproduction of N. crassa: 3-HETE induced conidiation in the dark, whereas 18-HODE induced conidiation in the light. The possible involvement of oxylipins in the regulation of the processes of sexual and asexual reproduction of N. crassa is discussed.     

Arachidonic acid metabolism in growth control of A549 human lung adenocarcinoma cells

The role of individual eicosanoids of the arachidonic acid (AA) cascade in the growth control of A549 human lung adenocarcinoma cells has been studied. Cyclooxygenase and lipoxygenase metabolites of [¹⁴C]AA incorporated were actively synthesized in the cultures of tumor cells with full confluence unaccomplished. In such cultures inhibitors of AA metabolism (indomethacin and esculetin) and also a lipoxygenase metabolite of AA, 15-hydroxyeicosatetraenoic acid (15-HETE), significantly suppressed the incorporation of [³H]thymidine and biosynthesis of prostaglandin E₂(PGE₂). Other lipoxygenase metabolites of AA (5-HETE and 12-HETE) had no effect on these parameters. The basic fibroblast growth factor (bFGF) had practically no affect on the growth of A549 cells and the PGE₂ production in cultures with 5% fetal calf serum (FCS); however, in the presence of 0.5% FCS this factor significantly increased the number of tumor cells. The growth-stimulating effect of bFGF was completely abolished by a cyclooxygenase inhibitor indomethacin. The data suggest a key role of PGE₂ in the growth control of A549 cells with an active synthesis of cyclooxygenase and lipoxygenase metabolites of AA, its importance in realization of the mitogenic effect of bFGF, and specific features of 15-HETE as a down-regulator of the PGE₂-dependent proliferation.     

13-Hydroxyoctadeca-9,11-dienoic acid (13-HODE) inhibits thromboxane A2 synthesis, and stimulates 12-HETE production in human platelets

The effect of 13-hydroxyoctadeca-9,11-dienoic acid (13-HODE), a major lipoxygenase product of endothelial cell linoleic acid metabolism on thrombin-induced platelet thromboxane B2 (TxB2), and 12-hydroxyeico-satetraenoic acid (12-HETE) production was evaluated. 13-HODE inhibited thrombin-induced TxB2 production in human platelets in a concentration-dependent manner. At concentrations of 10 and 30 microM, 13-HODE inhibited TxB2 production by 28 +/- 8% (1SE, n = 5; P less than 0.05) and 48 +/- 6% (P less than 0.01) respectively. 13-HODE (30 microM) also inhibited the production of platelet hydroxyheptadecatrienoic acid (38 +/- 5%, P less than 0.01). A concomitant stimulation of 12-HETE production by 13-HODE was observed (25 +/- 5% and 49 +/- 22% over control values at 10 and 30 microM respectively, P less than 0.01). Our results demonstrate a differential effect of 13-HODE on thrombin stimulated platelet cyclooxygenase and lipoxygenase metabolites.     

Fatty acid modulation of tumor cell adhesion to microvessel endothelium and experimental metastasis

Tumor cell interaction with the endothelium of the vessel wall is a rate limiting step in metastasis. The fatty acid modulation of this interaction was investigated in low (LM) and high (HM) metastatic B16 amelanotic melanoma (B16a) cells. 12(S)-HETE increased the adhesion of LM cells to endothelium derived from pulmonary microvessels. All other monohydroxy and dihydroxy fatty acids were ineffective. LTB₄ induced a modest stimulation but LTC₄, LTD₄, LTE₄ as well as LXA₄ and LXB₄ were ineffective. The 12(S)-HETE enhanced adhesion of B16a cells was inhibited by pretreatment with 13(S)-HODE but not by 13(R)-, 9(S)-HODE or 13-OXO-ODE. 13(S)-HODE decreased adhesion of HM B16a cells to endothelium. 12(S)-HETE enhanced surface expression of integrin αIIbβ₃ and monoclonal antibodies against this integrin but not against α₅β₁, blocked enhanced but not basal adhesion to endothelium. Intravenous injection of 12(S)-HETE treated LM cells resulted in increased lung colonization (experimental metastasis). This effect was specific for 12(S)-HETE and was inhibited by 13(S)-HODE but not by other HODE's. 12(S)-HETE also enhanced lung colonization by HM cells and 13(S)-HODE decreased lung colonization by HM cells. Our results suggest a highly specific bidirectional modulation of metastatic phenotype and lung colonization by 12(S)-HETE and 13(S)-HODE.     

Formation of leukotrienes and hydroxy acids by human neutrophils and platelets exposed to monosodium urate

Monosodium urate (MSU) crystals stimulate the production of arachidonic acid metabolites by human neutrophils and platelets. Neutrophils exposed to MSU generated leukotriene B₄(LTB₄). 6- -LTB₄, 12- -6- -LTB₄, and 5S, 12S DHETE from endogenous sources of arachidonate. In addition to these metabolites both monohyroxyeicosatetraenoic acids (i.e., 5-HETE) and w-oxidation products (i.e., 20-COOH LTB₄) were formed by neutrophils exposed to MSU. Addition of exogenous arachidonic acid led to increased formation of each of these metabolites. When neutrophils were treated with colchicine (10 uM), LTB₄ but 5-HETE formation was impaired. (1-¹⁴C) Arachidonate-labeled platelets exposed to MSU released (1-¹⁴C)-arachidonate. (¹⁴C)-12 HETE, (¹⁴C)-HHT and (¹⁴C)-thromboxane B₂. Results indicate that MSU stimulates arachidonic acid metabolism in both human neutrophils and platelets. Moreover, they suggest not only that metabolites of arachidonate may be considered as possible candidates for mediators of inflammation in crystal-associated diseases, but that colchicine blocks the formation of LTB₄.     

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.     

Tumor necrosis factor alpha stimulates arachidonic acid metabolism in human osteoblastic osteosarcomal cells

The effects of tumor necrosis factor alpha (TNF-α) on arachidonic acid (AA) metabolism were investigated by prelabeling the human osteoblastic osteosarcoma cell line, G292, with [³H]AA. TNF-α differentially stimulates cyclooxygenase and lipoxygenase pathways of AA metabolism in a dose response manner in the cells. The highest concentration of TNF-α (10⁻⁸ M) significantly increased the cyclooxygenase pathway, with prostaglandin E₂ (PGE₂) being a major product. However, at the lowest concentration (10⁻¹⁰ M) of TNF-α, 15-hydroxyeicosatetraenoic acid (HETE) production was significantly increased, with no significant effects on the other identifiable products. When the concentration of TNF-α was increased to 10⁻⁹ M leukotriene B₄ (LTB₄), 15-, 12-, and 5-HETE were significantly increased. The calcium ionophore A23187 (10⁻⁶ M) significantly increased 15-HETE production, without significantly affecting cyclooxygenase metabolites. However, a combination of TNF-α (10⁻⁸ M) and A23187 (10⁻⁶ M) caused an inhibitory effect on each agent-induced PGE₂ or 15-HETE production.     

An excised patch membrane sensor for arachidonic acid released in mouse hippocampal slices under stimulation of L-glutamate

An excised patch membrane sensor for arachidonic acid (AA) is described, whose response stems from AA-induced channel-type transport of ions across the excised patch membrane. The patch membrane sensor was prepared in situ by excising mouse hippocampal cell membranes with patch pipets having a tip diameter of < 0.5 microm. The sensor responds to AA, giving rise to a channel-type current, and its magnitude (apparent conductance) increased with increasing AA concentration in the range from 10 to 30 nM. The detection limit was 2.1 nM (S/N = 3). The induction of channel-type currents was selective to AA over fatty acids such as palmitic acid, stearic acid, oleic acid, gamma-linolenic acid, and docosahexaenoic acid and AA metabolites such as 12-HETE, 5-HETE, and prostaglandin D(2). The sensor was applied to quantification of AA released from various neuronal regions (CA1, CA3, and DG) of mouse hippocampus under stimulation of 100 microM L-glutamate. The release of AA from each region was observed 1 min after the stimulation and the concentration of AA 5 min after the stimulation varied among the neuronal sites, i.e., 8+/-1 nM (n = 5) for CA1, 15+/-3 nM (n = 3) for CA3, and 6+/-2 nM (n = 9) for DG. The L-glutamate-evoked release of AA was partly inhibited by ionotropic glutamate receptor antagonists (APV and DNQX) and completely blocked by phospholipase A2 (PLA2) inhibitor (MAFP), suggesting that the release of AA occurred by glutamate receptor-mediated activation of PLA2. The potential use of the present sensor for detecting local concentration of AA at various neuronal sites is discussed.     

Diacylglycerol as a component of the signal-coupling pathway during the initiation of endocytosis in Amoeba proteus

In an attempt to define the transmembrane-signal pathway used to couple external phagocytotic signals with effectors in the cell interior, the effects of diacylglycerol (DG) and related substances were examined in Amoeba proteus. DGs are highly chemotactic, readily attracting amoebae when presented in a glass micropipette. Addition of DG (10^-6 M) to the medium elicits rapid shape changes in the amoeba and the formation of large phagosomes. Monacylglycerol and 1,3-diacylglycerol were much less effective in eliciting phagosome formation. On the assumption that DG was stimulating phosphokinase C (PKC) activity in the amoeba, the effect of phorbol myristate acetate (PMA), a known activator of PKC activity i other cell systems, was assessed in the amoeba. PMA (10^-7 M) alone was capable of bringing about shape changes in amoebae as well as stimulating the formation of phagosomes. These observations suggest that PKC is involved in the signal-coupling associated with the onset of phagocytosis. On the other hand staurosporine and H-7, inhibitors of PKC activity in some cell systems, did not inhibit the phagocytic uptake of Tetrahymena by A. proteus. It may be then that DGs in the amoeba interact directly with elements of the cytoskeleton causing phagosome formation, although a role for PKC in the initiation of phagocytosis in the amoeba cannot be ruled out at this point.     

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.     

Multiple arachidonic acid metabolites inhibit sodium-dependent phosphate transport in OK cells

The cytochrome P450-dependent monoxygenase pathway represents a major route for the metabolism of arachidonic acid (AA) in the kidney. In turn, AA metabolites have been shown to affect renal electrolyte metabolism, including sodium transport. Specifically AA, 20-HETE and 12-HETE inhibit sodium-dependent (Na+-Pi) uptake into renal culture cells, and both 12-HETE and 14,15 EET have been shown to reduce renin release from renal cortical slices. Since the bulk of Pi transport occurs in the proximal tubule (PT), and the PT is a major site of AA metabolism, we studied the effect of AA and several of its metabolites on Na+-Pi uptake into PT-like opossum kidney (OK) cells. Incubation of OK cells in AA (10(-8) M) resulted in 17% inhibition of Pi uptake. Three metabolites of omega-hydroxylation of AA induced significant decreases in Pi uptake: 19R-HETE (10(-8) M) by 36% (P=0.008), 19S-HETE (10(-8) M) by 24% (P=0.002) and 20-COOH-AA (10(-8) M), a metabolite of 20-HETE, by 25% (P<0.0001). 14,15 EET (10(-8) M), a breakdown product of AA by the epoxygenase pathway, had the greatest effect on Pi uptake in OK cells. It decreased Pi uptake by 47% (P < 0.0001). Addition of the P450 inhibitor, 7-ER (10(-8) M), to OK cells resulted in a significant stimulation (28%) of Pi uptake (P=0.016). These results indicate that these AA metabolites have a significant inhibitory effect on Na+-Pi uptake in OK cells.     

12-Hydroxyeicosatetraenoic acid reduces prostacyclin production by endothelial cells

12-Hydroxyeicosatetraenoic acid (12-HETE), a lipoxygenase product released by activated platelets and macrophages, reduced prostacyclin (PGI₂) formation in bovine aortic endothelial cultures by as much as 70%. Maximal inhibition required 1 to 2 h to occur and after 2 hr, a concentration of 1 μM 12-HETE produced 80% of the maximum inhibitory effect. 5-HETE and 15-HETE also inhibited PGI₂ formation. The inhibition was not specific for PGI₂; 12-HETE reduced the formation of all of the radioactive eicosanoids synthesized from [1-¹⁴C]arachidonic acid by human umbilical vein endothelial cultures. Inhibition occurred in the human cultures when PGI₂ formation was elicited with arachidonic acid, ionophore A23187 or thrombin. These findings suggest that prolonged exposure to HETEs may compromise the antithrombotic and vasodilator properties of the endothelium by reducing its capacity to produce eicosanoids, including PGI₂.     

12-Hydroxy-5Z, 8Z, 10E, 14Z,eicosatetraenoic acid (12-HETE) stimulates cAMP production in normal human fibroblasts

We report here that the 12-lipoxygenase metabolite of arachidonic acid, 12-hydroxy-5Z, 8Z, 10E, 14Z, eicosatetraenoic acid (12-HETE), stimulates cAMP production in human fibroblasts among various cultured cell lines tested. Although 12-HETE seemed to stimulate the phospholipase C (PLC)-protein kinase C (PKC) system, inhibitors against PLC and PKC did not reduce the cAMP production induced by 12-HETE, indicating that the activation of PLC-PKC system is not positively coupled with the stimulation of cAMP production. On the other hand, the cAMP production induced by 12-HETE was dependent on the Ca²⁺/calmodulin system in the cells. The results suggest that 12-HETE specifically stimulates Ca²⁺/calmodulin-dependent adenylyl cyclase to increase cAMP level in the fibroblasts. J Cell Physiol 178:63–68, 1999. © 1999 Wiley-Liss, Inc.     

Metabolism of 18:2(n - 6), 18:3(n - 3), 20:4(n - 6) and 20:5(n - 3) by the fungus Gaeumannomyces graminis: identification of metabolites formed by 8-hydroxylation and by w2 and w3 oxygenation.

The present study was aimed at developing a cell-free preparation of Gaeumannomyces graminis to biosynthesize w2-hydroxy, w3-hydroxy and related metabolites of essential fatty acids. 14C-labelled linoleic acid (18:2(n - 6)), linolenic acid (18:3(n - 3)), arachidonic acid (20:4(n - 6)) and eicosapentaenoic acid (20:5(n - 3)) were incubated with the cytosolic and microsomal fractions and NADPH. Significant metabolism was only found in the cytosol. The main products were purified by high-performance liquid chromatography and identified by gas chromatography-mass spectrometry (GC-MS). 18:2(n - 6) was metabolized mainly to 8-hydroxy-9,12-octadecadienoic acid (8-HODE), while the w2 and the w3 alcohols were formed in relatively small amounts. The absolute configuration of the 8-hydroxyl was found to be R by ozonolysis of the diastereoisomeric (-)-menthoxycarbonyl derivative of 8-HODE and GC-MS analysis. In analogy, 18:3(n - 3) was converted to 8-hydroxy-9,12,15-octadecatrienoic acid and to smaller amounts of the 15,16-diol (15,16-DiHODE). In contrast, 8-hydroxy metabolites of 20:4(n - 6) or 20:5(n - 3) could not be detected. 20:4(n - 6) was efficiently converted to 18(R)-hydroxyeicosatetraenoic acid (18(R)-HETE) and 19(R)-HETE and to traces of 17-HETE, while 20:5(n - 3) was mainly metabolized to the 17,18-diol (17,18-DiHETE) and to smaller amounts of the w2 alcohol. In conclusion, the cytosol of G. graminis can be used for stereoselective biosynthesis of some hydroxy metabolites of essential fatty acids.