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Metabolism of arachidonic acid into hydroxyepoxides by garlic roots
Concurrent Sessions Wednesday 23 July
EFA & Eicosanoids 1997 - Edinburgh
205
W19
W20
Docosahexaenoic acid stimulates a phospholipase D pathway in h u m a n activated lymphocytes. Bechoua S., Dubois M., Lagarde M., Prigent A.F., INSERM U352, Biocllimie & Pharmacologie, INSA-Lyon, Villeurbanne, France.
The Anti-lnflammatory Effects of co3 Tetraenoic Fatty Acids Isolated from a Lipid Extract from the mussel, Perna canaliculus. T. A. Macrides ~, A. P. Treschow 1, N. Kalafatis~, P. F. A. Wright 1, P. M. Wynnez Department of Medical Laboratory Sciencel, Racing Analytical Services Ltd2 RMIT University, GPO Box 2476V, Melbourne, VIC, 3001, Australia.
Numerous evidences have indicated that n-3 fatty acids have beneficial therapeutic effects in inflammatory diseases through the modulation of immune cell function. The aim of the present study was to determine whether docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids may affect the early mitogen-induced production of diacylglycerol (DAG) and phosphatidic acid (PA) in human peripheral blood mononuclear cells (PBMC). These two lipid mediators, and particularly PA, have been shown to play a crucial role in the control of lymphocyte mitogenesis. The treatment of PBMC with 5 laM DHA, wlaich led to a measurable enrichment of cell phospholipids with DHA, increased the basal PA mass, measured by direct videodensitometry of TLC plates, by 57% as compared to non-enriched cells (18.6 + 2.2 ng vs. 11.8 -+ 1.4 ng/106cells, n=8, p<0.05). In the same experimental conditions. EPA had no effect. However, the marked increase of the PA mass which was routinely observed following ConA activation of control cells, was substantially attenuated in DHA-enriched cells (+ConA: 50.8 + 9.2 ng vs. +ConA+DHA: 37.3 + 5.4 ng/106cells, n=8, p<0.05). The basal DAG level was not modified in DHA-treated cells as well as in control cells stimulated by ConA (200 _+ 30 ng/106cells) but was 2fold increased in DHA-treated cells stinmlated by ConA (n=5, P<0.05). When ConA activation of DHA-enriched cells was performed in the presence of 1% butanol (BuOH), a significant amount of phosphatidylbutanel (PtBnOH) was observed, whereas no PtBuOH could be detected in non-enriched cells upon ConA activation. Furthermore, BuOH also suppressed the ConA-induced increase of DAG mass observed in DHA-enriched cells. Altogether, these results show that, in ConA-activated h u m a n PBMC, DHA stimulates both phospholipase D and PA phosphohydrolase activities, which ultimalely results in an increased DAG production.
The lipid extracts of Perna canaliculus, the green-lipped mussel of New Zealand, have been shown to have anti-inflammatory activity (AI) and the greatest activity is shown by the polyunsaturated free fatty acid (PUFA) class1. We have identified a homologous series of novel (o3, PUFAs with significant AI activity in an in vitro leukotriene inhibition assay. The proportion of novel PUFAs in the crude lipid extract was 0.12%w/w and in the total mussel material, 0.005%w/w. The total free FAs were isolated from a supercritical-COz lipid extract of the freeze-dried mussel powder by flash chromatography, followed by reversedphase HPLC. This technique involved a separation based on carbon number, folIowed by Ag-HPLC of the methyl esters based on the degree of unsaturation. Identification of FA components was performed using GLC with flame ionisation detection and individual structures were assigned from the pyrrolidide derivatives by GC-MS. Inhibition of Leukotriene Ba (LTB4) produced from stimulated human neutrophils was used as an in vitro screening method to test the efficacy of purified PUFAs for AI activity. The two most active fractions obtained from the separations inhibited LTB4 formation by 64% and 47% respectively (at 1:100 dilution). A structurally related family of o13 free fatty acids was identified in the most bioactive fraction which included C18:4, C19:4 and C20:4 PUFAs. Two C20:4o3 structural analogues of arachidonic acid have now been synthesised for testing in an in vivo adjuvant-induced polyarthritis model. 1. T. A. Macrides and N. Kalafatis (1995) Int. Patent PCT/AU 95/004/85
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W22
M E T A B O L I S M OF A R A C H I D O N I C A C I D I N T O HYDROXYEPOXIDES BY GARLIC ROOTS D. R e y n a u d , M. Ali a n d C.R. P a c e - A s c i a k Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, CANADA MSG 1X8 and Department of Biochemistry, Kuwait University, 13060 $afat, KUWAIT
Is Lipoxygenation of M e m b r a n e Lipids Implicated in the Irreversibility of Drought D a m a g e to Higher Plants? T. Schewe j, H. Kflhn 2, IUniv.Clinics Benjamin Franklin, Dept. Gynecology,2Univ. Clinics Charitr, Inst. B i o c h e m , Berlin, D
Fresh garlic bulbs were allowed to root for several weeks in water and the roots were harvested. The roots were homogenized in phosphate buffer and incubated with radiolabeled arachidonic acid of low specific activity. Products were isolated in the usual manner through acidified solvent extraction and thin layer chromatography (TLC). Two major products were formed, one migrating in the HETE region of the TLC, the other migrating in the polar trihydroxy region. The former was hydrogenated and shown by GCMS to be 15-HETE. The latter was shown to be a mixture of isomers resulting from 15-HPETE isomerization into hydroxy epoxide metabolites isolated as trihydroxy compounds. Trihydroxy compounds are formed from hydroxy epoxides both enzymatically (epoxide hydrolase activity) as well as nonenzymatically (acid extraction). Chiral phase HPLC analysis of A D A M derivatives with fluorescent detection showed the stereoselective and therefore enzymatic formation of 15(S)-HETE by the roots. Hydroxy epoxide enantiomers isolated through nonacidic workup were also shown to be enzymatically formed from 15(S)-HPETE as judged from incubations of 15(R/S)-HPETE with garlic roots vs heroin catalysis. This is similar to our reported findings with 12(R/S)HPETE and formation of the Hepoxilins in the mammalian system. These results showed the presence of an active 15-LOX pathway in garlic roots as well as HPETE enzymatic isomerization to produce structural isomers of the Hepoxilins. Data will be presented on the effects of a series of hydroxy epoxide analogs in the Hepoxilin series on the rooting patterns of garlic bulbs. Data will be also presented on the effect of LOX and C O X inhibitors on root growth, indicating that products in the 15-LOX pathway may represent important mediators of the plant rooting system.
Air-dried leaves from higher plants belonging to various families were found to contain oxygenated polyenoic fatty acids such as 9Sh y d r o x y - l O E , 1 2 Z , 1 5 Z - o c t a d e c a t r i e n o i c acid, 9 S - h y d r o x y - l O E , 1 2 Z octadecadienoic acid and the corresponding 9-keto derivatives in concentrations up to 100 ~tg/mg dry weight. Only small concentrations o f these compounds were detected in the corresponding freshly harvested plants. The chemical structures o f the compounds were established by co-chromatography with authentic standards in reverse-phase, straight-phase and chiral-phase HPLC, by uv spectroscopy and gas chromatography/mass spectrometry. Up to 90 % o f the oxygenated polyenoic fatty acids were detected in esterified lipids, whereas only 10 % were found in the free fatty acid fraction. The hydroxyoctadecanoids exhibited variable stereospecificity among various species. Investigation o f different stages o f senescence in one and the same leaf did not reveal any coincidence o f the formation o f octadecanoids with the degradation of chlorophyll. We conclude from these data that the formation o f esterified oxygenated polyenoic fatty, acids in higher plants is the consequence o f either drought damage or natural dehydration (abscission). In this process both lipoxygenase-mediated and non-enzymatic lipid peroxidation reactions appear to be involved. The alteration of the membrane structure by the oxygenation o f lipids m a y render a drought damage to plants irreversible.
EFA & Eicosanoids 1997 - Edinburgh
205
W19
W20
Docosahexaenoic acid stimulates a phospholipase D pathway in h u m a n activated lymphocytes. Bechoua S., Dubois M., Lagarde M., Prigent A.F., INSERM U352, Biocllimie & Pharmacologie, INSA-Lyon, Villeurbanne, France.
The Anti-lnflammatory Effects of co3 Tetraenoic Fatty Acids Isolated from a Lipid Extract from the mussel, Perna canaliculus. T. A. Macrides ~, A. P. Treschow 1, N. Kalafatis~, P. F. A. Wright 1, P. M. Wynnez Department of Medical Laboratory Sciencel, Racing Analytical Services Ltd2 RMIT University, GPO Box 2476V, Melbourne, VIC, 3001, Australia.
Numerous evidences have indicated that n-3 fatty acids have beneficial therapeutic effects in inflammatory diseases through the modulation of immune cell function. The aim of the present study was to determine whether docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids may affect the early mitogen-induced production of diacylglycerol (DAG) and phosphatidic acid (PA) in human peripheral blood mononuclear cells (PBMC). These two lipid mediators, and particularly PA, have been shown to play a crucial role in the control of lymphocyte mitogenesis. The treatment of PBMC with 5 laM DHA, wlaich led to a measurable enrichment of cell phospholipids with DHA, increased the basal PA mass, measured by direct videodensitometry of TLC plates, by 57% as compared to non-enriched cells (18.6 + 2.2 ng vs. 11.8 -+ 1.4 ng/106cells, n=8, p<0.05). In the same experimental conditions. EPA had no effect. However, the marked increase of the PA mass which was routinely observed following ConA activation of control cells, was substantially attenuated in DHA-enriched cells (+ConA: 50.8 + 9.2 ng vs. +ConA+DHA: 37.3 + 5.4 ng/106cells, n=8, p<0.05). The basal DAG level was not modified in DHA-treated cells as well as in control cells stimulated by ConA (200 _+ 30 ng/106cells) but was 2fold increased in DHA-treated cells stinmlated by ConA (n=5, P<0.05). When ConA activation of DHA-enriched cells was performed in the presence of 1% butanol (BuOH), a significant amount of phosphatidylbutanel (PtBnOH) was observed, whereas no PtBuOH could be detected in non-enriched cells upon ConA activation. Furthermore, BuOH also suppressed the ConA-induced increase of DAG mass observed in DHA-enriched cells. Altogether, these results show that, in ConA-activated h u m a n PBMC, DHA stimulates both phospholipase D and PA phosphohydrolase activities, which ultimalely results in an increased DAG production.
The lipid extracts of Perna canaliculus, the green-lipped mussel of New Zealand, have been shown to have anti-inflammatory activity (AI) and the greatest activity is shown by the polyunsaturated free fatty acid (PUFA) class1. We have identified a homologous series of novel (o3, PUFAs with significant AI activity in an in vitro leukotriene inhibition assay. The proportion of novel PUFAs in the crude lipid extract was 0.12%w/w and in the total mussel material, 0.005%w/w. The total free FAs were isolated from a supercritical-COz lipid extract of the freeze-dried mussel powder by flash chromatography, followed by reversedphase HPLC. This technique involved a separation based on carbon number, folIowed by Ag-HPLC of the methyl esters based on the degree of unsaturation. Identification of FA components was performed using GLC with flame ionisation detection and individual structures were assigned from the pyrrolidide derivatives by GC-MS. Inhibition of Leukotriene Ba (LTB4) produced from stimulated human neutrophils was used as an in vitro screening method to test the efficacy of purified PUFAs for AI activity. The two most active fractions obtained from the separations inhibited LTB4 formation by 64% and 47% respectively (at 1:100 dilution). A structurally related family of o13 free fatty acids was identified in the most bioactive fraction which included C18:4, C19:4 and C20:4 PUFAs. Two C20:4o3 structural analogues of arachidonic acid have now been synthesised for testing in an in vivo adjuvant-induced polyarthritis model. 1. T. A. Macrides and N. Kalafatis (1995) Int. Patent PCT/AU 95/004/85
W21
W22
M E T A B O L I S M OF A R A C H I D O N I C A C I D I N T O HYDROXYEPOXIDES BY GARLIC ROOTS D. R e y n a u d , M. Ali a n d C.R. P a c e - A s c i a k Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, CANADA MSG 1X8 and Department of Biochemistry, Kuwait University, 13060 $afat, KUWAIT
Is Lipoxygenation of M e m b r a n e Lipids Implicated in the Irreversibility of Drought D a m a g e to Higher Plants? T. Schewe j, H. Kflhn 2, IUniv.Clinics Benjamin Franklin, Dept. Gynecology,2Univ. Clinics Charitr, Inst. B i o c h e m , Berlin, D
Fresh garlic bulbs were allowed to root for several weeks in water and the roots were harvested. The roots were homogenized in phosphate buffer and incubated with radiolabeled arachidonic acid of low specific activity. Products were isolated in the usual manner through acidified solvent extraction and thin layer chromatography (TLC). Two major products were formed, one migrating in the HETE region of the TLC, the other migrating in the polar trihydroxy region. The former was hydrogenated and shown by GCMS to be 15-HETE. The latter was shown to be a mixture of isomers resulting from 15-HPETE isomerization into hydroxy epoxide metabolites isolated as trihydroxy compounds. Trihydroxy compounds are formed from hydroxy epoxides both enzymatically (epoxide hydrolase activity) as well as nonenzymatically (acid extraction). Chiral phase HPLC analysis of A D A M derivatives with fluorescent detection showed the stereoselective and therefore enzymatic formation of 15(S)-HETE by the roots. Hydroxy epoxide enantiomers isolated through nonacidic workup were also shown to be enzymatically formed from 15(S)-HPETE as judged from incubations of 15(R/S)-HPETE with garlic roots vs heroin catalysis. This is similar to our reported findings with 12(R/S)HPETE and formation of the Hepoxilins in the mammalian system. These results showed the presence of an active 15-LOX pathway in garlic roots as well as HPETE enzymatic isomerization to produce structural isomers of the Hepoxilins. Data will be presented on the effects of a series of hydroxy epoxide analogs in the Hepoxilin series on the rooting patterns of garlic bulbs. Data will be also presented on the effect of LOX and C O X inhibitors on root growth, indicating that products in the 15-LOX pathway may represent important mediators of the plant rooting system.
Air-dried leaves from higher plants belonging to various families were found to contain oxygenated polyenoic fatty acids such as 9Sh y d r o x y - l O E , 1 2 Z , 1 5 Z - o c t a d e c a t r i e n o i c acid, 9 S - h y d r o x y - l O E , 1 2 Z octadecadienoic acid and the corresponding 9-keto derivatives in concentrations up to 100 ~tg/mg dry weight. Only small concentrations o f these compounds were detected in the corresponding freshly harvested plants. The chemical structures o f the compounds were established by co-chromatography with authentic standards in reverse-phase, straight-phase and chiral-phase HPLC, by uv spectroscopy and gas chromatography/mass spectrometry. Up to 90 % o f the oxygenated polyenoic fatty acids were detected in esterified lipids, whereas only 10 % were found in the free fatty acid fraction. The hydroxyoctadecanoids exhibited variable stereospecificity among various species. Investigation o f different stages o f senescence in one and the same leaf did not reveal any coincidence o f the formation o f octadecanoids with the degradation of chlorophyll. We conclude from these data that the formation o f esterified oxygenated polyenoic fatty, acids in higher plants is the consequence o f either drought damage or natural dehydration (abscission). In this process both lipoxygenase-mediated and non-enzymatic lipid peroxidation reactions appear to be involved. The alteration of the membrane structure by the oxygenation o f lipids m a y render a drought damage to plants irreversible.