Oxidative metabolism of [1-14C] mono-trans isomers of linoleic and alpha-linolenic acids in the rat.

Trans polyunsaturated fatty acids are formed during processing of vegetable oils such as deodorization and frying. The oxidative metabolism of linoleic and alpha-linolenic acids and of their mono-trans isomers (9cis,12trans-18:2, 9trans,12cis-18:2 and 9cis, 12cis,15trans-18:3, 9trans,12cis,15cis-18:3, respectively) was studied in fasting rats. A single dose of 18.5 MBq of each [1-14C] labelled fatty acid (260 microg) was orally given to the animals. The 14CO2 expired was monitored during 24 h. Radioactive countings of the CO2-trapping agent were performed at regular intervals up to 24 h after oral administration of the radiolabelled fatty acid. Radioactive countings were also performed on several tissues (liver, heart, brain, kidneys, sus-epidydimal fat, gastrocnemian muscle, gastrointestinal tract and carcass). The 14CO2 production 24 h after oral administration of the fatty acid ranged from 55.5% to 68.7% of the radioactivity administered for the C18:2 isomers and from 69.7% to 73.5% for the C18:3 fatty acids. From 6 to 24 h, 14CO2 recovery was significantly higher after oral administration of 9cis, 12trans-18:2 than after giving both other octadecadienoic isomers. 14C retention per gram of tissue in the liver and in the heart was significantly lower after feeding 9cis,12trans-18:2 than after administration of both other C18:2 isomers. 14C retention per gram of tissue in the muscle was significantly lower after administration of both trans C18:2 isomers compared to linoleic acid. Neither 14CO2 recoveries nor 14C retentions were significantly different after administration of the three octadecatrienoic acids. The difference observed in 14CO2 recovery within the dienes was probably not due to a higher specificity of the enzymes involved in the beta-oxidation sequence for the Delta12trans double bond, as previously reported. Indeed, due to the labelling of the fatty acids on the carboxyl end, 14C values recorded in the CO2-trapping agent were only due to the first cycle of beta-oxidation.

Effects of three trans isomers of eicosapentaenoic acid on rat platelet aggregation and arachidonic acid metabolism.

Three trans isomers of eicosapentaenoic acid (EPA) were added to rat platelets stimulated with arachidonic acid (AA) in order to compare their effects on platelet aggregation and on AA oxygenation with those of EPA. The production of metabolites from radiolabelled 20:5delta 17trans was studied also. EPA induced an inhibition of platelet aggregation of 26.7 +/- 6.6% for a 20:5/20:4 ratio equal to 1. The 20:5delta 11trans and the 20:5delta 11trans,17trans were twice as antiaggregant. In contrast, the 20:5delta 17trans induced similar antiaggregant effect as its cis homologue. Each fatty acid showed a dose-dependent effect. In opposition to EPA, 20:5delta 17trans was also able to induce platelet aggregation (12 +/- 4.9% at 5 microM). With regards to the metabolism of AA, 20:5delta 11trans, 20:5delta 17trans and 20:5delta 11trans,17trans (20:5/20:4 = 1) reduced the formation of the cyclooxygenase metabolites (-63%, -37% and -68%, respectively) and enhanced that of 12-HETE (+67%, +38% and +74%, respectively) as compared to EPA. The analysis showed that radiolabelled 20:5delta 17trans was metabolized into five compounds which remained to be identified. The Rf of three of these compounds (X1, X2 and X4) were those of the metabolites of EPA. Experiments using baicalein induced an inhibition of the production of X2. This suggested that this compound was formed through the 12-lipoxygenase pathway. In the same way, using indomethacin as inhibitor, we observed that X1 and X4 were produced by the cyclooxygenase pathway. Our results suggest that the trans double bond in the delta 11 position may be responsible of the different physiological effects of the trans polyunsaturated fatty acids as compared to their cis homologue (EPA). Furthermore, 20:5delta 17trans seems to be recognised by the enzymatic system as 20:4 n-6.

Identification of novel trans isomers of 20:5n-3 in liver lipids of rats fed a heated oil.

Trans polyunsaturated n-3 fatty acids are formed as a result of the heat treatment of vegetable oils. It was demonstrated previously that the 18:3 delta 9cis, 12 cis, 15 trans containing a cis delta 9 ethylenic bond was converted to a geometrical isomer of 20:5n-3, the 20:5 delta 5 cis, 8 cis, 11 cis, 14 cis, 17 trans. In the present study, we have identified two new isomers of eicosapentaenoic acid, the delta 11 monotrans and the delta 11, 17 ditrans isomers in liver of rats fed a heated oil. These are formed as a result of the conversion of two of the main isomers of linolenic acid which are present in refined and frying oils, the 18:3 delta 9 trans, 12 cis, 15 cis and the 18:3 delta 9 trans, 12 cis, 15 trans.

Conversion of 18:3 delta 9cis, 12cis, 15trans in rat liver microsomes.

Several years ago, it was established that the delta 15 trans isomer of alpha-linolenic acid is converted in vivo into fatty acids containing 20 and 22 carbons (geometrical isomers of eicosapentaenoic and docosahexaenoic acids). The present study focused on the in vitro delta 6 desaturation, the first step of the biosynthesis of the n-3 long-chain polyunsaturated fatty acids from 18:3n-3. For that purpose, rat liver microsomes were prepared and incubated with radiolabeled 18:3 delta 9cis,12cis,15cis (18:3c,c,c) or 18:3 delta 9cis, 12cis, 15trans (18:3c,c,t) under desaturation conditions. The data show that 18:3c,c,t is converted at a lower rate compared with alpha-linolenic acid. The product of conversion of 18:3c,c,t may be 18:4 delta 6cis, 9cis, 12cis, 15trans resulting from a delta 6 desaturation of the trans substrate. Moreover, the conversion of radiolabeled 18:3c,c,t was strongly decreased by the presence of 18:3c,c,c (up to 48%) while the 18:3c,c,t only slightly decreased the conversion of radiolabeled 18:3c,c,c. Thus, the desaturation enzyme presented a higher affinity for the native all-cis n-3 substrate.

Nitrogen dioxide induces cis-trans-isomerization of arachidonic acid within cellular phospholipids. Detection of trans-arachidonic acids in vivo.

Oxygen free radicals oxidize arachidonic acid to a complex mixture of metabolites termed isoeicosanoids that share structural similarity to enzymatically derived eicosanoids. However, little is known about oxidations of arachidonic acid mediated by reactive radical nitrogen oxides. We have studied the reaction of arachidonic acid with NO2, a free radical generated by nitric oxide and nitrite oxidations. A major group of products appeared to be a mixture of arachidonic acid isomers having one trans-bond and three cis-double bonds. We have termed these new products trans-arachidonic acids. These isomers were chromatographically distinct from arachidonic acid and produced mass spectra that were nearly identical with mass spectra of arachidonic acid. The lack of ultraviolet absorbance above 205 nm and the similarity of mass spectra of dimethyloxazoline derivatives suggested that the trans-bond was not conjugated with any of the cis-bonds, and the C=C bonds were located at carbons 5, 8, 11, and 14. Further identification was based on comparison of chromatographic properties with synthetic standards and revealed that NO2 generated 14-trans-eicosatetraenoic acid and a mixture containing 11-trans-, 8-trans-, and 5-trans-eicosatetraenoic acids. Exposure of human platelets to submicromolar levels of NO2 resulted in a dose-dependent formation of 14-trans-eicosatetraenoic acid and other isomers within platelet glycerophospholipids. Using a sensitive isotopic dilution assay we detected trans-arachidonic acids in human plasma (50.3 +/- 10 ng/ml) and urine (122 +/- 50 pg/ml). We proposed a mechanism of arachidonic acid isomerization that involves a reversible attachment of NO2 to a double bond with formation of a nitroarachidonyl radical. Thus, free radical processes mediated by NO2 lead to generation of trans-arachidonic acid isomers, including biologically active 14-trans-eicosatetraenoic acid, within membrane phospholipids from which they can be released and excreted into urine.

Effects of a trans isomer of arachidonic acid on rat platelet aggregation and eicosanoid production.

The addition of a trans isomer of arachidonic acid (20:4 delta 14trans) to rat platelet suspensions inhibited the aggregation induced by 7.5 microM of arachidonic acid. This inhibitory effect of 20:4 delta 14trans was significant at concentrations of 7.5-22.5 microM and the range of inhibition was 20% at an inhibitor/substrate ratio (I/S) 1 to 66% when I/S reached 3. However, the addition of its structural homolog (20:3n-9) or the natural isomer (20:4n-6) did not induce any modification of the platelet aggregation. In parallel, adding 20:4 delta 14trans to the platelet significantly decreased thromboxane B2 and 12-hydroxyheptadecatrienoic acid production. In contrast, the 12-lipoxygenase pathway was stimulated, as 12-hydroxyeicosatetraenoic acid production increased up to 55% when the I/S reached 3. 20:3n-9, not being a substrate of the cyclooxygenase, did not induce any significant modification in the formation of thromboxane B2 and 12-hydroxyheptadecatrienoic acid. 20:4 delta 14t alone did not induce any platelet aggregation. However, this fatty acid was metabolized to a limited extent into two products that have still to be identified. One of them would be a product of the 12-lipoxygenase pathway.

In vitro desaturation or elongation of monotrans isomers of linoleic acid by rat liver microsomes.

Several nutritional studies have shown the in vivo conversion of the 9c, 12t-18:2 and 9t, 12c-18:2 into long chain polyunsaturated fatty acids (PUFA) containing 20 carbons (geometrical isomers of eicosadienoic and eicosatetraenoic acids). In the present work, some in vitro studies were carried out in order to have precise information on the conversion of these two isomers. In a first set of experiments, studies were focused on the in vitro delta6 desaturation, the first regulatory step of the biosynthesis of n-6 long chain PUFA, from 9c, 12c-18:2. Rat liver microsomes were prepared and incubated under desaturation conditions with [1-14C]-9c, 12c-18:2 in presence of unlabelled 9c, 12t-, 9t, 12c- or 9t, 12t-18:2. The data show that each trans isomer induced a decrease of the delta6 desaturation of the [1-14C]-9c, 12c-18:2, but the 9c, 12t-18:2 was the most potent inhibitor (up to 63%). Rat liver microsomes were also incubated with [1-14C]-9c, 12c-18:2, [1-14C]-9c, 12t-18:2 or [1-14C]-9t, 12c-18:2 under desaturation conditions. The results indicated that 18:2 delta9c, 12t is a much better substrate for desaturase than 9t, 12c-18:2. Moreover, the conversion levels of [1-14C]-9c, 12t-18:2 was similar to what was observed for its all cis homologue, at low substrate concentration only. In a second set of experiments, in vitro elongation studies of each mono-trans 18:2 isomer and 9c, 12c-18:2 were carried out. For that purpose, rat liver microsomes were incubated with [1-14C]-9c, 12c-18:2, [1-14C]-9c, 12t-18:2 or [1-14C]-9t, 12c-18:2 underelongation conditions. The data show that [1-14C]-9t, 12c-18:2 is betterelongated than 9c, 12c-18:2 while the amount of product formed from [1-14C]-9c, 12t-18:2 was lower than was produced from the 9c, 12c-18:2. Thus, the desaturation enzymes presented a higher affinity for the 9c, 12t-18:2 whereas the elongation enzyme presented a higher affinity for the 9t, 12c-18:2.