Oxidation-Induced Increase In Photoreactivity of Bovine Retinal Lipid Extract.

The mammalian retina contains a high level of polyunsaturated fatty acids, including docosahexaenoic acid (22:6) (DHA), which are highly susceptible to oxidation. It has been shown that one of the products of DHA oxidation-carboxyethylpyrrole (CEP), generated in situ, causes modifications of retinal proteins and induces inflammation response in the outer retina. These contributing factors may play a role in the development of age-related macular degeneration (AMD). It is also possible that some of the lipid oxidation products are photoreactive, and upon irradiation with blue light may generate reactive oxygen species. Therefore, in this work we analysed oxidation-induced changes in photoreactivity of lipids extracted from bovine neural retinas. Lipid composition of bovine neural retinas closely resembles that of human retinas making the bovine tissue a convenient model for studying the photoreactivity and potential phototoxicity of oxidized human retinal lipids. Lipid composition of bovine neural retinas Folch' extracts (BRex) was determined by gas chromatography (GC) and liquid chromatography coupled to an electrospray ionization source-mass spectrometer (LC-ESI-MS) analysis. Liposomes prepared from BRex, equilibrated with air, were oxidized in the dark at 37 °C for up to 400 h. The photoreactivity of BRex at different stages of oxidation was studied by EPR-oximetry and EPR-spin trapping. Photogeneration of singlet oxygen (1O2, 1Δg) by BRex was measured using time-resolved detection of the characteristic phosphorescence at 1270 nm. To establish contribution of lipid components to the analysed photoreactivity of Folch' extract of bovine retinas, a mixture of selected synthetic lipids in percent by weight (w/w %) ratio resembling that of the BRex has been also studied. Folch's extraction of bovine neural retinas was very susceptible to oxidation despite the presence of powerful endogenous antioxidants such as α-tocopherol and zeaxanthin. Non-oxidized and oxidized BRex photogenerated singlet oxygen with moderate quantum yield. Blue-light induced generation of superoxide anion by Folch' extract of bovine neural retinas strongly depended on the oxidation time. The observed photoreactivity of the studied extract gradually increased during its in vitro oxidation.

Cerebral plasmalogens and aldehydes in senescence-accelerated mice P8 and R1: a comparison between weaned, adult and aged mice.

In contrast with senescence-accelerated mice R1, SAM P8 show abnormal aging characteristics. Changes occurring during aging could be mainly caused by free radical reactions. The brain is a plasmalogen-rich tissue. These particular phospholipids may act as endogenous antioxidants, be oxidized and release long chain aldehydes and alpha-hydroxyaldehydes during oxidative stress. The aim of this study was to examine by GC/MS the age- and strain-related levels of plasmalogens, aldehydes and alpha-hydroxyaldehydes in brain homogenates of SAM P8 and R1 at weaning, 5 months and 9 months of age in order to better understand the differences between both strains. In SAM R1, the evolution of brain plasmalogen levels corresponded to characteristics of normal aging: an increase from weaned to adult mice followed by a decrease characterizing the normal loss of myelin. By contrast to SAM R1, there was no change in the plasmalogen content in SAM P8 brain. The levels of aldehydes and alpha-hydroxyaldehydes were similar for both strains, they remained constant between adult and aged mice. Specific changes in the aging of SAM P8 were not explained by cerebral levels of these oxidative products. Other mechanisms related to the toxicity of aldehydes and alpha-hydroxyaldehydes could be considered.

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.

Selection of methylation procedures for quantitation of short-chain glycerol-bound compounds formed during thermoxidation.

Five methylation procedures, including base- and acid-catalyzed methods, were tested in thermoxidized methyl linoleate and trilinolein, in order to quantitate major oxidation short-chain glycerol-bound compounds by gas chromatography. Results indicated that transmethylations using KOH in methanol or CH3ONa-CH3OH in tert.-butylmethyl ether were the most appropriate methods, given the excellent reproducibility and practically complete recovery obtained for the compounds of interest, mainly short-chain fatty acids and aldehydic acids. Also, formation of acids from aldehydes during thermoxidation as well as modifications of aldehydic functions under acidic conditions, such as conversion to acetals, were checked using dodecanal as model aldehyde.

Gas chromatographic separation and mass spectrometric identification of mixtures of oxyphytosterol and oxycholesterol derivatives application to a phytosterol-enriched food.

Pure individual phytosterols were prepared using reversed-phase HPLC in order to obtain the oxidized compounds of sitosterol, campesterol, stigmasterol and brassicasterol. 7-Hydroxy-, 7-keto-, 5,6-epoxy-, 4beta-hydroxy-, 4-ene-6-hydroxy-, 6-keto- and 5alpha,6beta-dihydroxyphytosterols were obtained as well as analogous compounds of cholesterol. The gas chromatographic properties as well as the electronic impact mass spectra of these compounds (as trimethylsilyl ether derivatives) were studied. These data were used to identify oxyphytosterols in a spread enriched in phytosterols: the oxyphytosterols represented no more than 68 microg/g of spread (about 0.08% of phytosterols were oxidised).

Sensitive and accurate quantitation of monoepoxy fatty acids in thermoxidized oils by gas-liquid chromatography.

A sensitive and accurate methodology for quantitation of monoepoxy fatty acid methyl esters (FAME) by gas-liquid chromatography is proposed. Analytical problems of interfering compounds, ie, methyl monoester of azelaic acid and methyl docosanoate, were solved by a second methylation step with diazomethane and by elimination of nonpolar FAME by adsorption chromatography, respectively. Six monoepoxy FAME were identified and quantitated in olive and sunflower oils heated at 180 degrees C for 15 h: trans-9,10- and cis-9,10-epoxystearate coming from oleate and trans-12,13-, trans-9,10-, cis-12,13- and cis-9,10-epoxyoleate coming from linoleate. Results demonstrated total recovery of monoepoxy compounds after nonpolar FAME elimination with the additional advantage of sample concentration, which allowed quantitation of monoepoxy FAME in the initial oils. Also, repeatability was excellent as relative standard deviations ranged from 2.2 to 5.1% for on-column injection and from 0.1 to 2.0% for automatic split injection.

Synthesis of (6Z,9Z,11E)-octadecatrienoic and (8Z,11Z,13E)-eicosatrienoic acids and their [1-(14)C]-radiolabeled analogs.

In order to study the metabolic pathway and the physiological effects of 9c,11t-18:2 (major isomer of conjugated linoleic acid) and its C(18:3) and C(20:3) metabolites, 6c,9c,11t-18:3 and 8c,11c,13t-20:3 and their [1-(14)C]-radiolabeled analogs were prepared stereoselectively by total synthesis. The 8c,11c,13t-20:3 was obtained in 11 steps. The synthesis involves a highly stereoselective Wittig reaction between 3-(t-butyldiphenylsilyloxy)propanal and the ylide of 7-(2-tetrahydropyranyloxy)heptanylphosphonium salt which gave (3Z)-1-(t-butyldiphenylsilyloxy)-10-(2-tetrahydropyranyloxy)dec-3-ene in a first step. Then the t-butyldiphenylsilyl derivative was deprotected selectively and the resulting alcohol function was converted via a bromide into a phosphonium salt. The second stereoselective Wittig condensation between the phosphonium salt and commercial (2E)-non-2-enal under cis-olefinic conditions using Lithium hexamethyldisilazide as base afforded the (7Z,10Z,12E)-1-(2-tetrahydropyranyloxy)nonadeca-7,10,12-triene in a very good isomeric purity. The intermediate product was brominated and transformed by reaction with magnesium into Grignard reagent, which was one-carbon elongated by unlabeled or labeled carbon dioxide to obtain the 8c,11c,13t-20:3 in good isomeric purity (95%) and high radiochemical purity for its [1-(14)C]-radiolabeled analog (99%). 6c,9c,11t-18:3 was synthesized in a similar way by using 5-(2-tetrahydropyranyloxy)pentanylphosphonium salt in place of 7-(2-tetrahydropyranyloxy)heptanylphosphonium salt in a first step. Other reactions were unchanged and products were obtained in similar yields. Similar to 8c,11c,13t-20:3, the 6c,9c,11t-18:3 was obtained in a very good isomeric purity (95%) and its [1-(14)C]-radiolabeled analog in a high radiochemical purity (95%).

Effects of conjugated linoleic acid isomers on the hepatic microsomal desaturation activities in vitro.

The influence of individual conjugated linoleic acid (CLA) isomers on the delta6 desaturation of linoleic and alpha-linolenic acids and on the delta9 desaturation of stearic acid was investigated in vitro, using rat liver microsomes. The delta6 desaturation of 18:2n-6 was decreased from 23 to 38% when the ratio of 9cis,11trans-18:2 to 18:2n-6 increased from 0.5 to 2. The compound 10trans,12cis-18:2 exhibited a similar effect only at the highest concentration. The delta6 desaturation of alpha-linolenic acid was slightly affected by the presence of CLA isomers. The sole isomer to induce an inhibitory effect on the delta9 desaturation of stearic acid was 10trans,12cis-18:2.

In vitro desaturation and elongation of rumenic acid by rat liver microsomes.

Various nutritional studies on CLA, a mixture of isomers of linoleic acid, have reported the occurrence of conjugated long-chain PUFA after feeding experimental animals with rumenic acid, 9c,11t-18:2, the major CLA isomer, probably as a result of successive desaturation and chain elongation. In the present work, in vitro studies were carried out to obtain information on the conversion of rumenic acid. Experiments were first focused on the in vitro delta6-desaturation of rumenic acid, the regulatory step in the biosynthesis of long-chain n-6 PUFA. The conversion of rumenic acid was compared to that of linoleic acid (9c,12c-18:2). Isolated rat liver microsomes were incubated with radiolabeled 9c,12c-18:2 and 9c,11t-18:2 under desaturation conditions. The data indicated that [1-(14)C]9c,11t-18:2 was a poorer substrate for delta6-desaturase than [1-(14)C]9c,12c-18:2. Next, in vitro elongation of 6c,9c,11t-18:3 and 6c,9c,12c-18:3 (gamma-linolenic acid) was investigated in rat liver microsomes. Under elongation conditions, [1-(14)C]6c,9c,11t-18:3 was 1.5-fold better converted into [3-(14)C]8c,11c,13t-20:3 than [1-(14)C]6c,9c,12c-18:3 into [3-(14)C]8c,11c,14c-20:3. Finally, in vitro delta5-desaturation of 8c,11c,13t-20:3 compared to 8c,11c,14c-20:3 was investigated. The conversion level of [1-(14)C]8c,11c,13t-20:3 into [1-(14)C]5c,8c,11c,13t-20:4 was 10 times lower than that of [1-(14)C]8c,11c,14c-20:3 into [1-(14)C]5c,8c,11c,14c-20:4 at low substrate concentrations and 4 times lower at the saturating substrate level, suggesting that conjugated 20:3 is a poor substrate for the delta5-desaturase.

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.

The effect of conjugated linoleic acid isomers on fatty acid profiles of liver and adipose tissues and their conversion to isomers of 16:2 and 18:3 conjugated fatty acids in rats.

Conjugated linoleic acid (CLA) is a collective term that describes different isomers of linoleic acid with conjugated double bonds. Although the main dietary isomer is 9cis,11trans-18:2, which is present in dairy products and ruminant fat, the biological effects of CLA generally have been studied using mixtures in which the 9cis,11trans- and the 10trans,12cis-18:2 were present at similar levels. In the present work, we have studied the impact of each isomer (9cis,11 trans- and 10trans,12cis-18:2) given separately in the diet of rats for 6 wk. The 10trans,12cis-18:2 decreased the triacylglycerol content of the liver (-32%) and increased the 18:0 content at the expense of 18:1 n-9, suggesting an alteration of the delta9 desaturase activity, as was already demonstrated in vitro. This was not observed when the 9cis,11trans-18:2 was given in the diet. Moreover, the 10trans,12cis-18:2 induced an increase in the C22 polyunsaturated fatty acids in the liver lipids. The 10trans,12cis-18:2 was mainly metabolized into conjugated 16:2 and 18:3, which have been identified. The 9cis,11trans isomer was preferentially metabolized into a conjugated 20:3 isomer. Thus, the 9cis,11trans- and the 10trans,12cis-CLA isomers are metabolized differently and have distinct effects on the metabolism of polyunsaturated fatty acids in rat liver while altering liver triglyceride levels differentially.

Beta-oxidation of conjugated linoleic acid isomers and linoleic acid in rats.

To assess the oxidative metabolism of conjugated linoleic acid (CLA) isomers, rats were force-fed 1.5-2.6 MBq of [1-14C]-linoleic acid (9c,12c-18:2), -rumenic acid (9c,11t-18:2), or-10trans,12cis-18:2 (10t,12c-18:2), and 14CO2 production was monitored for 24 h. The animals were then necropsied and the radioactivity determined in different tissues. Both CLA isomers were oxidized significantly more than linoleic acid. Moreover, less radioactivity was recovered in most tissues after CLA intake than after linoleic acid intake. The substantial oxidation of CLA isomers must be considered when assessing the putative health benefits of CLA supplements.

Effects of conjugated linoleic acid isomers on lipid-metabolizing enzymes in male rats.

Male weanling Wistar rats (n = 15), weighing 200-220 g, were allocated for 6 wk to diets containing 1% (by weight) of conjugated linoleic acid (CLA), either as the 9c,11 t-isomer, the 10t,12c-isomer, or as a mixture containing 45% of each of these isomers. The five rats of the control group received 1% of oleic acid instead. Selected enzyme activities were determined in different tissues after cellular subfractionation. None of the CLA-diet induced a hepatic peroxisome-proliferation response, as evidenced by a lack of change in the activity of some characteristic enzymes [i.e., acyl-CoA oxidase, CYP4A1, but also carnitine palmitoyltransferase-I (CPT-I)] or enzyme affected by peroxisome-proliferators (glutathione S-transferase). In addition to the liver, the activity of the rate-limiting beta-oxidation enzyme in mitochondria, CPT-I, did not change either in skeletal muscle or in heart. Conversely, its activity increased more than 30% in the control value in epididymal adipose tissue of the animals fed the CLA-diets containing the 10t,12c-isomer. Conversely, the activity of phosphatidate phosphohydrolase, a rate-limiting enzyme in glycerolipid neosynthesis, remained unchanged in adipose tissue. Kinetic studies conducted on hepatic CPT-I and peroxisomal acyl-CoA oxidase with CoA derivatives predicted a different channeling of CLA isomers through the mitochondrial or the peroxisomal oxidation pathways. In conclusion, the 10t,12c-CLA isomer seems to be more efficiently utilized by the cells than its 9c,11t homolog, though the Wistar rat species appeared to be poorly responsive to CLA diets for the effects measured.

Effect of growing area on tocopherols, carotenoids and fatty acid composition of Pistacia lentiscus edible oil.

In this investigation, we aim to study, for the first time, the effect of the growing area on tocopherols, carotenoids and fatty acid content of Pistacia lentiscus fixed oil. Fruits were harvested from eight different sites located in the north and the centre of Tunisia. Tocopherols, carotenoids and fatty acid content of the fixed oils were determined. The highest carotenoid content was exhibited by Feija oil (10.57 mg/kg of oil). Oueslatia and Tabarka oils displayed the highest α-tocopherol content (96.79 and 92.79 mg/kg of oil, respectively). Three major fatty acids were determined: oleic, palmitic and linoleic acids. Oleic acid was the main fatty acid presenting more than 50% of the total fatty acid content. Kebouche oil presented the highest oleic acid content (55.66%). All these results highlight the richness of carotenoids, tocopherols and unsaturated fatty acids in P. lentiscus seed oil and underscore the nutritional value of this natural product.

[Isoprostanes, biomarkers of lipid peroxidation in humans. Part 1. Nomenclature and synthesis]. / Les isoprostanes, biomarqueurs de peroxydation lipidique chez l'homme. Partie 1. Nomenclature et synthèse.

The synthesis of prostaglandins has been a subject of chemical attractiveness for the last 40 years with the successful strategy developed by Corey et al. via a formyl-lactone in the trans-PG series, derived from the action of cyclooxygenases (COXs). The non-enzymatic metabolic scheme of arachidonic acid, as a free radical catalyzed mechanism, has introduced new data concerning the reactivity of the arachidonyl radical in the absence of COXs and also a growing interest in the total synthesis of isoprostanes and analogues. The potent biological activity of these compounds has been attracting intense research interest since they were detected in humans as well as animal models in the early 1990s. The measurement of these isoprostanes has been regarded as one of the most useful non-invasive biomarkers for oxidative stress status. Two mechanisms for their biosynthesis have been proposed. In the first mechanism, a peroxyl radical undergoes successive 5-exo cyclizations analogous to the enzymatic mechanism proposed for prostaglandin biosynthesis. The second mechanism starts with a 4-exo cyclization of a peroxyl radical leading to an intermediate dioxetane. During the last two decades, several approaches towards the synthesis of isoprostanes and analogues have been reported in the literature by several groups of chemists. Finally, to date, two nomenclatures have been proposed by Taber et al. and Rokach et al., but only Taber's nomenclature have been approved by IUPAC.

[Isoprostanes, biomarkers of lipid peroxidation in humans. Part 2: Quantification methods]. / Les isoprostanes, biomarqueurs de peroxydation lipidique chez l'homme. Partie 2 : méthodes de quantification.

The isoprostanes are a new class of natural products produced in vivo by a non-enzymatic free-radical-induced peroxidation of polyunsaturated fatty acids. The quantification of these compounds represents a reliable and useful index of lipid peroxidation and oxidant stress in vivo. Then, a large amount of works has been done in the field of isoprostane analysis, but till now, no standardized method seems to emerge. Indeed, described methodologies differ either in the sample preparation steps or in the detection techniques or both. Extraction and purification procedures are often critical and time-consuming, requiring successive chromatographic steps and these procedures lead to a substantial loose of target compounds. Moreover, two main analytical approaches have been adopted for IsoP measurement: immunological methods or mass spectrometry. Some discussion about the methodology used for measurement of isoprostane is important. This review will aim to present and compare different methods developed nowadays for extraction, purification and analysis of F(2)-iPs in various biological samples.

[Isoprostanes, biomarkers of lipid peroxidation in humans. Part 3: Biomarkers and mediators in vascular physiology and disease]. / Les isoprostanes, biomarqueurs de peroxydation lipidique chez l'homme. Partie 3: biomarqueurs et médiateurs en physiologie et pathologie vasculaire.

The 15-series F(2)-isoprostanes mediate vasoconstriction in different vascular beds and species. This contraction is mediated by the thromboxane receptors stimulation, and may be modulated by the endothelium. Furthermore, 15-F(2t)-IsoP induces smooth muscle cells mitogenesis and monocyte adhesion to endothelial cells. Some 15-series E(2)-isoprostanes are more potent than F(2)-isoprostanes. In clinical studies, 15-F(2t)-IsoP levels are increased in vascular disorders involving atherosclerosis, ischemia-reperfusion and inflammation. F(2)-isoprostane levels correlate to the severity of heart failure and pulmonary hypertension, raising the potential prognostic interest of these biomarkers. Whether the effects observed in vitro are observed consistently in vivo at physiological concentrations and whether these effects contribute to pathological states in vivo is still debated.

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.