Dietary trans-10,cis-12 conjugated linoleic acid induces hyperinsulinemia and fatty liver in the mouse

Conjugated linoleic acids (CLA) are a class of positional, geometric, conjugated dienoic isomers of linoleic acid (LA). Dietary CLA supplementation results in a dramatic decrease in body fat mass in mice, but also causes considerable liver steatosis. However, little is known of the molecular mechanisms leading to hepatomegaly. Although c9,t11- and t10,c12-CLA isomers are found in similar proportions in commercial preparations, the respective roles of these two molecules in liver enlargement has not been studied. We show here that mice fed a diet enriched in t10,c12-CLA (0.4% w/w) for 4 weeks developed lipoatrophy, hyperinsulinemia, and fatty liver, whereas diets enriched in c9,t11-CLA and LA had no significant effect. In the liver, dietary t10,c12-CLA triggered the ectopic production of peroxisome proliferator-activated receptor gamma (PPARgamma), adipocyte lipid-binding protein and fatty acid transporter mRNAs and induced expression of the sterol responsive element-binding protein-1a and fatty acid synthase genes. In vitro transactivation assays demonstrated that t10,c12- and c9,t11-CLA were equally efficient at activating PPARalpha, beta/delta, and gamma and inhibiting liver-X-receptor. Thus, the specific effect of t10,c12-CLA is unlikely to result from direct interaction with these nuclear receptors. Instead, t10,c12-CLA-induced hyperinsulinemia may trigger liver steatosis, by inducing both fatty acid uptake and lipogenesis.     

植物乳杆菌ZS2058在磷酸盐缓冲液体系中生物转化共轭亚油酸

植物乳杆菌ZS2058是从泡菜中筛选到一株具有转化共轭亚油酸能力的乳酸菌。该菌株在MRS培养基中经0.5mg/mL的亚油酸诱导培养后,所获得的菌体细胞具有较强的转化能力。文中就植物乳杆菌ZS2058水洗细胞在磷酸盐缓冲液体系中生物转化共轭亚油酸进行了深入研究。在非厌氧条件下,植物乳杆菌ZS2058在亚油酸浓度为1mg/mL,湿细胞质量浓度约为150mg/mL,120r/min、37℃的条件下反应24h后,能将亚油酸转化为共轭亚油酸和羟基脂肪酸,其中c9,t11-CLA占所产生的CLA总量的96.4%,产量可高达312.4μg/mL,说明该菌株有很强的专一性。随着反应进一步进行,反应至36h时,c9,t11-CLA含量逐渐减少,伴随着大量羟基脂肪酸的产生;并且,以CLA(c9,t11-CLA和t10,c12-CLA的混合样品)为底物进行反应时,c9,t11-CLA被转化为羟基脂肪酸。由此可知,c9,t11-CLA可能是该菌株生物转化LA过程中的一个中间产物。     

Conjugated linoleic acid exhibits stimulatory and inhibitory effects on prostanoid production in human endothelial cells and platelets

In addition to their reported antitumorigenic properties, various conjugated linoleic acid (CLA) isomers have also been shown to decrease prostanoid synthesis as a result of inhibiting the cyclooxygenase (COX) enzyme. We have previously reported that several CLA isomers inhibited both platelet aggregation and formation of thromboxane A(2) (TXA(2)), a proaggregatory and vasoconstrictive agent. Since the interaction between platelets and vascular endothelial cells is essential to maintaining vascular homeostasis, we decided to investigate the effects of various CLA isomers on the production of endothelial prostacyclin (PGI(2)), a potent vasodilator and inhibitor of platelet function. Using interleukin 1-beta (IL1-beta)-stimulated human umbilical vein endothelial cells (HUVECs), we initially established that HUVECs of passage #2 should be used since these cells were most responsive to thrombin-induced conversion of endogenous arachidonic acid to PGI(2), as monitored by the formation of its stable, inactive metabolite, 6-ketoPGF(1alpha). In the first part of the study, the effects of CLA isomers in the free fatty acid form were tested. The 10(E), 12(Z)- and 9(Z), 11(E)-CLA isomers inhibited thrombin-induced 6-ketoPGF(1alpha) formation with I(50)'s of 2.6 and 5.5 microM, whereas the 9(Z), 11(Z)- and 9(E), 11(E)-CLA were ineffective at concentrations up to 60 microM. The inhibitory effect of the 10(E), 12(Z)-CLA was irreversible. Next, the effects of CLA incorporation into HUVECs on PGI(2) generation was determined. An average 8-fold stimulation of 6-ketoPGF(1alpha) formation was obtained with quiescent IL1-beta-exposed HUVECs pretreated for 18 h with 25 microM 9(Z), 11(Z)-CLA, whereas cells preincubated with the 10(E), 12(Z) isomer enhanced this eicosanoid 3-fold. Such IL1-beta-treated HUVECs prelabeled with 25 microM 9(Z), 11(Z)-CLA became refractory to thrombin stimulation, as measured by 6-ketoPGF(1alpha) production, whereas a small, statistically insignificant, inhibition was observed upon thrombin treatment of HUVECs prelabeled with the 10(E), 12(Z) isomer. Qualitative similar results were obtained with resting or thrombin-stimulated platelets containing these esterified CLA isomers indicating that these effects occur with cells that contain either the COX-1 or COX-2 isozymes. The results of this in vitro study indicate that the effects of CLA on cellular prostanoid formation in endothelial cells and platelets can be either inhibitory or stimulatory, and this seems to depend not only on the specific CLA isomer and whether or not the CLA is in the free fatty acid form or esterified into cellular lipids, but also whether cells are in the resting or stimulated state. These findings suggest that in vivo, CLA might have multiple, complex effects on vascular homeostasis.     

Molecular and cellular effects of cis-9, trans-11-conjugated linoleic acid in enterocytes: effects on proliferation, differentiation, and gene expression

It has been hypothesized that dietary conjugated linoleic acids (CLA) may inhibit colon tumorigenesis. The aim of our study was to investigate the cellular and molecular effects of cis-9 (9Z), trans-11 (11E)-CLA on the proliferation, differentiation, interaction with peroxisome proliferator-activated receptors (PPARs), and expression of genes relevant in the APC-beta-catenin-TCF4 signalling pathway in human HT-29 and Caco-2 colon cells. We found that 9Z,11E-CLA inhibited the proliferation of HT-29 and Caco-2 cells. Trans-vaccenic acid (VA) showed no antiproliferative effects at all. We determined that 9Z,11E-CLA induced cell differentiation as measured by intestinal alkaline phosphatase (IAP) enzyme activity in Caco-2 cells, mRNA expression of IAP, and activation of a 5' flanking region of IAP. The 9Z,11E-CLA activated human PPARdelta as measured in a reporter gene assay. Treatment of HT29 cells in the poliferation phase with 9Z,11E-CLA repressed mRNA-expression of proliferation genes such as c-myc, cyclin D1 and c-jun in a concentration dependent manner. The promoter activities of c-myc and AP1 were also inhibited after incubation with 9Z,11E-CLA. beta-Catenin mRNA and protein expression was also repressed by the treatment with 9Z,11E-CLA. In addition, the mRNA expression of PPARdelta was repressed by treatment of the HT-29 cells with 9Z,11E-CLA. We conclude that 9Z,11E-CLA has an antiproliferative effect at the cellular and molecular levels in human colon cells. The results indicate that the preventive effects of CLA in the development of colon cancer may be due to their downregulation of some target genes of the APC-beta-catenin-TCF-4- and PPARdelta signalling pathway.     

Antioxidant activity of conjugated linoleic acid isomers, linoleic acid and its methyl ester determined by photoemission and DPPH techniques

The chemiluminescent response of conjugated linoleic acid isomers (CLAs), linoleic acid (LA) and methyl linoleate (LAME) against the prooxidant t-butyl hydroperoxide (tBHP) was analyzed. The c9, t11-CLA and t10, c12-CLA isomers showed significant photoemission at the highest concentration used, while photoemission was not detected at any concentration of LA and LAME analyzed. These results show that CLAs are more susceptible to peroxidation than LA and LAME. Likewise, the effect of CLA, LA and LAME on lipid peroxidation of triglycerides rich in C20:5 omega3 and C22:6 omega3 (Tg omega3-PUFAs) was investigated. For that, chemiluminescence produced by triglycerides in the presence of tBHP, previously incubated with different concentrations of CLAs, LA and LAME (from 1 to 200 mM) was registered for 60 min. Triglycerides in the presence of t-BHP produced a peak of light emission (3151+/-134 RLUs) 5 min after addition. CLAs produced significant inhibition on photoemission, t10, c12-CLA being more effective than the c9, t11-CLA isomer. LA and LAME did not have an effect on lipid peroxidation of Tg omega3-PUFAs. CLA isomers, LA and LAME were also investigated for free radical scavenging properties against the stable radical (DPPH()). Both CLA isomers reacted and quenched DPPH() at all tested levels (from 5 to 25 mM), while LA and LAME did not show radical quenching activity even at the highest concentration tested. These data indicate that CLAs would provide protection against free radicals, but LA and LAME cannot.     

Slow Absorption of Conjugated Linoleic Acid in Rat Intestines,and Similar Absorption Rates of 9c,11t-Conjugated Linoleic Acid and 10t,12c-Conjugated Linoleic Acid

We have previously shown that the 9c,11t-conjugated linoleic acid (CLA) concentration was always significantly higher than the 10t,12c-CLA concentration following the administration of these compounds to mice and rats, and considered that structural differences between the conjugated double bonds in these isomers affected absorption in the small intestine. This study investigates the absorption of CLA in the rat intestine by a lipid absorption assay of lymph from the thoracic duct. In Study 1, we used safflower oil and a triacylglycerol form of CLA (CLA-TG), while in Study 2, we used 9c,11t-CLA and 10t,12c-CLA. The cumulative recovery of CLA was lower than that of linoleic acid until two hours after sample administration. There was no difference in the extent of lymphatic recovery of 9c,11t-CLA and 10t,12c-CLA after the administration of CLA-TG, 9c,11t-CLA, and 10t,12c-CLA to the rats, suggesting that geometrical and positional isomerism of the conjugated double bonds did not influence the absorption.     

共轭亚油酸生理功能及微生物合成调控研究进展

共轭亚油酸(conjugated linoleic acid,CLA)是一种新型功能性油脂,顺9,反11-十八碳二烯酸(c9,t11-CLA)和反10,顺12-十八碳二烯酸(t10,c12-CLA)由于具有比其他异构体更强的生理功能得到广泛关注和研究.微生物合成CLA具有安全性高、选择特异性强等特点,研究CLA产量提高...     

Troglitazone but not conjugated linoleic acid reduces gene expression and activity of matrix-metalloproteinases-2 and -9 in PMA-differentiated THP-1 macrophages

Gene expression and activity of matrix-metalloproteinases (MMP)-2 and -9 in macrophages are reduced through peroxisome proliferator-activated receptor gamma (PPARgamma)-dependent inhibition of NF-kappaB. Since conjugated linoleic acids (CLAs) are PPARgamma ligands and known to inhibit NF-kappaB via PPARgamma, we studied whether CLA isomers are capable of reducing gene expression and gelatinolytic activity of MMP-2 and -9 in PMA-differentiated THP-1 macrophages, which has not yet been investigated. Incubation of PMA-differentiated THP-1 cells with either c9t11-CLA, t10c12-CLA or linoleic acid (LA), as a reference fatty acid, resulted in a significant incorporation of the respective fatty acids into total cell lipids relative to control cells (P<.05). Treatment of PMA-differentiated THP-1 cells with 10 and 20 mumol/L troglitazone but not with 10 or 100 mumol/L c9t11-CLA, t10c12-CLA or LA reduced relative mRNA concentrations and activity of MMP-2 and MMP-9 compared to control cells (P<.05). DNA-binding activity of NF-kappaB and PPARgamma and mRNA expression of the NF-kappaB target gene cPLA(2) were not influenced by treatment with CLA. In contrast, treatment of PMA-differentiated THP-1 cells with troglitazone significantly increased transactivation of PPARgamma and decreased DNA-binding activity of NF-kappaB and relative mRNA concentration of cPLA(2) relative to control cells (P<.05). In conclusion, the present study revealed that CLA isomers, in contrast to troglitazone, did not reduce gene expression and activity of MMP-2 and -9 in PMA-differentiated THP-1 macrophages, which is probably explained by the observation that CLA isomers neither activated PPARgamma nor reduced DNA-binding activity of NF-kappaB. This suggests that CLA isomers are ineffective in MMP-associated extracellular matrix degradation which is thought to contribute to the progression and rupture of advanced atherosclerotic plaques.     

Potent inhibitory effect of trans9, trans11 isomer of conjugated linoleic acid on the growth of human colon cancer cells

This study compared the growth inhibitory effects of pure conjugated linoleic acid (CLA) isomers [cis(c)9,c11-CLA, c9,trans(t)11-CLA, t9,t11-CLA, and t10,c12-CLA] on human colon cancer cell lines (Caco-2, HT-29 and DLD-1). When Caco-2 cells were incubated up to 72 h with 200 μM, each isomer, even in the presence of 10% fetal bovine serum (FBS), cell proliferation was inhibited by all CLA isomers in a time-dependent manner. The strongest inhibitory effect was shown by t9,t11-CLA, followed by t10,c12-CLA, c9,c11-CLA and c9,t11-CLA, respectively. The strongest effect of t9,t11-CLA was also observed in other colon cancer cell lines (HT-29 and DLD-1). The order of the inhibitory effect of CLA isomer was confirmed in the presence of 1% FBS. CLA isomers supplemented in the culture medium were readily incorporated into the cellular lipids of Caco-2 and changed their fatty acid composition. The CLA contents in cellular lipids were 26.2±2.7% for t9,t11-CLA, 35.9±0.3% for c9,t11-CLA and 46.3±0.8% for t10,c12-CLA, respectively. DNA fragmentation was clearly recognized in Caco-2 cells treated with t9,t11-CLA. This apoptotic effect of t9,t11-CLA was dose- and time-dependent. DNA fragmentation was also induced by 9c,11t-CLA and t10,c12-CLA. However, fragmentation levels with both isomers were much lower than that with t9,t11-CLA. t9t11-CLA treatment of Caco-2 cells decreased Bcl-2 levels in association with apoptosis, whereas Bax levels remained unchanged. These results suggest that decreased expression of Bcl-2 by t9t11-CLA might increase the sensitivity of cells to lipid peroxidation and to programmed cell death, apoptosis.     

Purification of conjugated linoleic acid isomers through a process including lipase-catalyzed selective esterification

A mixture of conjugated linoleic acids (CLAs) was prepared by alkali conjugation of high purity linoleic acid. The preparation contained 45.1 wt% cis-9, trans-11 (c9,t11)-CLA, 46.8 wt% trans-10, cis-12 (t10,c12)-CLA, and 5.3 wt% other CLAs. A process comprising Candida rugosa lipase-catalyzed selective esterification with lauryl alcohol, molecular distillation, and urea adduct fractionation under strict conditions in ethanol was very effective for purification of c9,t11- and t10,c12-CLAs. In particular, the urea adduct fractionation efficiently eliminated CLAs except c9,t11- and t10,c12-isomers. Purification of c9,t11- and t10,c12-CLAs from 1.0 kg of the CLA mixture increased the c9,t11-CLA purity to 93.1% with 34% recovery of the initial content, and increased the t10,c12-CLA purity to 95.3% with 31% recovery.     

Accumulation of conjugated linoleic acid in <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> WU-P19 is enhanced by induction with linoleic acid and chitosan treatment

Production of conjugated linoleic acid (CLA) by the potential probiotic bacterium Lactobacillus plantarum WU-P19 was investigated with the aim of enhancing production. CLA produced using this bacterium may be used to supplement dietary intake. Cultures were fed linoleic acid for conversion to CLA and the CLA produced was measured. In some cases, chitosan was added to cultures to improve cellular uptake of linoleic acid. Under static conditions at 37 °C, the bacterium grew and produced CLA in the pH range of 5.5–6.5. At pH 6.0, a 36-h incubation period maximized the concentration of the dry biomass (0.82 g/L), the CLA content in the biomass (4.1 mg/g), and linoleic acid in the biomass (1.2 mg/g). In comparison with cultures grown without linoleic acid in the medium, supplementing the medium with linoleic acid at 600 μg/mL slowed the production of CLA, but the CLA content in the dry biomass increased to 12–14 mg/g and the linoleic acid content increased to 8–11 mg/g. Supplementing the culture medium with chitosan and linoleic acid enhanced production of CLA in the dry biomass to 21 mg/g within 36 h. Nearly 50% of the CLA was cis-9, trans-11-CLA, and the remainder was trans-10, cis-12-CLA. Linoleic acid content of the dry biomass was increased to 37 mg/g. Accumulation of CLA in the cells was enhanced by feeding linoleic acid. Supplementing the culture with linoleic acid and chitosan further increased accumulation of CLA.     

Differential action of 13-HPODE on PPARalpha downstream genes in rat Fao and human HepG2 hepatoma cell lines

In rats, oxidized fats activate the peroxisome proliferator-activated receptor alpha (PPARalpha), leading to reduced triglyceride concentrations in liver, plasma and very low density lipoproteins. Oxidation products of linoleic acid constitute an important portion of oxidized dietary fats. This study was conducted to check whether the primary lipid peroxidation product of linoleic acid, 13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE), might be involved in the PPARalpha-activating effect of oxidized fats. Therefore, we examined the effect of 13-HPODE on the expression of PPARalpha target genes in the rat Fao and the human HepG2 hepatoma cell lines. In Fao cells, 13-HPODE increased the mRNA concentration of the PPARalpha target genes acyl-CoA oxidase (ACO), cytochrome P450 4A1 and carnitine-palmitoyltransferase 1A (CPT1A). Furthermore, the concentration of cellular and secreted triglycerides was reduced in Fao cells treated with 13-HPODE. Because PPARalpha mRNA was not influenced, we conclude that these effects are due to an activation of PPARalpha by 13-HPODE. In contrast, HepG2 cells seemed to be resistant to PPARalpha activation by 13-HPODE because no remarkable induction of the PPARalpha target genes ACO, CPT1A, mitochondrial HMG-CoA synthase and delta9-desaturase was observed. Consequently, cellular and secreted triglyceride levels were not changed after incubation of HepG2 cells with 13-HPODE. In conclusion, this study shows that 13-HPODE activates PPARalpha in rat Fao but not in human HepG2 hepatoma cells.     

t10c12-CLA maintains higher bone mineral density during aging by modulating osteoclastogenesis and bone marrow adiposity

Conjugated linoleic acid (CLA) has been shown to positively influence calcium and bone metabolism. Earlier, we showed that CLA (equal mixture of c9t11-CLA and t10c12-CLA) could protect age-associated bone loss by modulating inflammatory markers and osteoclastogenesis. Since, c9t11-CLA and t10c12-CLA isomers differentially regulate functional parameters and gene expression in different cell types, we examined the efficacy of individual CLA isomers against age-associated bone loss using 12 months old C57BL/6 female mice fed for 6 months with 10% corn oil (CO), 9.5% CO + 0.5% c9t11-CLA, 9.5% CO + 0.5% t10c12-CLA or 9.5% CO + 0.25% c9t11-CLA + 0.25% t10c12-CLA. Mice fed a t10c12-CLA diet maintained a significantly higher bone mineral density (BMD) in femoral, tibial and lumbar regions than those fed CO and c9t11-CLA diets as measured by dual-energy-X-ray absorptiometry (DXA). The increased BMD was accompanied by a decreased production of osteoclastogenic factors, that is, RANKL, TRAP5b, TNF-alpha and IL-6 in serum. Moreover, a significant reduction of high fat diet-induced bone marrow adiposity was observed in t10c12-CLA fed mice as compared to that of CO and c9t11-CLA fed mice, as measured by Oil-Red-O staining of bone marrow sections. In addition, a significant reduction of osteoclast differentiation and bone resorbing pit formation was observed in t10c12-CLA treated RAW 264.7 cell culture stimulated with RANKL as compared to that of c9t11-CLA and linoleic acid treated cultures. In conclusion, these findings suggest that t10c12-CLA is the most potent CLA isomer and it exerts its anti-osteoporotic effect by modulating osteoclastogenesis and bone marrow adiposity.     

Activation of phosphatidylinositol-3 kinase,AMP-activated kinase and Akt substrate-160 kDa by trans-10, cis-12 conjugated linoleic acid mediates skeletal muscle glucose uptake

Conjugated linoleic acid (CLA), a dietary lipid, has been proposed as an antidiabetic agent. However, studies specifically addressing the molecular dynamics of CLA on skeletal muscle glucose transport and differences between the key isomers are limited. We demonstrate that acute exposure of L6 myotubes to cis-9, trans-11 (c9,t11) and trans-10, cis-12 (t10,c12) CLA isomers mimics insulin action by stimulating glucose uptake and glucose transporter-4 (GLUT4) trafficking. Both c9,t10-CLA and t10,c12-CLA stimulate the phosphorylation of phosphatidylinositol 3-kinase (PI3-kinase) p85 subunit and Akt substrate-160 kDa (AS160), while showing isomer-specific effects on AMP-activated protein kinase (AMPK). CLA isomers showed synergistic effects with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR). Blocking PI3-kinase and AMPK prevented the stimulatory effects of t10,c12-CLA on AS160 phosphorylation and glucose uptake, indicating that this isomer acts via a PI3-kinase and AMPK-dependent mechanism, whereas the mechanism of c9,t11-CLA remains unclear. Intriguingly, CLA isomers sensitized insulin-Akt-responsive glucose uptake and prevented high insulin-induced Akt desensitisation. Together, these results establish that CLA exhibits isomer-specific effects on GLUT4 trafficking and the increase in glucose uptake induced by CLA treatment of L6 myotubes occurs via pathways that are distinctive from those utilised by insulin.     

c9,t11-Conjugated linoleic acid ameliorates steatosis by modulating mitochondrial uncoupling and Nrf2 pathway

Oxidative stress, hepatic steatosis, and mitochondrial dysfunction are key pathophysiological features of nonalcoholic fatty liver disease. A conjugated linoleic acid (CLA) mixture of cis9,trans11 (9,11-CLA) and trans10,cis12 (10,12-CLA) isomers enhanced the antioxidant/detoxifying mechanism via the activation of nuclear factor E2-related factor-2 (Nrf2) and improved mitochondrial function, but less is known about the actions of specific isomers. The differential ability of individual CLA isomers to modulate these pathways was explored in Wistar rats fed for 4 weeks with a lard-based high-fat diet (L) or with control diet (CD), and, within each dietary treatment, two subgroups were daily administered with 9,11-CLA or 10,12-CLA (30 mg/day). The 9,11-CLA, but not 10,12-CLA, supplementation to CD rats improves the GSH/GSSG ratio in the liver, mitochondrial functions, and Nrf2 activity. Histological examination reveals a reduction of steatosis in L-fed rats supplemented with both CLA isomers, but 9,11-CLA downregulated plasma concentrations of proinflammatory markers, mitochondrial dysfunction, and oxidative stress markers in liver more efficiently than in 10,12-CLA treatment. The present study demonstrates the higher protective effect of 9,11-CLA against diet-induced pro-oxidant and proinflammatory signs and suggests that these effects are determined, at least in part, by its ability to activate the Nrf2 pathway and to improve the mitochondrial functioning and biogenesis.     

Distribution of conjugated linoleic acid and metabolites in different lipid fractions in the rat liver.

Conjugated linoleic acid (CLA) is known to provide certain health benefits in experimental animal models. The major CLA isomer in food is c 9,t11-CLA. A primary objective of this study was to investigate the uptake of c 9,t11-CLA and its downstream metabolites into various lipid fractions in the liver of rats fed either a high or low CLA diet (containing 0.1 or 0.8 g CLA/100 g diet, respectively). As expected, the levels of all conjugated diene (CD) fatty acids (CD 18:2 + CD 18:3 + CD 20:3 + CD 20:4) were elevated about 8-fold in the high CLA diet group. However, there was no change in the distribution of CLA and CLA metabolites into various lipid fractions due to CLA intake. Unlike linoleic acid or gamma-linolenic acid, which were distributed mainly in phospholipids, CD 18:2, CD 18:3, and CD 20:3 were incorporated primarily in neutral lipid. Furthermore, the incorporation of all nonconjugated unsaturated fatty acids was not perturbed by CLA. Regardless of the level of CLA in the diet, CD 20:4 was predominantly enriched in phosphatidylserine and phosphatidylinositol. In contrast, arachidonic acid was primarily enriched in phosphatidylcholine and less so in phosphatidylethanolamine. The above findings may have potential implication regarding the role of CLA in modulating eicosanoid metabolism.     

Sequential substitution of 1,2-dichloro-ethene: a convenient stereoselective route to (9Z,11E)-, (10E,12Z)- and (10Z,12Z)-

Conjugated linoleic acid (CLA) isomers are present in human foods derived from milk or ruminant meat. To study their metabolism, (9Z,11E)-, (10E,12Z)- and (10Z,12Z)-[1-(14)C]-octadecadienoic acids with high radiochemical and isomeric purities (>98%) were prepared by stereoselective multi-step syntheses involving sequential substitution of 1,2-dichloro-ethene. In the case of the (9Z,11E) isomer, a first metal-catalyzed cross-coupling reaction between (E)-1,2-dichloro-ethene and 2-non-8-ynyloxy-tetrahydro-pyran, obtained from 7-bromo-heptan-1-ol, gave a conjugated chloroenyne. A second coupling reaction with hexylmagnesium bromide provided a heptadecenynyl derivative. Stereoselective reduction of the triple bond and bromination afforded (7E,9Z)-17-bromo-heptadeca-7,9-diene. Formation of the Grignard reagent and carbonation with 14CO(2) gave (9Z,11E)-[1-(14)C]-octadeca-9,11-dienoic acid (overall yield from 7-bromo-heptan-1-ol, 14.4%). (10E,12Z)- and (10Z,12Z)-[1-(14)C]-octadeca-10,12-dienoic acids were synthesized by the same methodology using 1-heptyne, 8-bromo-octan-1-ol and, respectively, (E)-1,2-dichloro-ethene and its (Z) isomer (overall yield from 8-bromo-octan-1-ol, 13.1% (10E,12Z); 17.2% (10Z,12Z)). Impurities (<2% if present) were identified as being (E,E) CLA isomers and were removed by RP-HPLC. Metabolism studies in animal are in progress.     

Conjugated linoleic acid reduction of murine mammary tumor cell growth through 5-hydroxyeicosatetraenoic acid

Conjugated linoleic acid (CLA) is a dietary fatty acid that has been shown to reduce tumorigenesis and metastasis in breast, prostate and colon cancer in animals. However, the mechanism of its action has not been clarified. The goal of this study was to determine whether CLA altered mouse mammary tumor cell growth and whether specific metabolites of the lipoxygenase pathway were involved in CLA action. Both t10, c12-CLA and a lipoxygenase inhibitor, but not c9, t11-CLA or linoleic acid (LA), reduced mouse mammary tumor cell viability and growth by inducing apoptosis and reducing cell proliferation. t10, c12-CLA reduced the production of the 5-lipoxygenase metabolite, 5-hydroxyeicosatetraenoic acid (5-HETE). That effect was not seen with c9, t11-CLA or LA. Adding 5-HETE back to tumor cells reduced the t10, c12-CLA effect on both apoptosis and cell proliferation. These data suggest that t10, c12-CLA reduction of tumor cell growth may involve the suppression of the 5-lipoxygenase metabolite, 5-HETE, with subsequent effects on apoptosis and cell proliferation.