Cytotoxic effect of conjugated trienoic fatty acids on mouse tumor and human monocytic leukemia cells

The cytotoxicity of fatty acids from seed oils containing conjugated linolenic acids (CLN) was studied. Fatty acids from pomegranate, tung, and catalpa were cytotoxic to human monocytic leukemia cells at concentrations exceeding 5 μM for pomegranate and tung and 10 μM for catalpa, but fatty acids from pot marigold oil had no effect at concentrations ranging up to 163 μM. The main conjugated fatty acids of pomegranate, tung, catalpa, and pot marigold were cis(c)9, trans(t)11, c13-CLN (71.7%), c9,t11,t13-CLN (70.1%), t9,t11,c13-CLN (31.3%), and t8,t10,c12-CLN (33.4%), respectively. Therefore, the cytotoxicities of fatty acids from pomegranate, tung, and catalpa were supposed to be due to 9,11,13-CLN isomers. To elucidate the cytotoxicity of these CLN, we separated each CLN isomer from the fatty acid mixtures by high-performance liquid chromatography and analyzed its cytotoxicity. The cytotoxicities of c9,t11,c13-CLN, c9,t11,t13-CLN, and t9,t11,c13-CLN were much stronger than that of t8,t10,c12-CLN. Therefore, the higher cytotoxicity of fatty acids from pomegranate, tung, and catalpa than those from pot marigold would be derived from the different activities of 9,11,13-CLN and 8,10,12-CLN. Since there was little difference in the cytotoxicities of c9,t11,c13-CLN, c9,t11,t13-CLN, and t9,t11,c13-CLN, it is suggested that the cis/trans configuration of 9,11,13-CLN isomers had little effect on their cytotoxic effects. The mechanism of the cytotoxicity of the four fatty acids above may involve lipid peroxidation, because the order of toxicity of the fatty acids was consistent with their susceptibility to peroxidation in aqueous phase. This was supported by the decrease in the cytotoxicity of the fatty acids by addition of butylated hydroxytoluene.     

Absorption and Metabolism of <Emphasis Type="Italic">cis</Emphasis>-9,<Emphasis Type="Italic">trans</Emphasis>-11-CLA and of Its Oxidation Product 9,11-Furan Fatty Acid by Caco-2 Cells

Furan fatty acids (furan-FA) can be formed by auto-oxidation of conjugated linoleic acids (CLA) and may therefore be ingested when CLA-containing foodstuff is consumed. Due to the presence of a furan ring structure, furan-FA may have toxic properties, however, these substances are toxicologically not well characterized so far. Here we show that 9,11-furan-FA, the oxidation product of the major CLA isomer cis-9,trans-11-CLA (c9,t11-CLA), is not toxic to human intestinal Caco-2 cells up to a level of 100 μM. Oil-Red-O staining indicated that 9,11-furan-FA as well as c9,t11-CLA and linoleic acid are taken up by the cells and stored in the form of triglycerides in lipid droplets. Chemical analysis of total cellular lipids revealed that 9,11-furan-FA is partially elongated probably by the enzymatic activity of cellular fatty acid elongases whereas c9,t11-CLA is partially converted to other isomers such as c9,c11-CLA or t9,t11-CLA. In the case of 9,11-furan-FA, there is no indication for any modification or activation of the furan ring system. From these results, we conclude that 9,11-furan-FA has no properties of toxicological relevance at least for Caco-2 cells which serve as a model for enterocytes of the human small intestine.     

Occurrence of mixtures of geometrical isomers of conjugated octadecatrienoic acids in some seed oils: Analysis by open-tubular gas liquid chromatography and high performance liquid chromatography

Analytical methods to obtain the detailed compositions of the fatty acids in oils containing more than one conjugated octadecatrienoic acid by open-tubular gas liquid chromatography (GLC) and by reversed-phase high performance liquid chromatography (HPLC) were established. Effective GLC separations ofcis,trans,trans-9,11,13-octadecatrienoic acid (ctt-9,11,13–18∶3),ctc-9,11,13–18∶3,ttc-9,11,13–18∶3,ttt-9,11,13–18∶3,ttc-8,10,12–18∶3, andttt-8,10,12–18∶3 were obtained with an opentubular column coated with the nonpolar liquid phase OV-1 using an instrument having all-glass carrier gas pathways. The HPLC method also gave satisfactory separations for the isomeric conjugated octadecatrienoates on the basis of number of thecis andtrans double bonds. Two or three minor conjugated trienoic acids were found along with the principal conjugated trienoic acid in tung oil, and seed oils of cherry,Prunus sp., Momordica charantia, Trichosanthes anguina, Punica granatum, Catalpa ovata, andCalendula officinalis. The mechanism for the formation of the conjugated trienoic acid mixtures in the seed oils is discussed. TheC. ovata seed oil also containedct andtt-9,12-octadecadienoic acids. Thett isomer is presumed to be a precursor ofttc-9,11,13–18∶3, the main conjugated trienoic acid in this oil.     

Characterization of fatty acid isomers in dehydrated castor oil by gas chromatography and gas chromatography-mass spectrometry techniques

Fatty acid isomers present in dehydrated castor oil were analyzed by gas chromatography and gas chromatography-mass spectrometry (GC-MS) of their dimethyl oxazoline derivatives. Conjugated linoleic acid 9,11 and 10,12 isomers were identified by GC based on equivalent chainlengths. Segmental peak analysis of GC-MS total ion chromatogram mass fragmentation pattern revealed the presence of 7,9 and 8,10 conjugated linoleic acids along with 9,11 and 10,12 conjugated linoleic acids. Interestingly, a reverse elution of trans,trans-conjugated linoleic acids was observed, viz. 10,12 was followed by 9,11, which was in turn followed by the 8,10 and 7,9 isomers. The observed reverse order of elution was in contrast to the conventional elution pattern of both nonconjugated and conjugated cis,cis-, cis,trans- and trans,cis-isomers.     

Oleic Acid Attenuates trans-10,cis-12 Conjugated Linoleic Acid-Mediated Inflammatory Gene Expression in Human Adipocytes

The weight loss supplement conjugated linoleic acid (CLA) consists of an equal mixture of trans-10,cis-12 (10,12) and cis-9,trans-11 (9,11) isomers. However, high levels of mixed CLA isomers, or the 10,12 isomer, causes chronic inflammation, lipodystrophy, or insulin resistance. We previously demonstrated that 10,12 CLA decreases de novo lipid synthesis along with the abundance and activity of stearoyl-CoA desaturase (SCD)-1, a δ-9 desaturase essential for the synthesis of monounsaturated fatty acids (MUFA). Thus, we hypothesized that the 10,12 CLA-mediated decrease in SCD-1, with the subsequent decrease in MUFA, was responsible for the observed effects. To test this hypothesis, 10,12 CLA-treated human adipocytes were supplemented with oleic acid for 12?h to 7?days, and inflammatory gene expression, insulin-stimulated glucose uptake, and lipid content were measured. Oleic acid reduced inflammatory gene expression in a dose-dependent manner, and restored the lipid content of 10,12 CLA-treated adipocytes without improving insulin-stimulated glucose uptake. In contrast, supplementation with stearic acid, a substrate for SCD-1, or 9,11 CLA did not prevent inflammatory gene expression by 10,12 CLA. Notably, 10,12 CLA impacted the expression of several G-protein coupled receptors that was attenuated by oleic acid. Collectively, these data show that oleic acid attenuates 10,12 CLA-induced inflammatory gene expression and lipid content, possibly by alleviating cell stress caused by the inhibition of MUFA needed for phospholipid and neutral lipid synthesis.     

Determination of CLA <Emphasis Type="Italic">trans</Emphasis>,<Emphasis Type="Italic">trans</Emphasis> Positional Isomerism in CLA-Rich Soy Oil by GC–MS and Silver Ion HPLC

A ~20% CLA-rich soy oil with low saturated fat and no cholesterol was produced by photoisomerizing soy oil linoleic acid. The oil is predominately trans,trans CLA, with the oil containing 17% trans,trans CLA. Recent studies have shown that trans,trans CLA-rich soy oil significantly reduces heart disease and diabetes risk factors in obese rats. However, the positional isomerism of these geometrical isomers has not been identified. The objectives of the study were to determine trans,trans CLA positional isomerism of CLA fatty acids in CLA-rich soy oil and determine the resolution of trans,trans CLA positional isomers by silver ion chromatography. GC–MS studies of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) derivatives of CLA-rich oil showed that 9,11 CLA and 10,12 CLA were the major positional isomers. These were hypothesized to be the trans,trans CLA isomers, which was confirmed by silver ion chromatography and subsequent GC–FID fatty acid and ATR-FTIR geometrical isomer analysis of the collected fractions. The identification of 9,11 trans,trans CLA and 10,12 trans,trans CLA as the major CLA isomers in CLA-rich oil then allowed the deduction of the synthetic mechanism of the photoisomerism of soy oil linoleic acid to trans,trans CLA.     

<Emphasis Type="Italic">Trans</Emphasis>-Fatty Acid-Stimulated Mammary Gland Growth in Ovariectomized Mice is Fatty Acid Type and Isomer Specific

We previously reported that the trans-18:2 fatty acid trans-10, cis-12 conjugated linoleic acid (t10,c12-CLA) stimulates mammary gland development independent of estrogen and its receptor. Given the negative consequences of dietary trans-fatty acids on various aspects of human health, we sought to establish whether other trans-fatty acids could similarly induce ovary-independent mammary gland growth in mice. Prepubertal BALB/cJ mice were ovariectomized at 21 days of age then were fed diets enriched with cis-9, trans-11 CLA (c9,t11-CLA), or mixtures of trans-18:1 fatty acids supplied by partially hydrogenated sunflower, safflower, or linseed oil. The resultant mammary phenotype was evaluated 3 weeks later and compared to the growth response elicited by t10,c12-CLA, or the defined control diet. Whereas partially hydrogenated safflower oil increased mammary gland weight, none of the partially hydrogenated vegetable oils promoted mammary ductal growth. Similarly, the c9,t11-CLA supplemented diet was without effect on mammary development. Taken together, our data emphasize a unique effect of t10,c12-CLA in stimulating estrogen-independent mammary gland growth manifest as increased mammary ductal area and elongation that was not recapitulated by c9,t11-CLA or the partially hydrogenated vegetable oil diets.     

Comparison of silver-ion high-performance liquid chromatographic quantification of free and methylated conjugated linoleic acids

Silver-ion high-performance liquid chromatography was used to fractionate a mixture of conjugated linoleic acid (CLA) isomers (as the free fatty acids, CLAFFA) in commercial CLA mixtures and biological samples. Due to the unchanged retention mechanism, it was assumed that the elution order of the isomers remained the same as that of methyl esters separated on the same column. The most abundant isomers, cis/trans 10,12-18:2 and cis/trans 9,11-18:2, were separated better as free acids on a single column than in the methyl ester form. Quantification of the CLA standard was used as the reference profile to evaluate different methylation methods commonly used to prepare CLA methyl esters for quantitation. Acid- and vuigi base-catalyzed derivatization methods resulted in CLA intraisomerization and losses in total conjugated dienes content. Acid (HCl and BF₃) methylations significantly elevated the level of trans,trans isomers and significantly reduced the cis/trans isomers. Base methylation, tetramethylguanidine/methanol, resulted in loss of trans,trans isomers, and a substantial loss of total underivatized conjugated dienes. Other catalysts such as the trimethylsilyldiazomethane produced additional peaks of unidentified artifacts. The analysis of CLAFFA appears to provide more accurate quantification of CLA isomers in commercial and biological samples.     

Conjugated Linoleic Acid Isomers Reduce Cholesterol Accumulation in Acetylated LDL-Induced Mouse RAW264.7 Macrophage-Derived Foam Cells

Synthetic activators of peroxisome proliferator-activated receptors (PPAR)-alpha and -gamma are capable of reducing macrophage foam cell cholesterol accumulation through the activation of genes involved in cholesterol homeostasis. Since conjugated linoleic acids (CLA) were also demonstrated to activate PPARalpha and PPARgamma in vivo and in vitro, we tested the hypothesis that CLA are also capable of reducing macrophage foam cell cholesterol accumulation. Thus, mouse RAW264.7 macrophage-derived foam cells were treated with CLA isomers, c9t11-CLA and t10c12-CLA, and linoleic acid (LA), as reference fatty acid, and analyzed for the concentrations of free and esterified cholesterol, cholesterol efflux and expression of genes involved in cholesterol homeostasis (CD36, ABCA1, LXRalpha, NPC-1, and NPC-2). Treatment with c9t11-CLA and t10c12-CLA, but not LA, lowered cholesterol accumulation, stimulated acceptor-dependent cholesterol efflux, and increased relative mRNA concentrations of CD36, ABCA1, LXRalpha, NPC-1, and NPC-2 (P < 0.05). In conclusion, the present study showed that CLA isomers reduce cholesterol accumulation in RAW264.7 macrophage-derived foam cells presumably by enhancing lipid acceptor-dependent cholesterol efflux.     

13C nuclear magnetic resonance spectroscopic analysis of seed oils containing conjugated unsaturated acids

¹³C Nuclear magnetic resonance spectroscopy has been used in a nondestructive investigation of conjugated unsaturated acids in seed oil triacylglycerols. Spectra of seven seed oils, fromPunica granatum, Cucurbita palmata, Jacaranda mimosifolia, Centranthus ruber, Catalpa bignonioides, Chilopsis linearis andCalendula officinalis, containing among them six isomeric trienoic acids,cis,trans,cis- andtrans,trans,cis-8,10,12-,cis,trans,cis-, cis,trans,trans-, trans,trans,cis- andtrans,trans,trans-9,11,13-octadecatrienoic acids, and of the oil ofImpatiens balsamina containingcis,trans,trans,cis-9,11,13,15-octadecatetraenoic acid, have been examined. Structures of component acids were derived from shifts of double bond carbons and of carbons close to the double bond systems. Compositions of the oils were obtained from signal intensities. Results were similar to those obtained by older methods. Only oil ofCentranthus ruber contained more than one major conjugated acid; bothcis,trans,trans- andtrans,trans,trans-9,11,13-octadecatrienoic acids were found. The latter acid is now thought to occur naturally. Presented in part at the International Society for Fat Research/American Oil Chemists' Society World Congress, New York, 1980. NRCC no. 20405.     

Influence of heat and refining on formation of CLA isomers in sunflower oil

The aims of this study were to determine whether CLA are formed during refining of vegetable oils and to study the level and composition of CLA during heating. The effects of three refining steps (neutralization, bleaching, and deodorization) were analyzed with respect to their effect on CLA content. Two temperatures (180 and 220°C) were used for heating; CLA appeared only after deodorization. The level of CLA was positively influenced by temperature. More CLA were present after treatment at 220°C than at 180°C (1.3 and 0.2% of total FA, respectively). The high temperature modified the relative proportions of the CLA isomers. The main CLA isomers in fresh or heated oils were the trans,trans ones (mainly 9,11 and 10,12 isomers).     

Dietary t10,c12-CLA but not c9,t11 CLA Reduces Adipocyte Size in the Absence of Changes in the Adipose Renin–Angiotensin System in <Emphasis Type="Italic">fa/fa</Emphasis> Zucker Rats

In obesity, increased activity of the local renin–angiotensin system (RAS) and enlarged adipocytes with altered adipokine production are linked to the development of obesity-related health problems and cardiovascular disease. Mixtures of conjugated linoleic acid (CLA) isomers have been shown to reduce adipocyte size and alter the production of adipokines. The objective of this study was to investigate the effects of feeding individual CLA isomers on adipocyte size and adipokines associated with the local adipose RAS. Male fa/fa Zucker rats received either (a) control, (b) cis(c)9,trans(t)11-CLA, or (c) t10,c12-CLA diet for 8 weeks. The t10,c12-CLA isomer reduced adipocyte size and increased cell number in epididymal adipose tissue. RT-PCR and Western blot analysis revealed that neither CLA isomer altered mRNA or protein levels of angiotensinogen or AngII receptors in adipose tissue. Likewise, levels of the pro-inflammatory cytokines TNF-α and IL-6 or the anti-inflammatory cytokine IL-10 were unchanged in adipose tissue. Similarly, neither CLA isomer had any effect on phosphorylation nor DNA binding of NF-κB. Our results suggest that although the t10,c12-CLA isomer had beneficial effects on reducing adipocyte size in obese rats, this did not translate into changes in the local adipose RAS or associated adipokines.     

Synthesis and isolation of <Emphasis Type="Italic">trans</Emphasis>-7, <Emphasis Type="Italic">cis</Emphasis>-9 octadecadienoic acid and other CLA isomers by base conjugation of partially hydrogenated γ-linolenic acid

CLA is of considerable interest because of reported potentially beneficial effects in animal studies. CLA, while not yet unambiguously defined, is a mixture of octadecadienoic acids with conjugated double bonds. The major isomer in natural products is generally considered to be cis-9,trans-11-octadecadienoic acid (c9, t11), which represents >75% of the total CLA in most cases. Other isomers are drawing increased attention. The t7,c9 isomer, which is often the second-most prevalent CLA in natural products, has been reported to represent as much as 40% of total CLA in milk from cows fed a high-fat diet. The need for a reference material became apparent in a recent study directed specifically at measuring t7,c9-CLA in milk, plasma, and rumen. A suitable standard mixture was produced by stirring 0.5 g of γ-linolenic acid (all cis-6,9, 12-C_18∶3) with 100 mL of 10% hydrazine hydrate in methanol for 2.5 h at 45°C. The solution was diluted with H₂O and acidified with HCl. The resulting partially hydrogenated FA were extracted with ether/petroleum ether, dried with Na₂SO₄, and conjugated by adding of 6.6% KOH in ethlylene glycol and heating for 1.5 h at 150–160°C. Approximately 20 mg each of cis-6, trans-8; trans-7, cis-9; cis-9, trans-11; and rans-10, cis-12 were obtained along with other FA. Methyl esters (FAME) of these four cis/trans isomers were resolved by Ag⁺HPLC (UV 233) and partially resolved by GC/(MS or FID) (CP-Sil 88). Treatment of these FAME with I₂ yielded all possible cis/trans (geometric) isomers for the four positions 6,8; 7,9; 9,11; and 10,12.     

Thermally induced formation of conjugated isomers of linoleic acid

Chemical pathways responsible of the conjugation of linoleic acid during heat treatments such as refining (deodorization), frying or cooking processes have been investigated. For this purpose, methyl linoleate was submitted to oxidative and non‐oxidative thermal conditions. The resulting degradation products were mainly composed of geometrical and conjugated fatty acid isomers. Oxidative conditions were obtained using tert‐butyl hydroperoxide under inert atmosphere, and air. The obtained results from both thermal oxidative conditions were compared to non‐oxidative thermal treatment. Higher levels of conjugated linoleic acid were found when linoleate was heated under oxidative conditions. Two distinct mechanisms responsible for the formation of CLA isomers are proposed and discussed. Evidence of formation of 9,11‐C18:2 and 10,12‐C18:2 acids from 9,12‐C18:2 by a free‐radical chain reaction is provided. The first step consists in the formation of a free radical by abstraction of an active bis‐allylic hydrogen. By delocalization of the initial free radical, two allylic free radicals were stabilized and converted into the corresponding CLA isomers via the abstraction of a hydrogen radical from other linoleic acid or oxygenated species. Kinetic observations confirmed the significance of the bimolecular mechanism. Moreover, the proposed mechanism is supported by several pieces of information from the literature on peroxidation of linoleic acid. Under pure thermal conditions and/or for diluted samples, a second pathway to the formation of CLA from heat‐treated linoleic acid is proposed via an intramolecular rearrangement of the pentadienyl structure. This thermal [1,3]‐sigmatropic rearrangement results in a mixture of 9,11 and 10,12 CLA isomers. The formed cis/trans CLA isomers were readily rearranged by a [1,5]‐sigmatropic shift to yield trans‐8,cis‐10 and cis‐11,trans‐13 CLA isomers, respectively.     

Furan fatty acids determined as oxidation products of conjugated octadecadienoic acid

The objective of this study was to identify oxidation products of conjugated linoleic acid (CLA), a series of octadecadienoic acids with conjugated double bonds, which have been reported to have antioxidant and anticarcinogenic properties. Reference materials of CLA were oxidized in different concentrations of water/methanol; for example, 0.5 g octadecadienoic acid was dissolved in 50 mL methanol, and 100 mL water was added; this suspension was heated at 50°C and continuously aerated. Aliquots of 5 mL were taken over time, extracted with ether, treated with diazomethane and examined by gas chromatography/mass spectrometry and/or gas chromatography with flame-ionization detection. Products identified included the following furan fatty acids (FFAs): 8,11-epoxy-8,10-octadecadienoic; 9,12-epoxy-9,11-octadecadienoic; 10,13-epoxy-10,12-octadecadienoic; and 11,14-epoxy-11,13-octadecadienoic. Conjugated dienes should be considered as a possible source of FFAs, and CLA may have products common to furans in their overall oxidative scheme.     

Modulation of arachidonic acid distribution by conjugated linoleic acid isomers and linoleic acid in MCF-7 and SW480 cancer cells

The relationship between growth and alterations in arachidonic acid (AA) metabolism in human breast (MCF-7) and colon (SW480) cancer cells was studied. Four different fatty acid preparations were evaluated: a mixture of conjugated linoleic acid (CLA) isomers (c9,t11, t10,c12, c11,t13, and minor amounts of other isomers), the pure c9,t11-CLA isomer, the pure t10,c12-CLA isomer, and linoleic acid (LA) (all at a lipid concentration of 16 microg/mL). 14C-AA uptake into the monoglyceride fraction of MCF-7 cells was significantly increased following 24 h incubation with the CLA mixture (P < 0.05) and c9,t11-CLA (P < 0.02). In contrast to the MCF-7 cells, 14C-AA uptake into the triglyceride fraction of the SW480 cells was increased while uptake into the phospholipids was reduced following treatment with the CLA mixture (P < 0.02) and c9,t11-CLA (P < 0.05). Distribution of 14C-AA among phospholipid classes was altered by CLA treatments in both cell lines. The c9,t11-CLA isomer decreased (P < 0.05) uptake of 14C-AA into phosphatidylcholine while increasing (P < 0.05) uptake into phosphatidylethanolamine in both cell lines. Both the CLA mixture and the t10,c12-CLA isomer increased (P < 0.01) uptake of 14C-AA into phosphatidylserine in the SW480 cells but had no effect on this phospholipid in the MCF-7 cells. Release of 14C-AA derivatives was not altered by CLA treatments but was increased (P < 0.05) by LA in the SW480 cell line. The CLA mixture of isomers and c9,t11-CLA isomer inhibited 14C-AA conversion to 14C-prostaglandin E2 (PGE2) by 20-30% (P < 0.05) while increasing 14C-PGF2alpha by 17-44% relative to controls in both cell lines. LA significantly (P < 0.05) increased 14C-PGD2 by 13-19% in both cell lines and increased 14C-PGE2 by 20% in the SW480 cell line only. LA significantly (P < 0.05) increased 5-hydroperoxyeicosatetraenoate by 27% in the MCF-7 cell line. Lipid peroxidation, as determined by increased levels of 8-epi-prostaglandin F2alpha (8-epi-PGF2alpha), was observed following treatment with c9,t11-CLA isomer in both cell lines (P < 0.02) and with t10,c12-CLA isomer in the MCF-7 cell line only (P < 0.05). These data indicate that the growth-promoting effects of LA in the SW480 cell line may be associated with enhanced conversion of AA to PGE2 but that the growth-suppressing effects of CLA isomers in both cell lines may be due to changes in AA distribution among cellular lipids and an altered prostaglandin profile.     

Differential Inflammatory Responses in Cultured Endothelial Cells Exposed to Two Conjugated Linoleic Acids (CLAs) under a Pro-Inflammatory Condition

Conjugated linoleic acid (CLA) isomers have been shown to possess anti-atherosclerotic properties, which may be related to the downregulation of inflammatory pathways in different cell types, including endothelial cells (ECs). However, whether different CLA isomers have different actions is not entirely clear, with inconsistent reports to date. Furthermore, in cell culture studies, CLAs have often been used at fairly high concentrations. Whether lower concentrations of CLAs are able to affect EC responses is not clear. The aim of this study was to evaluate the effects of two CLAs (cis-9, trans-11 (CLA9,11) and trans-10, cis-12 (CLA10,12)) on the inflammatory responses of ECs. ECs (EA.hy926 cells) were cultured under standard conditions and exposed to CLAs (1 to 50 μM) for 48 h. Then, the cells were cultured for a further 6 or 24 h with tumour necrosis factor alpha (TNF-α, 1 ng/mL) as an inflammatory stimulant. ECs remained viable after treatments with 1 and 10 μM of each CLA, but not after treatment with 50 μM of CLA10,12. CLAs were incorporated into ECs in a concentration-dependent manner. CLA10,12 increased the levels of ICAM-1, IL-6, and RANTES in the culture medium, while CLA9,11 had null effects. Both CLAs (1 μM) decreased the appearance of NFκB1 mRNA, but only CLA9,11 maintained this downregulation at 10 μM. CLA10,12 had no effect on THP-1 cell adhesion to ECs while significantly decreasing the percentage of ECs expressing ICAM-1 and also levels of ICAM-1 expression per cell when used at 10 µM. Although CLA9,11 did not have any effect on ICAM-1 cell surface expression, it reduced THP-1 cell adhesion to the EA.hy926 cell monolayer at both concentrations. In summary, CLA10,12 showed some pro-inflammatory effects, while CLA9,11 exhibited null or anti-inflammatory effects. The results suggest that each CLA has different effects in ECs under a pro-inflammatory condition, highlighting the need to evaluate the effects of CLA isomers independently.     

Biosynthesis of conjugated linoleic acid in humans

This paper deals with the reanalysis of serum lipids from previous studies in which deuterated fatty acids were administered to a single person. Samples were reanalyzed to determine if the deuterated fatty acids were converted to deuterium-labeled conjugated linoleic acid (CLA, 9c, 11t-18∶2) or other CLA isomers. We found 11-trans-octadecenoate (fed as the triglyceride) was converted (Δ9 desaturase) to CLA, at a CLA enrichment ofca. 30%. The 11-cis-octadecenoate isomer was also converted to 9c, 11c-18∶2, but at <10% the concentration of the 11t-18∶1 isomer. No evidence (within our limits of detection) for conversion of 10-cis-or 10-trans-octadecenoate to the 10,12-CLA isomers (Δ12 desaturase) was found. No evidence for the conversion of 9-cis, 12-cis-octadecadienoate to CLA (via isomerase enzyme) was found. Although these data come from isomerase enzyme) was found. Although these data come from four single human subject studies, data from some 30 similar human studies have convinced us that the existence of a metabolic pathway in one subject may be extrapolated to the normal adult population.     

Conjugated Linoleic Acids Have Anti-Inflammatory Effects in Cultured Endothelial Cells

Conjugated linoleic acid (CLA) isomers may have a role in preventing atherosclerosis through the modulation of inflammation, particularly of the endothelium. However, whether low concentrations of CLAs are able to affect basal unstimulated endothelial cell (EC) responses is not clear. The aim of this study was to evaluate the effects of two CLAs (cis-9, trans-11 (CLA9,11) and trans-10, cis-12 (CLA10,12)) on the basal inflammatory responses by ECs. EA.hy926 cells (HUVEC lineage) were cultured under standard conditions and exposed to individual CLAs for 48 h. Both CLAs were incorporated into ECs in a dose-dependent manner. CLA9,11 (1 μM) significantly decreased concentrations of MCP-1 (p < 0.05), IL-6 (p < 0.05), IL-8 (p < 0.01) and RANTES (p < 0.05) in the culture medium. CLA10,12 (10 μM) decreased the concentrations of MCP-1 (p < 0.05) and RANTES (p < 0.05) but increased the concentration of IL-6 (p < 0.001). At 10 μM both CLAs increased the relative expression of the NFκβ subunit 1 gene (p < 0.01 and p < 0.05, respectively), while decreasing the relative expression of PPARα (p < 0.0001), COX-2 (p < 0.0001) and IL-6 (p < 0.0001) genes. CLA10,12 increased the relative expression of the gene encoding IκK-β at 10 μM compared with CLA9,11 (p < 0.05) and increased the relative expression of the gene encoding IκBα at 1 and 10 μM compared with linoleic acid (both p < 0.05). Neither CLA affected the adhesion of monocytes to ECs. These results suggest that low concentrations of both CLA9,11 and CLA10,12 have modest anti-inflammatory effects in ECs. Thus, CLAs may influence endothelial function and the risk of vascular disease. Nevertheless, at these low CLA concentrations some pro-inflammatory genes are upregulated while others are downregulated, suggesting complex effects of CLAs on inflammatory pathways.